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Analysis of ret/PTC Gene Rearrangements Refines the Fine Needle Aspiration Diagnosis of Thyroid Cancer

Departments of Pathology and Laboratory Medicine (C.C.C., B.C., Y.C.B., S.A.) and Medicine (Endocrinology) (S.E.), The Freeman Center for Endocrine Oncology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5G 2M9

Address all correspondence and requests for reprints to: Dr. Sylvia Asa, University Health Network, 610 University Avenue, 4-302, Toronto, Ontario, Canada M5G 2M9. E-mail: sylvia.asa{at}uhn.on.ca

	Abstract

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Papillary carcinoma (PC) represents the most common malignancy of the thyroid gland. Therefore, the assessment of fine needle aspiration biopsies of thyroid nodules rests heavily on the identification of nuclear features of PC. The ret/PTC oncogene, formed by several gene rearrangements, is specific for PC among thyroid tumors. In this study we examined thyroid aspirates for the presence of ret/PTC gene rearrangements by RT-PCR and Southern hybridization. We prospectively collected thyroid aspirates in Cytolyt solution and prepared slides for cytological examination using the ThinPrep method. All remaining material was then used for nucleic acid extraction with subsequent RT-PCR for the housekeeping gene PGK-1 to ensure ribonucleic acid integrity, for thyroglobulin to ensure the presence of follicular epithelial cells, and for the three most common ret/PTC gene rearrangements (ret/PTC-1, -2, and -3). The results of the first 73 cases with surgical follow-up were correlated with the cytological diagnosis and final histopathology. ret/PTC gene rearrangements were detected in 17 of 33 samples (52%) that were PC on histopathology; the presence of gene rearrangements was confirmed by molecular analysis of corresponding surgically resected frozen tissue. There were no false positives. The identification of ret/PTC gene rearrangements refined the diagnosis of PC in 9 of 15 specimens (60%) that would otherwise have been considered indeterminate and in 2 of 6 that were considered insufficient for cytological diagnosis. The results indicate that RT-PCR for ret/PTC is a specific marker that can be applied to fine needle aspiration biopsies and improves the diagnosis of malignancy when used as an adjunct to traditional cytology.

	Introduction

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THE EVALUATION of a thyroid nodule is a very common clinical problem. Up to 20% of the general population has a palpable thyroid nodule; with ultrasonography, that figure becomes 70% (1). However, only approximately 5% of clinically apparent thyroid nodules actually harbor malignancy (2). The challenge facing general practitioners, endocrinologists, surgeons, and pathologists is to arrive at an accurate preoperative diagnosis of malignancy and to ensure that the patient receives timely and appropriate treatment. An inaccurate or uncertain preoperative diagnosis results in delays, less than definitive surgery, or unnecessary surgical intervention with the risks that it entails.

The information used in the evaluation of patients presenting with thyroid nodules includes serum TSH, thyroid scintigraphy, computed tomography/magnetic resonance imaging, ultrasonography, and fine needle aspiration (FNA). Of these, FNA is the only test that can provide a definitive preoperative diagnosis of malignancy (2). The sensitivity and specificity of FNA are reported to be 68–98% and 56–100%, respectively (3); however, these data often exclude aspirates that yield insufficient material for definitive diagnosis and those with indeterminate diagnoses; together, they account for almost half of all FNA specimens (2, 3). This results in repeated aspirations and delays diagnosis. Of specimens that are adequate, approximately one quarter are labeled indeterminate. This leads to increased rates of unnecessary surgery, as only about one quarter of indeterminate cases will receive a postoperative malignant diagnosis by histological examination (2). Moreover, patients with this diagnosis usually undergo hemithyroidectomy, and a malignant diagnosis in 25% leads to second stage completion thyroidectomy in most centers (3); this two-stage surgery has higher morbidity than initial total thyroidectomy that is undertaken with a definitive malignant diagnosis on FNA (4).

The most common malignancy of the thyroid gland is papillary carcinoma (PC); it represents 95% of thyroid malignancy in North America. The evaluation of a thyroid nodule is, therefore, primarily a search for PC. At the molecular level, more than half of PCs harbor one of several chimeric oncogenes called ret/PTC, which result from gene rearrangements involving the ret protooncogene on chromosome 10 (5, 6, 7). The generation of these novel fusion transcripts provides us with a molecular marker that is specific for PC (8) and is identified in almost half of these lesions in most populations (6, 9, 10).

We hypothesized that the application of RT-PCR for ret/PTC fusion transcripts on FNA specimens of thyroid would improve the diagnostic yield of this technique, especially in nondiagnostic cases.

	Materials and Methods

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Inclusion criteria

Thyroid aspirates eligible for this study satisfied all of the following requirements: material undergoing molecular analysis contained thyroid follicular epithelial cells as determined by the presence of thyroglobulin messenger ribonucleic acid (mRNA) on RT-PCR, the extracted RNA was of appropriate integrity and quality to undergo RT-PCR as determined by the presence of PGK-1 mRNA by RT-PCR, and a definitive histological diagnosis from a thyroidectomy specimen was available as the gold standard against which the cytology and molecular results would ultimately be measured.

Overview and general methodology

We prospectively collected thyroid aspirates in Cytolyt fixative (Cytyc Corp., Boxborough, MA), and samples were prepared using the ThinPrep method for cytological evaluation. In some cases, conventional smears were also available for cytological examination. Samples were not used for molecular analysis until the routine cytology examination was complete and reported to avoid any compromise of patient care. After completion of cytological reporting, usually after 3 days but within 1 week of the biopsy procedure, the remaining material that had been stored at room temperature was used for RNA extraction for RT-PCR (see below). Cytological, molecular, and histological analyses of all samples were performed independently and in a completely blinded fashion.

Cytological evaluation

Cytological diagnoses were grouped into four diagnostic categories: insufficient, benign, indeterminate, or malignant. The definition of adequacy requires at least six clusters of thyroid follicular epithelial cells on two slides (11); in the absence of an adequate specimen, the diagnosis reported was insufficient. The ThinPrep processor (Cytyc Corp.) constantly monitors the rate of flow during the cell collection process, indicating whether a specimen is dilute. All specimens in this category were collected to the limit of the technology for cytological evaluation; the analysis of ret/PTC rearrangements was carried out on the remainder of the material. The benign category included the following diagnoses: benign unspecified, colloid nodule, or thyroiditis. The malignant category included papillary carcinoma, malignant not otherwise specified, and anaplastic carcinoma. The diagnostic criteria for PC require the identification of at least the minimal six features of this disorder (12): syncytial tissue fragments, enlarged nuclei with fine dusty chromatin, chromatin ridge (also known as nuclear grooves), single/multiple micro/macronucleoli, and intranuclear inclusions. The indeterminate category included follicular lesion, Hurthle cell lesion, cellular atypia (defined as having up to two of the features of PC), and suspicious for malignancy (defined as having three to five features of PC).

Histology and immunohistochemistry

After surgical resection, tissues fixed in 10% neutral buffered formalin were completely embedded in 3- to 4-mm paraffin blocks and 4-µm-thick sections were stained with hematoxylin and eosin for histological examination. The nuclear and architectural features were carefully evaluated. For confirmation of diagnosis, sections were immunostained for HBME-1, CK19, and Ret as previously described (13) using the ultrastreptavidin detection system (Signet, Dedham, MA) and the following primary antibodies/antisera: monoclonal antibody HBME-1 (DAKO Corp., Carpinteria, CA; 1:100), monoclonal anti-CK-19 (clone b170, Novocastra, Newcastle upon Tyne, UK; 1:150), and polyclonal antiserum to Ret (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:200). Sections stained for Ret or HBME-1 underwent pretreatment with 44% formic acid, and sections stained for CK-19 were pretreated with pepsin.

RNA extraction and RT-PCR

RNA was extracted using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). The final pellet was resuspended in 10 µL diethylpyrocarbonate water. RT was performed on one fifth of the sample as previously described (14). Each RT sample underwent PCR for the housekeeping gene PGK-1 to ensure RNA integrity (14) and for thyroglobulin mRNA to ensure the presence of thyroid follicular epithelial cells (15). PCR for ret/PTC-1, -2, and -3 was performed as described previously (14). Negative controls performed with each RT-PCR reaction omitted either template or reverse transcriptase. The products were resolved on a 1.2% agarose gel containing ethidium bromide and visualized under UV light.

Southern hybridization

PCR products were transferred to nylon membranes (Roche, Laval, Canada) by upward capillary action in 20 x SSC (standard saline citrate) followed by UV cross-linking. Complementary DNA probes (provided by Dr. S. Jhiang, Columbus, OH) were labeled with digoxigenin as described previously (7). Labeling, hybridization, and detection were performed according to manufacturer’s protocol (Roche).

	Results

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The results of the study are summarized in Table 1. Of the first 75 cases collected, 73 cases fulfilled the inclusion criteria; 2 cases lacked detectable thyroglobulin mRNA. The final histopathological diagnoses were 33 PCs, 11 follicular adenomas, 25 nodular hyperplasias, 3 Hashimoto’s thyroiditis, and 1 anaplastic carcinoma. Among the 33 PCs confirmed at histopathological examination, 21 contained ret/PTC gene rearrangements identified by RT-PCR of frozen tissue, and 20 of these exhibited at least focal immunohistochemical positivity for ret.

View larger version (17K): [in this window] [in a new window]   Figure 1. Representative RT-PCR for ret/PTC-1 in thyroid aspirates. Two of five PC (lanes 6–10) have detectable ret/PTC-1 transcripts; none of five benign lesions (lanes 1–5) is positive. Kb, Size ladder; -, negative control without reverse transcriptase; +, positive control.

The molecular analysis proved most useful in 9 of 15 cases of PC that would otherwise have been classified as indeterminate based on the presence of some atypical features on the cytological evaluation, but these cases did not fulfil the criteria for a cytological diagnosis of malignancy.

On final analysis, 21 of the 33 PCs had ret/PTC gene rearrangements on frozen tissue; only 17 of these were detected in the cytological specimen.

	Discussion

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The use of ThinPrep methodology for the cytological evaluation of thyroid has been controversial (16, 17, 18). Our institution has used this technique, and the results of our cytological evaluation are comparable to those in the literature. Although the transition to this methodology requires adaptation by the technologists and pathologists who evaluate the material, it appears to be a valuable investment. We show that good quality mRNA can be extracted from thyroid cells fixed in Cytolyt, making possible the addition of molecular diagnostics in this field that suffers from a high yield of indeterminate and insufficient outcomes.

We detected the presence of a ret/PTC fusion transcript in 17 of 33 FNA samples, all of which were histologically proven papillary carcinomas; the most frequent rearrangement was ret/PTC-1, ret/PTC-3 was found in 3 cases, and ret/PTC-2 was present in only 1 case. Our method would also have detected ret/PTC-4 and ret/PTC-5, but these rearrangements were not identified. These results are consistent with other published reports documenting the frequency of such rearrangements in these tumors (7). In all cases where ret/PTC was detected in FNA samples, the presence of the gene rearrangement was confirmed in the corresponding surgically resected tumor. There were 4 cases where a ret/PTC rearrangement was present in the surgically resected tumor, but could not be detected in the FNA sample. This may be due to sampling error. As we used thyroglobulin as the positive control for the presence of thyroid follicular cells in our specimens, this could reflect failure to biopsy tumor and inclusion of nontumorous thyroid in the aspirate. Alternatively, this could represent a limitation of the technique, attributable to variable levels of ret/PTC expression in different areas of a given tumor.

No false positive results were reported in this study. One paper in the literature has reported the identification of ret/PTC rearrangements in Hashimoto’s thyroiditis (19). Our series contained three cases of Hashimoto’s thyroiditis; none contained ret/PTC rearrangements. However, this small number cannot exclude the possibility of this potential source of false positive results. It should be noted that a false positive diagnosis of papillary carcinoma occurs in a small percentage of patients using cytological evaluation alone (12). The lack of false positive results in this initial study is encouraging. It confirms that ret/PTC is a specific and useful tool in the diagnosis of papillary thyroid carcinoma. It paves the way for larger prospective studies using this and other genetic defects in the diagnosis of thyroid carcinoma.

Superficially, it would seem that RT-PCR is more sensitive because it detected more cases than cytology alone (17 vs. 12). However, examining the distribution of these cases is much more revealing. Based on our data, RT-PCR of FNA is most informative for aspirates that would otherwise have been nondiagnostic. In 2 of 6 histologically proven papillary carcinomas that contained insufficient material for diagnosis by cytological examination, the correct diagnosis was made by RT-PCR for ret/PTC on FNA samples. There were 15 samples considered indeterminate by cytological examination that were histologically proven to be papillary carcinomas; in 9 of these cases we were able to demonstrate their malignant nature using molecular analysis of the aspirated material. On the other hand, of the 12 histologically proven papillary carcinomas that were correctly identified by cytology alone, only 6 were identified by RT-PCR alone. This underscores the fact that RT-PCR for ret/PTC should not and cannot replace cytology; it can, however, be a very useful adjunct to traditional cytology in refining the yield from the FNA approach. In this series, the combination of techniques allowed an increased diagnostic yield from 12 cases definitively diagnosed by cytological examination alone to 23 cases diagnosed by cytology and RT-PCR.

Our study has examined one of several potential molecular markers of malignancy in thyroid. We suggest a new approach to the handling and evaluation of FNA specimens; as new molecular genetic markers are identified, molecular diagnostics on FNA material will continue to expand and play a role in the management of patients with suspected endocrine neoplasms.

Received November 16, 2000.

Revised January 17, 2001.

Accepted February 1, 2001.

	References

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