[CANCERRESEARCH57.2144-2147. June I, 1997) Advances in Brief Telomerase Activity: A Marker to Distinguish Follicular Thyroid Adenoma from Carcinoma' Christopher B. Umbricht, Motoyasu Saji, William H. Westra, Robert Udelsman, Martha A. Zeiger, and Saraswati Sukumar@ Breast Cancer Program. Oncology Center (C. B. U., S. SI. Division of Surgical Oncology and Endocrine Surgery (M. S.. R. U., M. A. 1], and Department of Pathology 1W. H. WI, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Abstract Finally, activation of one of the three ras oncogenes appears to be a fairly common and early event (5) but is detected in adenomas as well The inability to distinguish microinvasivefollicular thyroid cancer as in carcinomas. More importantly, less than 50% of follicular from benign follicular tumors preoperatively presents an important sur carcinomas in any published series show these changes (6, 7). Thus, gical dilemma. We examined 44 folilcular tumors and found telomerase no molecular markers that can reliably distinguish adenomas from activity in all 11 folllcular carcinomas and in S of 33 benign fofficular tumors. It was undetectable in 22 normal thyroid tissues adjacent to the carcinomas have yet been found. Recently, activation of the ribonu tumors. Telomerase activity may thus provide a diagnostic marker dis cleoprotein telomerase has been found in a wide variety of carcinomas tinguishing benign from malignant follicular thyroid tumors. The ability (8—li). Telomerase is an enzyme that maintains the stability and to identify invasive follicular thyroid tumors could avert over 14,000 integrity of chromosomal ends composed of telomeres (12). Although thyroidectomies annually in the United States, thereby significantly de telomerase activity is repressed in almost all nonneoplastic somatic creasing morbidity and health care costs. cells, reactivation of the enzyme appears to be a necessary event for the sustained growth of most human malignant neoplasms (13). Ac Introduction cordingly, detection oftelomerase activity may be a useful strategy for distinguishing benign from malignant lesions, especially when the Estimates of the prevalence of thyroid nodules in the general distinction is not straightforward on morphological grounds alone (14, population range from 4% as detected by palpation to 67% as detected 15). We have analyzed telomerase activity in follicular thyroid lesions by high resolution ultrasonography (1). The introduction of FNA3 for to determine whether this assay could be useful in distinguishing the evaluation of thyroid nodules has had a major impact on clinical between benign follicular lesions and follicular carcinomas. management, leading to a 35—75%reduction in the number of patients requiring surgery (1). Nevertheless, 10% of FNAs are classified as Materials and Methods suspicious for carcinoma, and the patients need to undergo surgery to clarify their diagnosis. Of 28,992 thyroid resections performed in the Tissue Samples. All cases undergoing thyroidectomy for suspected follic United States in 1995, as many as 17,557 were for benign uninodular ular thyroid cancer for which frozen tissue was available in our tissue bank thyroid tumors (2). Follicular neoplasms represent the majority of were collected for this study. None of the tumors recurred after thyroidectomy. suspicious FNAs, 80% of which are postoperatively diagnosed as Tissue blocks from the center of thyroid nodules were obtained at the time of benign (1, 3). The difficulty in the diagnosis of follicular neoplasms of surgery by a staff pathologist and stored at —80°Cwitha coded identifier. All the thyroid stems from the fact that microinvasive follicular carcino cases with a final pathological diagnosis of medullary, papillary, or mixed mas differ from follicular adenomas only in that they exhibit either thyroid cancer were excluded from this analysis. Adjacent normal thyroid capsular or vascular invasion, a feature impossible to assess on FNA. tissue was obtained when possible. The tissue blocks were embedded in OCT. and two consecutive 10-pm cryostat sections were obtained and immediately Even intraoperatively, frozen section evaluation of follicular neo frozen in liquid nitrogen and stored at —80°C.Oneslide was stained with H&E plasms often cannot distinguish benign from malignant (4). Because and reviewed with the study pathologist in conjunction with the paraffin of this, patients with asymptomatic follicular adenomas of the thyroid embedded sections for evidence of invasive growth and the presence of often undergo unnecessary surgery, and conversely, patients with lymphocytic infiltrates. The tissue of the other cryostat section was suspended follicular carcinomas may initially undergo inadequate surgery. in 50 @.tlof telomerase lysis buffer [1 X TLB = 0.5% (3-[(3-cholamidopropyl Therefore, markers capable of differentiating benign from malignant dimethyl-ammonio].l-propane sulfonate, 10 mist Tris-HC1 (pH 7.5), 1 mist follicular neoplasms would have a significant impact on the manage MgCl2, 1 tflM EGTA, 5 mist f3-mercaptoethanol, 0.1 mM (4-(2-aminoethyl)- ment of these patients. Follicular neoplasms of the thyroid have been benzenesulfonyl fluoride hydrochiorine, and 10% glycerol], and extracts were evaluated by cytogenetic and loss of heterozygosity studies (5—7). prepared as described (16). Protein concentrations were determined by Brad Four of five investigations on follicular carcinomas showed some ford assay (17). Cytological samples from FNAs were obtained after adequate diagnostic samples had been made by expelling the residual aspirate on a glass abnormality of the short arm of chromosome 3, whereas two studies slide and snap freezing the sample in liquid nitrogen. Extracts were then of follicular adenomas showed abnormalities in both 3p and llq. prepared by overlaying the frozen samples with 50 @a1ofTLB and recovering the lysate by aspiration. Received 2/27/97; accepted 4/20/97. Telomerase Assay. Aliquots of tissue extract containing 0.5 @gofprotein, The costs of publication of this article were defrayed in part by the payment of page with and without inactivation with RNase A (16), were used for the telomerase charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. assays. Standard TRAP assays were performed as described (16). All assays I Supportedin part by JohnsHopkinsOncologyCenterfunds(to S .5.) andby Grant contained 10ag ofthe l50-bp ITAS for detection ofTaq polymerase inhibitors 9610 from the Susan 0. Komen Foundation (to C. B. U.). (18). When ITAS inhibition was detected, the assay was modified to include an 2 To whom requests for reprints should be addressed, at Johns Hopkins Oncology extraction with phenol:chloroform:isoamyl alcohol at 25:24:1, followed by Center, 370 Ross Building. 720 Rutland Avenue, Baltimore, MD 21205-2196. Phone: (410) 614-2484; Fax: (410) 614-4073; E-mail: [email protected]. ethanol precipitation (19) before the PCR amplification step. PCR products 3 The abbreviations used are: FNA, fine-needle aspiration; TRAP, telomere repeat were electrophoresed in a 10% nondenaturing polyacrylamide gel. The DNA amplification protocol; ITAS. internal telomerase assay standard; ag, attogram (I0 “g). ladders were visualized using a Molecular Dynamics Phosphorimager after a 2144 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1997 American Association for Cancer Research. TELOMERASE AC1'Wll'Y IN FOWCIJLAR ThYROID TUMORS @,@ 12-hexposure. Detectable telomerase activity was defined as a hexanucleotide , FC ni FC ni FC ni PC ni ladder of three or more bands not present in matched buffer controls. The assays were performed without knowledge of sample diagnoses. RNAseA: + + + + + + + + Statistical Analysis. The tissue samples were divided into two groups: 1 2 3 4 5 6 7 8 9 10111213141516 Bu samples with no detectable telomerase activity; and samples with detectable telomeraseactivity.The followingparameterswereusedfor univariateanal ITAS@ @ ysis: presence of invasive carcinoma,tumor size, presence of oxyphilic .5 1 (Huerthie cell) histological subtype, patient age, and sex. All Ps refer to either Fisher's Exact test for tables or Wilcoxon's Rank Sum test for nonparametric data, where appropriate (20). The analysis was performed using the JMP statistical software package (SAS Institute, Inc.) on a Macintosh microcom puter. Results A total of 66 frozen samples of follicular neoplasms, hyperplastic nodules, and normal thyroid tissue were obtained to prepare whole cell extracts. Standard TRAP assays were performed as described (16, c- 18)on44thyroidtissues,theFNAsof whichshowedafollicular lesion (11 follicular carcinomas, 23 follicular adenomas, and 10 b hyperplastic nodules), and on 22 matched normal thyroid tissue spec miens obtained from 8 cases of carcinoma and 14 cases of benign ‘I. follicular lesions. The data are summarized in Table I, and represent ative results of TRAP assays are presented in Figs. 1 and 2. All 11 ••1 carcinomas were positive for telomerase activity, showing the char @ acteristic ladder of hexamer telomere repeats. Five of 23 follicular Telomerase + + + adenomas (22%) and 3 of 10 hyperplastic nodules (30%) were also activity: positive. All normal thyroid tissue samples were negative. As illus Fig. 1. Telomerase activity in follicular thyroid cancer. Clarified tissue lysates (0.5 @g trated in Fig. 2, no appreciable difference was apparent between