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Departments of Endocrinology (S.-Y.C., S.K.R., M.S.), Clinical Pathology (M.K.G.), General Surgery (M.M., A.S.), and Anatomic Pathology (J.B.), The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Address all correspondence and requests for reprints to: Manjula K. Gupta, Department of Clinical Pathology (L-30), The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195. E-mail: guptam{at}ccf.org.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: Our objective was to assess the diagnostic value of circulating TSHR mRNA for preoperative detection of DTC in patients with thyroid nodules.
Patients: We measured TSHR mRNA levels by RT-PCR in 258 subjects: 51 healthy subjects and 207 patients (thyroid nodules, n = 180; recurrent thyroid cancer, n = 27) with fine-needle aspirations (FNA) and/or thyroid/neck surgery. Eighty-nine patients also had d-1 postoperative levels assessed.
Outcome Measures: TSHR mRNA levels were compared with FNA cytology for cancer detection preoperatively and serum thyroglobulin and/or whole-body 131I scans postoperatively.
Results: Based on cytology/pathology, 88 patients had DTC and 119 had benign thyroid disease. The TSHR mRNA levels in cancer patients were significantly higher than in benign disease (P < 0.0001). At a cutoff value of 1.02 ng/µg total RNA, the TSHR mRNA correctly classified 78.7% of patients preoperatively (sensitivity = 72.0%; specificity = 82.5%). Of 131 patients with FNA and surgery, 51 were FNA positive (all cancer), 17 were FNA negative (15 benign, two cancer), and 63 were indeterminate. TSHR mRNA correctly diagnosed DTC in 16 of 24 (67%) and benign disease in 29 of 39 (74%) patients with indeterminate FNA (combined sensitivity = 90%; specificity = 80%). Combining TSHR mRNA and ultrasound features for follicular lesions correctly classified all follicular cancers and could have spared surgery in 31% of these patients with benign disease. TSHR mRNA has a short life in circulation, and normalized levels on postoperative d 1 correlated with disease-free status, whereas elevated levels predicted residual/metastatic disease.
Conclusions: TSHR mRNA measured with FNA enhances the preoperative detection of cancer in patients with thyroid nodules, reducing unnecessary surgeries, and immediate postoperative levels can predict residual/metastatic disease.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Molecular markers have been explored as alternative methods for detecting thyroid cancer with variable success and suffers from the lack of specificity (5, 6, 7, 8). The variable results in these studies may relate to the differences in methods used including RNA isolation, use of different primers, and the normalization process. Based on FNA cytology, it is virtually impossible to differentiate follicular cancers (FC) from benign follicular adenomas (FA), and several investigators have focused on molecular markers for this purpose with variable success (9, 10, 11, 12, 13). Because thyroid/thyroid cancer cells express functional TSH receptors (TSHR) (7, 14), we have exploited this marker for the detection of thyroid cancer cells in peripheral blood. With the use of a carefully selected primer pair and qualitative RT-PCR we have shown that TSHR mRNA can be a highly sensitive and specific marker for recurrent or metastatic thyroid cancer (15, 16). The objective of the current prospective study was to expand the potential clinical usefulness of this assay by the quantitative detection of TSHR mRNA and to explore its role in the preoperative diagnosis of cancer among patients with thyroid nodules and in monitoring the disease after surgery.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The study consisted of 258 patients including 51 healthy subjects without a history of thyroid disease (female to male ratio, 1.7; age range, 25–60 yr) and 207 patients with thyroid disease. All 207 patients with thyroid disease were prospectively recruited during 2002–2005 at their initial presentation for the evaluation of thyroid nodules or for recurrent thyroid cancer at the Cleveland Clinic. Of the 207 patients, 182 (88%) underwent FNA biopsies as part of the evaluation of their thyroid nodules. Of the remaining 25 patients, 18 proceeded directly to surgery (Fig. 1
) for enlarging multinodular goiters (MNG). The remaining seven patients had recurrent thyroid cancer that did not require surgery or FNA.
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Ultrasound (U/S) and U/S-guided FNA were performed as a routine diagnostic work-up of patients with thyroid nodular disease. U/S features of thyroid nodules considered suspicious for possible thyroid malignancy included irregular shape, ill-defined margins, solid structure, hypervascularity, fine internal calcifications, and size greater than 3.5 cm (17). The FNA results were reviewed by one of our pathologists. Indeterminate FNA included the following cytological categories: hypercellular follicular nodule (oxyphilic or nonoxyphilic), atypical epithelial cells without further qualification or with the comment "cannot exclude follicular neoplasm or papillary thyroid cancer," and hemorrhagic cyst contents with few follicular cells. The status of residual/metastatic disease was assessed postoperatively by serum Tg (Nichols Institute Diagnostics, San Juan Capistrano, CA) and Tg-Ab (Tosoh Bioscience, Inc., South San Francisco, CA) measurements and/or by whole-body 131I scans (WBS).
TSHR mRNA measurement by real-time RT-PCR
Briefly, venous blood (5–7 ml) was collected, and the mononuclear cells were separated by Ficoll-Hypaque gradient. Total RNA was extracted with TRIzol reagent (Life Technologies, Rockville, MD). RNA integrity was tested in every sample by performing RT-PCR for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For RT-PCR, we used an in-cycle fluorescent detection system (Rotorgene 3000; Corbett Research, Sydney, Australia) and one-step Quantitect SYBR Green kit (QIAGEN Inc., Valencia, CA). The assay was performed in duplicate using 1 µg RNA according to the manufacturer’s recommendations. The primer sequences for TSHR mRNA and for GAPDH have been described previously (15, 16). Each sample was also quantified for GAPDH mRNA in a separate reaction tube within the same run. For quantitation, total RNA extracted from papillary thyroid carcinoma (PTC) tissue was used as a reference preparation to produce a standard (calibration) curve consisting of four concentrations ranging from 0.144–144 ng total thyroid cancer RNA. The intraassay (n = 16) and interassay (n = 31) coefficients of variations (CV) at two dilutions of standards were 0.9 and 5.6% for the threshold cycles and were 13.0 and 19.7% for concentrations. A positive and negative control was included in each amplification reaction, and the interassay CV for the positive control was 15.9% (n = 8). The subject samples were normalized for the amount of RNA loaded into each reaction tube (1 µg). Results are reported as reference preparation equivalent TSHR mRNA ng/µg of total RNA. The estimated functional assay sensitivity is 0.14 ng/µg of total RNA with a CV of 9%.
Statistical analysis
Statistical analysis was performed using JMP 5.1 (SAS Institute, Inc., Cary, NC). All subject groups demonstrated positively skewed distributions; hence, data are expressed as medians and interquartile ranges (25–75th percentiles) unless otherwise specified. The Kruskal-Wallis test and the pairwise Wilcoxon rank sum test with Bonferroni correction were used to investigate the group differences at an overall 
= 0.05.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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According to FNA cytology and/or final surgical pathology, 88 of the 207 patients had differentiated thyroid cancer (DTC) and 119 patients had benign thyroid disease. The characteristics of these patients are summarized in Table 1. The final breakdown of patients based on FNA cytology and surgical results is illustrated in Fig. 1
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The median TSHR mRNA values showed significant increase in various disease groups: recurrent DTC greater than newly diagnosed DTC greater than benign thyroid disease greater than healthy controls (P < 0.001). The median levels and ranges are listed in Table 1 and illustrated in Fig. 2. All cancer patients were classified according to lymph node status and the presence of distant metastasis. TSHR mRNA levels in node-positive patients (n = 43) were higher than node-negative patients (n = 38) but were not statistically significant [median 1.45 (0.5–11.4) and 8.1 (0.7–45.4); P = 0.079]. Also, there was no significant difference between node-positive patients and patients with distant metastasis (n = 6) [median 8.1 (0.7–45.40) and 28.4 (17.9–57.5); P = 0.156]. However, the levels in patients with distant metastases were significantly higher than node-negative patients (P = 0.005) (Fig. 2). There was no significant correlation between tumor size and TSHR mRNA levels in node-negative patients.
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). Using this cutoff, 63 of 88 patients with thyroid cancer were positive by the TSHR mRNA assay, and of these, 38 had newly diagnosed thyroid cancer and 25 had recurrent thyroid cancer, giving the test a sensitivity of 62% (38 of 61) in newly diagnosed DTC (Fig. 3B) and 93% (25 of 27) in recurrent cancer. In the group with benign disease, our assay correctly predicted the diagnosis in 75% (52 of 69) of the patients with surgical confirmation and in 74% (37 of 50) of those with only a cytological diagnosis of benign disease. All healthy subjects were below this cutoff level of 1.02 ng/µg. Overall, our assay correctly classified 203 of 258 subjects tested yielding an overall accuracy of 78.7%.
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As shown in Fig. 1, FNA was performed in 182 of 207 patients, and 131 (77 DTC and 54 benign disease) of these had surgical confirmation. Among these 131, 20 were recurrent cancers and 111 (57 DTC and 54 benign) were new patients presented with thyroid nodule(s). FNA was positive in 51 (16 recurrent cancers), negative in two (one recurrent cancer), and indeterminate in 24 (three recurrent cancers) of surgically confirmed DTC [surgical diagnoses included 17 PTC, five FC, and two Hürthle cell (HC) carcinomas]. TSHR mRNA correctly predicted the diagnosis in 16 of 24 (67%) patients with indeterminate FNA, including four of five cases of FC and both cases of HC carcinoma. FNA was negative in 17 (27%) and was indeterminate in 39 (73%) of benign disease patients. In this group, we correctly predicted the diagnosis in 29 of 39 (74%) patients including seven of 10 cases of FA, 14 of 18 patients with colloid nodules/MNG, four of seven patients with benign hyperplastic nodules, two of two with Hashimoto’s disease, and both of the patients with an oxyphilic nodule and a HC adenoma.
Overall, 67 of 182 (37%) of all patients and 63 of 131 (48%) of patients with surgically confirmed diagnosis had indeterminate FNA. If indeterminate FNA are considered to be nondiagnostic, the ability of FNA to correctly classify patients (diagnostic efficacy/accuracy) was only 48% with a sensitivity of 64% and a specificity of only 28% (Table 2). There was 70% concordance between FNA and RNA assay results. These patients were analyzed to determine the diagnostic synergy of combining both the FNA and TSHR mRNA in the preoperative diagnosis of thyroid disease in all 131 patients or in 111 new patients, and there was a significant improvement in the overall diagnostic efficacy (Table 2 and Fig. 4A). Eight patients were false negative by TSHR mRNA; three with cytological diagnosis of hyperplastic follicular lesions (two FC and one micro-PTC follicular variant), five with cytological diagnosis of atypical cells PTC (two microcancers). Eleven patients were false positives, seven (64%) with hyperplastic follicular lesions (three FA and four hypercellular follicular nodules) and four with large MNG and cytological diagnosis of atypical cells.
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Ten of 11 cases of surgically confirmed FA and five of seven cases of FC had indeterminate FNA, and our assay correctly diagnosed benign disease in eight of 11 (73%) patients with FAs and was positive in five of seven (71%) patients with FC. Altogether, it correctly predicted the diagnosis in 13 of 18 patients (72%) with follicular lesions.
Overall, there were 33 patients with indeterminate FNA specifying hypercellular follicular nodule/lesion with or without HC features, and 29 of these had U/S characteristics available including eight of 11 FA and five of seven FC. We found that by including U/S characteristics with TSHR mRNA in a decision-making algorithm, all thyroid cancer patients would have been captured for appropriate treatment including all five FC patients. Of these 29, 13 (seven cancer and six benign) were TSHR positive; all would have been candidates for surgery. The remaining 16 (five thyroid cancer and 11 benign) patients were negative, and seven of these (five cancer and two FA) had suspicious U/S findings including nodule size greater than 3.5 cm and one additional unfavorable U/S feature or two or more unfavorable features; all would have required surgery. The remaining nine (31%) patients were TSHR mRNA negative and had single or no unfavorable U/S finding, and all had benign thyroid disease and would have been spared surgery. This model would have captured all thyroid cancer patients including five FC patients (100%) (false-negative rate of 0%) at the expense of a false-positive rate of 27.5% and would have spared surgery in nine of 17 (53%) patients with benign disease (31% of all patients with indeterminate FNA) (Table 2 and Fig. 4B). In comparison, the use of U/S alone showed a false-negative rate of 50% and a false-positive rate of 60%.
First-day postsurgery TSHR mRNA levels as a predictor of residual/metastatic disease
Of 89 patients who had first-day postoperative samples drawn, 45 had DTC and all but two underwent near-total thyroidectomy or radical neck dissection; 44 had benign thyroid disease, of which 28 had near-total thyroidectomy and 16 had lobectomy. The status of residual/metastatic disease in these patients was assessed within 8–9 months after surgery by stimulated Tg levels (
2 ng/ml) and or WBS. There was a significant decrease in postoperative levels [median 1.55 (0.5–33.7) and 0.13 (0.05–0.61); P < 0.001] in the cancer patients but not in benign disease patients [0.38 (0.18–0.85) and 0.32 (0.17–0.57); P = 0.29]. Thirty cancer patients and nine benign disease patients were positive preoperatively, and all of these except seven DTC (six thyroidectomies, one lobectomy) and two benign thyroid disease patients (one thyroidectomy and one isthmusectomy) had normal d-1 postoperative levels (Fig. 5).
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All patients in whom the TSHR mRNA levels were normalized after surgery remained disease free as evidenced by follow-up serum Tg and or WBS. All patients who were negative preoperatively (n = 50) remained negative the day after surgery including seven who had additional follow-up studies.
TSHR mRNA levels in Tg-Ab-positive patients
Sixteen of 61 (26%) patients with newly diagnosed thyroid cancer and eight of 27 (29%) with recurrent thyroid cancer were Tg-Ab positive. Of these, 10 Tg-Ab-positive patients with newly diagnosed thyroid cancer and all eight patients with recurrent thyroid cancer were positive by the assay. There was no significant difference between the sensitivity of the assay in detecting thyroid cancer in the Tg-Ab-positive or -negative patients (P = 0.1).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We have previously demonstrated that the selection of both TSHR and Tg mRNA primers specific to thyroid cells is of paramount importance in the assay (16, 21). Here, in this prospective study, using a quantitative assay, we attempted to validate and expand the potential clinical usefulness of this assay. In this study, we were able to quantify TSHR mRNA in the blood of healthy subjects. However, unlike a previous study measuring Tg mRNA, the levels of TSHR mRNA detected were extremely low (22). Furthermore, our results show significant gradation of TSHR mRNA levels in various disease groups: recurrent DTC greater than newly diagnosed DTC greater than benign thyroid disease greater than healthy controls. This is in contrast to previous quantitative reports, mostly for Tg mRNA, which failed to find significant differences between healthy subjects and patients with DTC, with most authors concluding that the clinical use of this test in the follow-up of patients with thyroid cancer was limited (8, 23, 24). Using a ROC curve, we were able to establish an optimal cutoff value of TSHR mRNA for diagnosing thyroid cancer. However, using this cutoff value on the same data set to estimate sensitivity and specificity may have an inherit bias toward overestimate, and future studies using independent data sets are required to confirm these estimates. Furthermore, the sensitivity of our assay in Tg-Ab-positive patients was similar to that in the Tg-Ab-negative patients. This, combined with its high sensitivity in detecting recurrent cancer, suggests that there may be a benefit of using the assay to monitor Tg-Ab-positive patients for cancer recurrence postoperatively. Future long-term monitoring studies are required to confirm these findings.
By prospectively recruiting patients with thyroid nodules before surgery, we were also able to explore the relationship between TSHR mRNA levels and the stage of disease. To our knowledge, aside from nine patients studied prospectively by Ringel et al. (22), no other quantitative study has attempted to correlate Tg or TSHR mRNA levels preoperatively with the extent of disease. In our study, although there was a trend toward a higher TSHR mRNA levels in patients with distant metastasis greater than node-positive patients greater than node-negative patients, only patients with distant metastasis had statistically significant increase compared with organ-localized disease. However, we failed to find any relationship with TSHR mRNA levels and tumor size, suggesting that factors other than the tumor size may influence the TSHR mRNA expression and will require additional investigations.
Although our assay is unlikely to replace the role of the FNA in the preoperative diagnosis of thyroid nodules, its main advantage lies in the evaluation of thyroid nodules with indeterminate FNA. In our study, the incidence of indeterminate FNA is higher (37%), which may reflect the referral bias of our tertiary institution. This may also explain the lower sensitivity and specificity of the FNA, calculated using only clearly positive and negative FNA results, than those reported by other groups. However, the incidence of DTC in the group with indeterminate FNA was 35%, which is consistent with previous studies suggesting that the rate of malignancy in indeterminate FNA is 20–40% (2). In this particularly problematic group, our assay correctly predicted the diagnosis in 45 of 63 (71%) cases, thus significantly enhancing the diagnostic performance of FNA alone. Although false negatives were seen in some microcancers (37%), the false positives primarily included patients with indeterminate hypercellular follicular lesions (64%) and large MNG. Inability of FNA to differentiate benign follicular lesions from FC is well recognized and has been the subject of recent investigations, all in search of novel molecular markers (10, 12, 13). In our series, 89% of follicular lesions had indeterminate FNA, and our simple blood test correctly classified 13 of 18 patients with follicular lesions and detected both HC cancers with indeterminate FNA. Furthermore, we found that inclusion of U/S features in the algorithm would have allowed detection of all cancers among indeterminate FNA specified as hypercellular follicular nodules including all FC. This could potentially translate into a reduced number of unnecessary surgeries for patients with benign thyroid nodules and indeterminate FNA.
One intriguing finding is that TSHR mRNA levels normalized in most patients within 1 d after thyroidectomy. This suggests that the TSHR mRNA/thyroid cancer cells have a short life in the circulation. Furthermore, we found that all patients with normalized levels had no evidence of disease and that elevated levels correlated with the presence of residual/metastatic disease as evidenced by follow-up WBS and serum Tg measurement. These findings suggest that immediate postoperative levels can accurately predict residual/metastatic disease and can be used to monitor disease activity immediately after surgical resection and even for long-term monitoring.
In conclusion, we have shown that our quantitative TSHR mRNA assay is helpful in the preoperative diagnosis of thyroid nodules, particularly in the subgroup of patients with indeterminate FNA. The immediate postsurgical levels can serve as a sensitive marker for detecting residual/metastatic disease. Furthermore, it detects recurrent cancer with high sensitivity and can be valuable as an alternative to serum Tg measurement in patients who harbor Tg antibodies. Future studies are required to establish its role in long-term monitoring.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online November 21, 2006
Abbreviations: CV, Coefficients of variation; DTC, differentiated thyroid cancer; FA, follicular adenoma; FC, follicular cancer; FNA, fine-needle aspiration; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MNG, multinodular goiter; PTC, papillary thyroid carcinoma; ROC, receiver operating characteristic; Tg, thyroglobulin; Tg-Ab, Tg antibody; TSHR, TSH receptor; U/S, ultrasound; WBS, whole-body 131I scans.
Received September 25, 2006.
Accepted November 15, 2006.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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