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Original Article |
Endocrine, Diabetes, and Metabolism Service (V.J.B., Y.V., H.B.B.), Department of Clinical Investigation (J.A., B.S.), Department of Pathology (C.F.A.), Walter Reed Army Medical Center, Washington, D.C. 20307; and Endocrinology Section (M.S., K.D.B., M.D.R.), Washington Hospital Center, MedStar Research Institute, Washington, D.C. 20010
Address all correspondence and requests for reprints to: Victor J. Bernet, M.D., LTC, MC, USA, Assistant Chief, Endocrinology Service, Endocrinology, Diabetes and Metabolism Service, 7D, 6900 Georgia Avenue Northwest, Walter Reed Army Medical Center, Washington, D.C. 20307-5001. E-mail: VICTOR.BERNET{at}NA.AMEDD.ARMY.MIL.
Abstract
Galectin-3, a lectin-family protein that appears to be involved in malignant transformation, has been reported to be an accurate immunohistochemical marker for thyroid cancer. However, immunohistochemistry is a subjective method that can be difficult to apply to cytologic specimens. Therefore, we sought to develop an objective and quantitative assay to measure galectin-3 mRNA in thyroid tissue to enhance potential clinical use of galectin-3 in the molecular analysis of thyroid nodules. In this study, total RNA from 37 snap-frozen thyroid tissue specimens was isolated from eight papillary and nine follicular thyroid cancers, six follicular adenomas, seven adenomatoid nodules, and seven normal thyroid lobes from patients undergoing thyroidectomy. Normalized levels of galectin-3 mRNA, expressed as picograms per nanogram GAPDH mRNA, were higher in papillary carcinomas (3327 pg/ng) and follicular adenomas (1314 pg/ng) than in thyroid normal tissue (426 pg/ng; P = 0.0012 and 0.032, respectively). Galectin-3 mRNA levels were also higher in papillary cancers than in adenomatoid nodules (P = 0.0012). However, galectin-3 mRNA levels were not statistically greater in follicular carcinomas than either normal tissue or follicular adenomas (P = 0.068 and 0.12, respectively). In summary, in comparison to galectin-3 immunohistochemistry, quantitative measurement of galectin-3 mRNA appears useful in the identification of papillary thyroid cancers (PTCs) but does not appear to be useful in distinguishing follicular carcinomas from follicular adenomas.
FINE-NEEDLE ASPIRATION (FNA) is the first line tool for the evaluation of solitary, nonautonomous thyroid nodules. Although a cytologist can accurately distinguish a benign thyroid nodule from papillary thyroid cancer (PTC) by FNA, in the instance of follicular neoplasms, cytology from the FNA alone does not allow classification of a nodule as benign or malignant. In such cases, thyroid surgery is required to distinguish a benign follicular adenoma from a malignant follicular carcinoma (1). However, approximately 85% of these cytologically indeterminant nodules when surgically removed are indeed benign on histology (2, 3). Therefore, unnecessary thyroid surgeries could be avoided if a more reliable marker for the presence of thyroid cancer were available for preoperative evaluation.
Galectin-3 has been found to be a potential marker for various cancers, including those of the thyroid (4). The ability to identify and quantify its presence in a thyroid nodule could potentially be very useful in the management of patients with inconclusive FNA results. To this end, we decided to study galectin-3, a 30-kDa protein that is a member of the endogenous ß-galactoside binding family of lectins (5). Galectins, as a group, appear to be involved with several cell functions, including cell adhesion, recognition, proliferation, malignant transformation, immunomodulation, and metastasis (6, 7, 8, 9, 10). Galectin-1 expression has been found to be elevated in oncogene-transformed cell lines in comparison to normal rat thyroid cells (11, 12). Galectin-3 expression has been found to be altered in carcinoma of the breast (13), colon (14), stomach (15), and in glioblastomas (16).
A review of the literature revealed a limited number of studies addressing galectin-3 in thyroid tissue. By using immunohistochemical and immunoblotting techniques with anti-galectin antibodies, Xu et al. (17) found that thyroid malignancies, both papillary and follicular, express high levels of galectin-1 and galectin-3. In contrast, none of the 32 benign thyroid tissues examined (including goiter, adenoma, or adjacent normal thyroid tissue) were found to express galectin-1 or galectin-3 (17). By means of immunohistochemical staining, Orlandi et al. (18) compared levels of galectin-3 expression in presurgical thyroid FNA samples and the respective histological specimens. Galectin-3 expression was found in all 18 papillary thyroid specimens in both the cytology cell blocks from FNA and histologic samples. Moreover, in follicular thyroid cancer (FTC) specimens, 14 of 17 were positive for galectin-3 in the cytology cell blocks from FNA, whereas all 17 follicular cancer histology samples displayed galectin-3 expression. Benign follicular adenomas displayed no galectin-3 staining in 26 of 29 samples. Review of the three adenoma samples with galectin-3 staining revealed the presence of cells with nuclear changes potentially consistent with malignancy but no evident invasion of surrounding tissue. It is unclear whether these three cases represented false-positive staining for galectin-3 or early malignancy that could not be clearly identified by histology. Cvejic et al. (19) also studied galectin-3 expression by immunohistochemistry in thyroid tumors. This group found evidence for galectin-3 expression in 100% of 20 PTCs and 10 anaplastic cancers, as well as in 73% of 15 FTCs. In contradistinction, only 36% of 14 follicular adenomas and 0 of 15 normal thyroid tissue samples had galectin-3 staining identified by the use of monoclonal antibody. Of note, galectin-3 expression was primarily found in the cytoplasm, but staining could also be seen in the nuclear and/or membrane region in some cells. Inohara et al. (20) also evaluated galectin-3 expression in 172 specimens. Immunohistochemical analysis revealed significant staining for galectin-3 in all cases of papillary (primary, n = 45; metastasis, n= 20), follicular (primary, n= 8; metastasis, n = 2), and anaplastic (primary, n = 5; metastasis, n = 3) cancer in both primary tumors as well as metastasis. Most recently, Bartolazzi et al. (21) reported on the detection of galectin-3 and CD44v6 by immunostaining in thyroid nodules from 1009 tissue specimens or cytologic cell-blocks plus 226 cytology specimens obtained by ultrasound-guided FNA. The investigators from this study reported a sensitivity of 99% and specificity of 98% in distinguishing benign from malignant thyroid nodules by the presence of staining for galectin-3.
Although the immunohistochemical detection of galectin-3 appears to be useful to distinguish benign from malignant thyroid lesions, the interpretation of such staining may be hindered by its somewhat subjective nature, and immunostaining of cytologic samples can be technically difficult. We, therefore, elected to examine whether a more objective method of galectin-3 measurement could be developed. We assessed possible differences in mRNA levels of galectin-3 between various types of benign and malignant thyroid tissues with the hypothesis that variations in galectin-3 mRNA might be suitable for use as a tumor marker. The goal of the present study was to develop a quantitative assay to measure levels of galectin-3 mRNA in thyroid tissue and assess the level of expression in various forms of benign and malignant thyroid nodules.
Materials and Methods
Experimental subjects
This study was reviewed and approved by the Walter Reed Army Medical Center (WRAMC) research review committee of the Department of Clinical Investigation. Surgical tissue samples for the study were obtained under approved protocol. Consent was obtained from all subjects who assented to participate in the Endocrine Service’s frozen thyroid tissue bank. Patients undergoing thyroidectomy for standard clinical indications consented to have their removed tissues be snap-frozen in liquid nitrogen and stored at -70 C. All samples were assigned unique identifiers to protect patient confidentiality and were obtained from patients 18 yr of age or older who were eligible for care in the WRAMC health care system. Tissue samples included the following histological subtypes: normal extra-nodular tissue (NL), benign nodules (BN), PTC, FTC, and follicular adenoma (FA). Pathology reports were used to identify final nodule diagnoses to allow the selection of a variety of thyroid conditions. Normal tissue specimens were obtained from an extra- nodular portion of the thyroid that grossly appeared to be without any pathologic change. In total, 37 frozen tissue specimens were obtained, consisting of NL (n = 7), BN (n = 7), PTC (n = 8), FTC (n = 9) and FA (n = 6).
Galectin-3 RT-PCR
Total RNA was recovered from frozen specimens, using the acid guanidinium isothiocyanate-phenol chloroform method (TriReagent, Molecular Research Center, Inc., Cincinnati, OH), and quantitated by scanning UV spectrophotometry. A quantitative RT-PCR assay was developed for galectin-3 using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA), in which real-time PCR product accumulation is detected using a sequence-specific fluorescent oligonucleotide probe. One microgram of total RNA was reverse transcribed using random hexamer primers and Multiscribe Reverse Transcriptase (PE Applied Biosystems). Quantitative PCR was performed using intron-spanning galectin-3-specific forward and reverse primers. A 103-bp-long galectin-3 cDNA segment, spanning the junction of exons 4 and 5 (GenBank accession no. NM-002306), was amplified using the forward primer 5'ACGGTGAAGCCCAATGCA-3' and reverse primer 5'TGACTCTCTCCTGTTGTTCTCATTGAA-3'. The antisense probe 5'-AATGATGTTGCCTTCCACTTTAACCCACG-3' was labeled (Synthegen, Houston, TX) with 5'-reporter dye and the 3'- quencher dye. RNA was pretreated with Dnase 1 to remove any contaminating genomic DNA. Specificity and size of the PCR-amplified product were confirmed by agarose gel electrophoresis and cycle sequencing.
Samples were assayed in duplicate using the TaqMan Universal Master Mix (PE Applied Biosystems) according to the manufacturer’s protocol. Cycling conditions for TaqMan PCR were 2 min at 50 C, 10 min at 95 C, followed by 40 cycles of 15 sec at 95 C and 1 min at 60 C. Duplicate six point standard calibration curves, prepared by serial dilution of human thyroid poly-A mRNA (CLONTECH Laboratories, Inc. Palo Alto, CA) were assayed with the samples under identical conditions. The standard curve displayed a strong linear relationship when plotted as threshold cycle vs. concentration, with a correlation coefficient (r = 0.994).
Galectin-3 assay results were normalized to expression of the glyceraldehyde-3-phosphate dehydrogenase (GADPH) using TaqMan primers and probe identical to those suggested by the manufacturer (PE Applied Biosystems). For each sample, the amount of target (galectin-3) and endogenous reference (GAPDH) was determined from the calibration curves. The target amount was then divided by the reference amount to obtain normalized values and was reported as picograms galectin-3 mRNA per nanogram GAPDH mRNA. Negative control specimens for the RT-PCR were performed for detection of assay contamination.
Immunohistochemical staining
Five samples were selected for immunohistochemical staining with a galectin-3-specific antibody using a diaminobenzidine detection system. Two PTC samples were chosen, one with and one without lymphocytic infiltration, plus one sample from the FTC, FA, and BN groups. Slides were first deparaffinized using the automated Ventana Discovery system (Ventana Medical Systems, Tucson, AZ). The slides were then conditioned in a citrate buffer with a pH of 6.0. An enzyme protease was added and incubated for 4 min. The primary antibody used was a monoclonal mouse IgG galectin-3 antibody from Research Diagnostics Inc. (Flanders, NJ). This antibody was diluted to 1:100 and incubated with the sample for 60 min. The secondary antibody was an antimouse antibody added at a dilution of 1:1000. The slides were counterstained with hematoxylin, and a bluing agent was applied as a post counterstain. The slides were then evaluated and rated for evidence of galectin-3 staining by an experienced pathologist.
Statistical analysis
Data were analyzed using SPSS statistical software (SPSS, Inc., Chicago, IL). Mean levels for the galectin-3 mRNA amplification (normalized to GAPDH) by quantitative RT-PCR were obtained for each group of specimens, and nonparametric analyses were used to compare differences between the mean galectin-3 mRNA level for each group.
Results
Total RNA was isolated from the 37 thyroid surgical samples including: NL (n = 7), BN (n = 7), PTC (n = 8), FTC (n = 9) and FA (n = 6). Levels of galectin-3 mRNA were determined by real-time quantitative RT-PCR using intron-spanning galectin-3 primers and an internal fluorescent probe and were normalized to simultaneously measured GAPDH mRNA. A standard calibration curve for the quantitative RT-PCR was obtained and yielded a correlation coefficiency of r = 0.994. The following mean levels (±SE) of galectin-3 mRNA were found: NL, 426 ± 81 pg/ng GAPDH mRNA; BN, 434 ± 93 pg/ng GAPDH mRNA; PTC, 3327 ± 1039 pg/ng GAPDH mRNA; FA, 1314 ± 420 pg/ng GAPDH mRNA; and FTC, 1179 ± 378 pg/ng GAPDH mRNA (Table 1
). Levels of galectin-3 mRNA, expressed as picograms per nanogram GAPDH mRNA, were higher in papillary cancers and FA than normal tissue with significance values of P less than 0.0012 and P less than 0.032, respectively (Table 2). Galectin-3 mRNA levels in PTC lesions were higher than BN (P < 0.0012) but not in comparison to FTC (P < 0.068) and FA (P < 0.12). Furthermore, two of the eight PTC lesions were found to have a lymphocytic infiltrate present. The two samples with documented lymphocytic infiltration had an average normalized galectin-3 mRNA result of 7117 ± 1658 in comparison to the other six specimens, which had an average galectin-3 level of 4020 ± 1276 and which were not found to be statistically different (P = 0.15).
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, A and B).
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The increasing use of FNA for thyroid nodule evaluation resulted in a reduction in the number of patients undergoing thyroidectomy for benign nodules. In addition, a concomitant increase in yield of malignancy at thyroidectomy was noted (1). The cytologic findings from FNA clearly allow more accurate selection of patients requiring surgical excision of a thyroid nodule. However, in present practice, patients having the cytologic diagnosis of follicular neoplasm will undergo thyroid surgery with the reality that up to 85% of these procedures are unnecessary because follicular cancer will only be found in approximately 15% of cases (22). The ultimate goal in the preoperative evaluation of thyroid nodules would be to accurately designate lesions as benign or malignant, and thereby allow cancerous lesions to be targeted for surgical intervention. Clinical features can be used to help predict an increase or decrease in the likelihood of cancer in a nodule, but these parameters have limited utility (22, 23). Unfortunately, standard evaluation of thyroid nodules by Diff-Quik and/or Papanicolou staining, even under the guidance of a highly trained cytopathologist, cannot definitively distinguish benign from malignant thyroid nodules in all cases. Useful thyroid cancer markers are therefore a prime focus of interest to investigators as a potential means to enhance the preoperative evaluation of thyroid nodules.
As previously mentioned, several groups have reported the potential usefulness of galectin-3 as a marker for thyroid cancer. Using immunohistochemical staining techniques, galectin-3 was used to distinguish between nodules of follicular cancer and benign adenomas using cell blocks obtained from FNA cytology (18). Immunohistochemical data has revealed primarily cytoplasmic staining for galectin-3, although nuclear and/or membrane staining can also be seen (19). However, the interpretation of immunohistochemical staining is very subjective, is open to possible interpretation errors, and can be technically difficult with cytologic samples.
Because galectin-3 seemed to hold promise as a marker for thyroid cancer, our group decided to determine whether galectin-3 mRNA expression in thyroid nodules could be measured by quantitative RT-PCR techniques. Moreover, we hypothesized that quantitative measurement of galectin-3 mRNA could be used as an objective way to distinguish benign from malignant thyroid nodules. Our investigation of galectin-3 mRNA by quantitative RT-PCR yields a different picture of galectin-3 expression in thyroid lesions than that found in immunohistochemical testing. In contradistinction to prior immunohistochemical data, galectin-3 mRNA expression was detected not just in malignant tissue, but in all the tissue specimens, including: NL, BN, PTC, FA, and FTC. Expression of galectin-3 mRNA was not statistically different between FA and FTC lesions, but, in agreement with previously reported immunohistochemical studies by other groups, our data would support that PTC lesions do indeed express a higher level of galectin-3 mRNA than normal thyroid tissue and benign nodules.
Telomerase, another promising marker for thyroid cancer, is limited by the fact that it is also expressed in lymphocytes, and so benign thyroid lesions with lymphocytic infiltration might exhibit telomerase levels in the same range as cancerous thyroid lesions (24). In the present study, comparison of galectin-3 mRNA expression in samples with and without lymphocytic infiltration was made. No statistical difference was found in the levels of galectin-3 mRNA between samples with and without a lymphocyte presence, but the limited number of samples prevents conclusive determination of whether such infiltration has an impact on galectin-3 mRNA measurement. In addition, a follow-up comparison of immunohistochemical staining between two selected PTC lesions was performed, one with and one without lymphocytic infiltration. In both cases, galectin-3 staining was demonstrated in the thyroid cancer tissue, whereas the areas with lymphocyte infiltration did not demonstrate galectin-3 staining.
The ideal thyroid malignancy marker would be both very specific and sensitive for the presence of cancerous thyroid tissue, and permit clinicians to accurately categorize a follicular neoplasm as a follicular adenoma or carcinoma preoperatively. In addition, any potential thyroid cancer marker should be widely available, be of reasonable expense, and be a simple test to collect and perform. Molecular markers could potentially be the diagnostic tool that would allow thyroidologists to get closer to this final goal. To date, there is no marker or set of markers that definitively distinguishes benign from malignant lesions. A myriad of tumor markers are under investigation for a large variety of cancers. Some markers that have been investigated for thyroid cancer include: thyroglobulin, anti-thyroid peroxidase antibodies, TSH receptor, PAX-8, thyroid-specific transcription factors, NIS and deiodinase I, 
-fetofibronectin, high mobility group I-Y protein, telomerase, human telomerase reverse transcriptase (hTERT), c-Met/hepatocyte growth factor, human milk fat globule antigen G1, and galectin-3 among others (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). Various changes in expression have been reported for these markers, including increased or reduced expression within malignancy compared with benign tissue or evidence of antigenic changes, as has been reported for the enzyme thyroperoxidase. The measurement of hTERT would appear to hold some promise for distinguishing between benign follicular adenomas and FTC, as would immunostaining for the c-Met/hepatocyte growth factor receptor as well as galectin-3 (21, 31, 35). The latter two markers also may be useful for distinguishing PTC and follicular variant of PTC from benign lesions.
The measurement of galectin-3 mRNA by quantitative RT-PCR provides an objective method by which to measure expression of galectin-3 mRNA in thyroid lesions. In instances of diagnostic uncertainty following cytopathologic review of a thyroid lesion, the measurement of galectin-3 mRNA in FNA cytology samples may potentially be an accurate method for distinguishing benign nodules from those harboring PTC. The usefulness of quantitative RT-PCR measurement of mRNA galectin-3 in thyroid lesions does not seem to be hindered by the presence of lymphocytes in contradistinction to telomerase. Because lymphocytic infiltration is more associated with PTC than FTC, the simultaneous quantification of galectin-3 and telomerase/hTERT in thyroid nodules might provide an accurate adjunct for diagnosing PTC and FTC, respectively.
In conclusion, the results from our study indicate that measurement of galectin-3 mRNA by quantitative RT-PCR appears to be a potential technique for differentiating between normal/benign thyroid nodules and PTC. Our results do not support the use of quantitative RT-PCR measurement of galectin-3 mRNA to distinguish benign follicular adenomas from FTC because expression of galectin-3 mRNA was similar in these types of nodules.
Acknowledgments
We acknowledge the following people for their invaluable assistance: Dr. Diarmuid Nicholson, for sequencing the RT-PCR galectin-3 product; Brian Reinhardt, for assistance with immunostaining efforts; Dr. Craig Shriver, for support in collection of thyroid tissue samples; and Robin Howard, for help with statistical analysis.
Footnotes
The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.
Abbreviations: BN, Benign nodules; FA, follicular adenoma; FNA, fine-needle aspiration; FTC, follicular thyroid cancer; GADPH, glyceraldehyde-3-phosphate dehydrogenase; hTERT, human telomerase reverse transcriptase; NL, normal extra-nodular tissue; PTC, papillary thyroid cancer.
Received March 13, 2002.
Accepted July 15, 2002.
References
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