The Journal of International Medical Research
2009; 37:927–938
Comparative Analysis of Protein Expression in Differentiated Thyroid Tumours:a Multicentre Study
H-S L IANG 1,3, Y-H Z HONG 1, Z-J L UO 4, Y H UANG 5, H-D L IN 6, M L UO 7, S-Z HAN 2,
H-X S U 1, S-B Z HOU 1AND K-Q X IE 3
Department of Endocrinology, and 2Department of Pathology, Ninth Affiliated Hospital, Guangxi Medical University, Beihai, China; 3Department of Endocrinology, Nanfang Hospital, Southern Medical University, Guangzhou, China; 4Department of Endocrinology, First Affiliated Hospital, Guangxi Medical University, Nanning, China; 5Department of Hepatobiliary and Endocrine Surgery, Guangxi People’sHospital, Nanning, China; 6
Department of Hepatobiliary and Endocrine Surgery, Guangxi Pingnan People’sHospital, Pingnan, China; 7Shanghai Endocrine and Metabolism Research Institute, Shanghai, China This study compared clinical features and protein expression profiles in differentiated thyroid tumours to identify protein markers with the potential for indicating malignancy status. Tissue microarrays were constructed using 119thyroid tumour samples (45papillary carcinomas, 26follicular carcinomas, 48adenomas). Generally, there was overexpression of proliferating cell nuclear antigen (PCNA),p53, matrix metalloproteinase (MMP)-7,Hector Battifora mesothelial-1(HBME-1),MMP-2, pituitary tumour-transforming gene (PTTG)and human telomerase reverse transcriptase (hTERT)in malignant
thyroid carcinomas, and overexpression of fragile histidine triad (FHIT),p16and E-cadherin in thyroid adenomas. Multiple factor binary logistic regression analysis indicated that MMP-2, HBME-1, p16and FHIT were independently related to differentiated thyroid tumours. Receiver–operating characteristics for these four factors showed HBME-1as best for diagnostic accuracy. Sensitivity and specificity were enhanced using an HBME-1and p16cluster. HBME-1expression was not significantly different for papillary and follicular carcinomas, whereas p16expression was significantly specific.
1
KEY WORDS:D IFFERENTIATED THYROID TUMOURS ; C ARCINOMAS ; A DENOMAS ; D IAGNOSIS ; S ENSITIVITY ;
S PECIFICITY ; I MMUNOHISTOCHEMISTRY ; P ROTEIN EXPRESSION
Introduction
Thyroid neoplasms are very common endocrine tumours, however it is sometimes difficult to distinguish between benign and malignant neoplasms because similar clinical manifestations are frequently encountered during intumescence colour
imaging investigations. Numerous researchers have demonstrated the value of histopathological criteria in predicting whether thyroid neoplasms are benign or malignant, however difficulties remain in distinguishing between some malignant and benign differentiated thyroid tumours. 1
Protein expression in differentiated thyroid tumours
The diagnostic value of a number of immunohistochemical stains has been studied in thyroid neoplasms, for example fragile histidine triad (FHIT),metalloproteinase (MMP)-2,MMP-7, p53, p16, pituitary tumour-transforming gene (PTTG)and proliferating cell nuclear antigen (PCNA).These stains have been validated as simple, reliable and repeatable markers for use in the diagnosis of malignant and benign thyroid tumours in humans. 2–5Some have also been shown to be prognostically relevant in other types of tumour. 2However, no consistent correlations of diagnostic value have been identified between these cancer/anti-cancerproteins and tumour histopathology. 3,5In addition, multi-protein profiling using immunohistochemical analysis of tissue has been shown to be an efficient, reproducible and valid method to study cancer or anti-cancer proteins in endocrine neoplasms. 6–8
The main objective of this study was to use immunohistochemical analyses in thyroid carcinoma and thyroid adenoma tissue samples to identify protein phenotypes of potential value for differentiating between benign and malignant thyroid neoplasms.
histopathology slides were available for review. Paraffin-embedded tissue blocks were available for all patients. A further cohort of patients, treated surgically for thyroid adenomas between 2002and 2008, were also identified. Patients with non-thyroid tumours, undifferentiated thyroid tumours, thyroid hyperplasia, goitre or metastasis to the thyroid gland, and those who had only undergone fine-needle aspiration or biopsy of the thyroid were excluded from the study. All available clinical, pathological, treatment and follow-up data were reviewed and updated for all patients.
TREATMENT
Primary treatment was carried out at each of the four hospitals from which records were obtained based on standard care carried out at the Ninth Affiliated Hospital, Guangxi Medical University, involving thyroidectomy with or without concomitant solid organ (pancreas,spleen, kidney and/orliver) resection as necessary to achieve complete resection of all grossly evident disease. Adjuvant treatment in the form of radiotherapy or chemotherapy was administered as part of standard care or as part of clinical trials.
Patients and methods
PATIENTS
Consecutive patients with histologically confirmed differentiated thyroid carcinoma treated between 2001and 2008were identified from the Pathology Departments of the following four hospitals:Ninth Affiliated Hospital, Guangxi Medical University, Beihai, China; First Affiliated Hospital, Guangxi Medical University, Nanning, China; Guangxi People’sHospital, Nanning, China; and Guangxi Pingnan People’sHospital, Pingnan, China. All patients had undergone resection of a primary thyroid carcinoma for which
PATHOLOGICAL AND CLINICAL DATA
All available operative reports and data included in the institutional pathology database and pathology reports for the primary tumours were reviewed to confirm completeness of the resection procedure. All available autopsy reports were also reviewed. A mean of five haematoxylin–eosin slides were evaluated per patient in conjunction with the corresponding pathology record, but there was blinding with respect to the clinical data. All histologically confirmed thyroid adenomas
Protein expression in differentiated thyroid tumours
and carcinomas in this study were included in the analysis.
Patients’clinical data included age, sex and radiological tumour size.
following protein markers:FHIT, peroxisome proliferator-activated receptor gamma (PPAR-γ), p16, PCNA, p53, Ki-67, MMP-2, PTTG, human telomerase reverse transcriptase (hTERT),Hector Battifora mesothelial-1(HBME-1),MMP-7, E-cadherin and basic fibroblast growth factor (bFGF)(Table1).
Tissues known to express the antigens of interest were used as positive controls and normal tissues were used as baseline controls. Sections from the tissue arrays were deparaffinized, rehydrated in graded alcohols and processed using avidin–biotinimmunoperoxidase. The sections were then placed in 0.01M citrate buffer, pH 6.0, and heated for 15min in a microwave oven. This procedure was followed for all antibodies used in the study, with the exception of those that were incubated in pre-heated 0.05%
TUMOUR SAMPLES
Tissue microarrays were constructed using 1mm diameter tumour cores taken from appropriate areas of the formalin-fixed paraffin-embedded tissue blocks. The tissue cores were arrayed into a recipient paraffin block using a tissue arraying instrument (BeecherInstruments, Silver Springs, MD, USA). Serial sections 4µmthick were cut from the tissue microarray blocks and mounted on glass slides.
IMMUNOHISTOCHEMISTRY
Using the tissue microarrays, immunohisto-chemical expression was investigated for the
Protein expression in differentiated thyroid tumours
trypsin, 0.05%CaCl 2in 0.05M Tris–HCl(pH7.6) for 5min at 37°Cbefore microwave treatment (PTTG,PCNA and MMP-7). Following antigen retrieval, the slides were incubated in 10%normal bovine serum for 30min, and then incubated for a further period of time in appropriately diluted primary antibody at the dilution factors, incubation times and temperatures shown in Table 1. Diaminobenzidine was used as the chromogen and haematoxylin as the nuclear counterstain. Rates of lost cases attributable to tissue damage ranged between 1%and 10%for the different protein markers.
light brown; and 3, dark brown and the positive cell rate within the same cell type was graded as follows:0, 75%positive cells. The staining intensity and positive cell rate scores were than multiplied together to give the following overall scores, as follows:0, negative (–);1–4, weakly positive (+);5–8, medially positive (++);and 9–12, strongly positive (+++).
STATISTICAL ANALYSIS
Statistical evaluation of the data was carried out using the Statistical Package for Social Sciences (SPSSversion 13.0; SPSS Inc., Chicago, IL, USA). Continuous data were compared using analysis of variance or the rank sum test and paired comparisons. Categorical data were compared using the χ2test. Factors were evaluated using logistic regression analysis, where tumour type was the dependent variable, Y , and clinical and immunohistochemical data were the independent, X , variables (Table2). The factors that showed statistical significance in a single-factor ordinal logistic regression analysis were entered into a multiple-factor
CLASSIFICATION
Immunoreactivity was classified as continuous data (0%undetectable through to 100%homogeneous staining) for all markers. A pathological image analysis system (DMR+Q550,Leica, Wetzlar, Germany) was used to examine and score slides independently by estimating the percentage of tumour cells that showed characteristic staining; the stained sections were all examined at high magnification (400×).Staining intensity was graded as follows:0, achromatism; 1, light yellow; 2,
Protein expression in differentiated thyroid tumours
binary logistic regression analysis. Factors were eliminated using a forward-stepwise method, with entry level set at α=0.05and elimination level set at α=0.10. Predictive performance for variables was assessed using the receiver–operatingcharacteristic (ROC)curve. The ROC used the relationship between sensitivity and specificity to calculate the best value for each variable for predicting thyroid tumour status. The variable with the best value was selected and, using successive partitioning, the predictive accuracy and best cut point for the best selected variable was calculated. A P -value
follicular carcinomas) and 48patients with thyroid adenomas.
PROTEIN MARKER IMMUNOREACTIVITY
Of the 13protein markers evaluated, significant differences in immunoreactivity were observed between the thyroid carcinoma and thyroid adenomas groups for FHIT, p16, PCNA, p53, MMP-7, HBME-1, E-cadherin, MMP-2, PTTG and hTERT (P
Results
PATIENTS
The study included tissue specimens and collected data from 119patients:71patients with thyroid carcinoma (45with thyroid papillary carcinomas, 26with thyroid
MULTIPLE FACTOR BINARY
LOGISTIC REGRESSION ANALYSIS
Multiple factor binary logistic regression analysis indicated that MMP-2, HBME-1, p16and FHIT were related to differentiated
Protein expression in differentiated thyroid tumours
A
B
C
D
E
F
G
FIGURE 1:Expression of Hector Battifora mesothelial-1(HBME-1)in (A)papillary thyroid carcinoma, (B)follicular thyroid carcinoma and (C)thyroid adenoma. Expression of p16in (D)thyroid adenoma; (E)follicular thyroid carcinoma (p16positive), (F)follicular thyroid carcinoma (p16negative) and (G)papillary thyroid carcinoma. The chromogen used in all images was diaminobenzidine. Scale bars 50
µm
Protein expression in differentiated thyroid tumours
thyroid tumours; FHIT and p16were negatively correlated and HBME-1and MMP-2were positively correlated (Table4). Goodness-of-fit indicated that the observed proportion of patients with tumours without carcinoma was similar to the predicted proportion in the derivation group (χ2, 10.666; degrees of freedom, 8; P =0.221). The calibration curves for the derivation data demonstrated good calibration of the prediction rule.
85.0%,respectively. In an attempt to increase the sensitivity of HBME-1, p16was selected on the basis that its ROC curve area was greater than the areas for MMP-2and FHIT. Sensitivity of the cluster, HBME-1and p16, was 95.8%and the specificity was 95.5%.
HBME-1AND P 16EXPRESSION IN DIFFERENT THYROID CARCINOMAS
The expressions of HBME-1and p16in thyroid papillary and follicular carcinomas are given Table 6. HBME-1expression was not significantly different for papillary and follicular carcinomas, whereas expression of p16was significantly specific for differentiated thyroid carcinomas (P =0.026). The expression of p16in papillary carcinomas was mainly either weak or not detected, with no cases being strongly positive, whereas in some cases of follicular carcinoma p16expression was strongly positive.
PREDICTIVE VALUES OF TISSUE MARKER EXPRESSIONS
The four variables (MMP-2,HBME-1, p16and FHIT) that attained a statistically significant level of significance in the multivariate analysis were analysed using the ROC curve, to identify the appropriate cut-off points for the prediction of malignancy. A threshold that yielded an appropriate trade-off between sensitivity and specificity (i.e.probability of 0.5for a tumour without carcinoma) was used and the results are given in Table 5. The best selected variable was HBME-1, with results for sensitivity, specificity, accuracy, positive predictive value and negative predictive values of 70.8%,91.5%,83.2%,82.3%and
Discussion
The development of advanced cross-sectional imaging has led to earlier diagnosis of malignant thyroid tumours, thereby increasing the challenge of differentiating
benign from malignant pathology on the basis of histomorphological features alone. Most thyroid adenomas can be distinguished from carcinomas by permanent section microscopy based on well-defined, uniformly agreed-upon, pathological criteria. 9However, the specific histological diagnosis of thyroid neoplasms is still challenging, particularly as some cases are difficult to distinguish clearly on clinical and pathological features. Diagnostic difficulties lead to misdiagnosis in some patients and create difficulties with regard to the choice of therapeutic schedules. Therefore, the identification of a simple measure to distinguish the various types of differentiated thyroid tumour will, therefore, have meaningful clinical value. 10
This study investigated 13factors that were likely to be linked to the differential diagnosis of differentiated thyroid tumours:FHIT, PPAR-γ, p16, PCNA, p53, Ki-67, MMP-2, PTTG, hTERT, HBME-1, MMP-7, E-cadherin and bFGF. However, there is some controversy over the usefulness of immunohistochemical analysis of biomarkers in cases that are indeterminate. The purpose of this study was to determine whether immunohistochemical protein profiling of thyroid tissue may identify markers that are useful in distinguishing benign from malignant differentiated thyroid tumours.
The investigation of cell-cycle-related proteins by immunohistochemical analysis has been used to study a variety of endocrine neoplasms. The most frequently detected mutations in human malignancy involve the cell-cycle control factors p53, PCNA and Ki-67. 11,12In the present study there was significantly higher expression of p53and PCNA in thyroid carcinomas compared with thyroid adenomas, however the expression of Ki-67was not significantly different
between the two groups; immunopositivity for Ki-67among the carcinomas was highly variable, ranging from +to +++.This result may be related to antibody selection, inability of the antibody to recognize a protein with altered configuration, or antigen retrieval methodology but it makes it unlikely that Ki-67alone will be of practical use in differentiating benign from malignant differentiated thyroid tumours. Up-regulation of FHIT, p16and PPAR-γhave also been shown to result in cell-cycle arrest and apoptosis. 13–15Previous studies have demonstrated low to absent p16or FHIT immunoreactivity in thyroid carcinomas 4,16and, in the present study, their expression in thyroid carcinomas was found to be low. Conversely their expression has been found to be uniformly high in other benign endocrine tumours. 17In the present study, there was no difference in expression of PPAR-γbetween thyroid carcinomas and adenomas, which underlines the high variability in expression, although PPAR-γhas been found by others to be overexpressed in thyroid carcinomas, but only in thyroid follicular carcinomas and not in thyroid papillary or undifferentiated carcinomas. 10This suggests that PPAR-γmay play a fundamental role in thyroid follicular tumours.
Both PTTG and hTERT have anti-degradation and anti-apotosis effects on cells and appear to be involved in cell proliferation or apotosis inactivation in human malignancy. 18–20In previous studies, underexpression of PTTG and hTERT was found to be associated with benign tumour biology. 18,21,22There were significant differences in the positivity scores for PTTG and hTERT between thyroid adenomas and carcinomas in the present study that indicated their potential for use in differential diagnosis; the immunoreactivity for PTTG and hTERT was significantly higher in thyroid carcinoma compared with thyroid adenoma. These data suggest that PTTG and hTERT may be specific for the diagnosis of thyroid malignancy and for predicting malignant risk.
The expression of proteins associated with tumour cell invasion showed, in the present study, that MMP-2and MMP-7were more frequently identified in thyroid carcinoma than in thyroid adenoma, supporting the findings of previous studies and validating the value of these protein markers in distinguishing benign from malignant differentiated thyroid tumours. 23,24The association between E-cadherin underexpression and endocrine tumours has been reported previously, 25and the present results indicated that E-cadherin is also underexpressed in thyroid carcinomas.
Although a previous study has indicated that tumour vessel proliferation and the number of adverse pathological features in differentiated thyroid tumours are associated with bFGF immunostaining, 26there was no significant difference between bFGF expression in thyroid carcinomas and expression in thyroid adenomas in the present study. This suggests that bFGF is not a useful tool for diagnosing malignancy in differentiated thyroid tumours. The present study demonstrated overexpression of HBME-1in thyroid carcinomas, with 92%of cases showing positive expression compared with 29%for thyroid adenomas. This indicates that HBME-1could be considered a good marker for distinguishing between benign and malignant differentiated thyroid tumours. The number of adverse morphological features displayed by differentiated thyroid tumours correlated significantly with under-expression of FHIT, p16and E-cadherin, and with overexpression of MMP-2, PCNA, PTTG, p53, hTERT, HBME-1and MMP-7. To the best of our knowledge, the association of these 10factors in differentiated thyroid tumours has not been reported previously; the alliance of these factors may be a useful tool in distinguishing between benign and malignant differentiated thyroid tumours. Immunohistochemical staining of the 13protein markers investigated in this study and the three factors of tumour classification and size, patients’gender, and age, were subject to multiple factor binary logistic regression analysis which indicated that MMP-2, HBME-1, p16and FHIT were factors likely to be related to differentiated thyroid tumours. The correlations between malignant differentiated thyroid tumours were negative for FHIT and p16, and positive for HBME-1and MMP-2, with HBME-1and p16showing the highest potential for differentiation between thyroid
carcinomas and adenomas. It has previously been suggested that HBME-1is a highly sensitive and reliable diagnostic marker for the pre-operative identification of thyroid carcinomas. 10,27In a study by Liu et al ., 9the accuracy of HBME-1immunodetection in the differential diagnosis of benign and malignant thyroid lesions was 88%,which compares with 83.2%in the present study. Although HBME-1has frequently been suggested as a very sensitive marker for papillary carcinoma, some studies have indicated that it shows low reactivity in follicular carcinoma. 28In the present study, HBME-1expression was not significantly higher in papillary carcinomas compared with follicular carcinomas. In comparison, a greater number of follicular carcinomas showed strong positivity to p16compared with papillary carcinomas. Thus, the use of HBME-1immunohistochemistry in differentiated thyroid carcinomas may increase diagnostic accuracy but, in the present study, p16had a slightly higher sensitivity. Cluster analysis with p16and HBME-1in the present study found that the sensitivity increased to 95.8%and the specificity increased to 95.5%,indicating enhanced diagnostic accuracy.
In conclusion, the under-or over-expression of cell-cycle-related proteins and tumour-invasive proteins can aid the differential diagnosis of benign and malignant tumours. In the present study, expressions of the protein markers, FHIT, p16, E-cadherin, MMP-2, PCNA, PTTG, p53, hTERT, HBME-1and MMP-7were found to be significantly different between differentiated thyroid carcinomas and thyroid adenomas, indicating their utility as tools for differential diagnosis. In particular, HBME-1was found to be a useful marker for distinguishing between benign and malignant differentiated thyroid tumours, and its
H-S Liang, Y-H Zhong, Z-J Luo et al .
Protein expression in differentiated thyroid tumours
combined use with p16was found to increase specificity and sensitivity and, hence, diagnostic accuracy.
Guangxi Province Science Foundation for Youth (No.0728108). The authors are grateful to Xiujiu Liang for technical assistance and to Su Xuan for helping with the manuscript preparation.
Acknowledgements
This study was supported by a grant from the Applied Basic Research Programs of Science and Technology Commission Foundation of Guangxi Province (No.0339080) and
Conflicts of interest
The authors had no conflicts of interest to declare in relation to this article.
•Received for publication 15January 2009•Accepted subject to revision 20January 2009
•Revised accepted 22May 2009
Copyright 2009Field House Publishing LLP
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Author’saddress for correspondence
Dr Hua-Sheng Liang
Department of Endocrinology, Ninth Affiliated Hospital, Guangxi Medical University,
Beihai 536000, China.
E-mails:flochaos@fimmu.com;flowchaos@yahoo.com.cn