1Department of Endocrinology - Endocrine Oncology, Theagenio Cancer Hospital, Thessaloniki, Greece, 2Department of Medical Sciences, Endocrine Oncology, Uppsala University, Uppsala, Sweden, 3Department of Pathology, Medical School, Aristotle University of Thessaloniki, Greece, 4Department of Medical Sciences, Clinical Chemistry, Uppsala University, Uppsala, Sweden
OBJECTIVE: Peptide receptor radionuclide therapy (PRRT) is dependent upon binding of radiolabelled peptides to their respective receptor expressing cells. The main objective of this study was to characterize the expression of somatostatin receptor (SSTR) subtypes in non-medullary thyroid cancers in order to be able to recommend the use of PRRT as a treatment option in patients with progressive local or metastatic disease. DESIGN: We constructed tissue microarrays from paraffin blocks prepared from 47 cases of non-medullary thyroid carcinomas and related normal thyroid tissue. Immunohistochemical staining was performed with five different polyclonal SSTR antibodies. RESULTS: SSTR subtypes sst2 and sst3 were expressed in all non-medullary thyroid carcinomas, sst1 and sst5 in 75%, and sst4 in 38%. Coexpression of more than three subtypes was detected in 36 of the 47 cases. The expression of SSTR subtypes in normal thyroid tissue was low or absent. CONCLUSIONS: Non-medullary thyroid carcinomas frequently express all SSTR subtypes. This expression provides a basis for further studies with the aim of exploring PRRT as a possible new treatment for iodine-131 refractory metastatic non-medullary thyroid carcinomas.
Thyroid carcinoma, Somatostatin receptor, Tissue microarray, Immunohistochemistry
INTRODUCTION
Thyroid carcinoma is the most frequent type of endocrine cancer. The prognosis for follicular cell-derived differentiated thyroid carcinoma (DTC) (papillary/follicular thyroid cancer) is generally good.1 Conventional therapy involves thyroidectomy, thyrotropin hormone suppressive therapy with thyroxin, and iodine-131 administration.2 Approximately 20% of patients with DTC do not, however, undergo remission but experience progressive local or metastatic disease,3 and for some of these patients additional radioiodine therapy is ineffective because of the loss of their capacity for radioiodine uptake by the dedifferentiated tumour cells.3,4 New therapeutic approaches are needed for these patients.
Somatostatin receptor scintigraphy (SRS) has become an efficient method for detection of neuroendocrine tumours and radiolabelled somatostatin analogues are also used for tumour targeting therapy.5 SRS depends upon binding of labelled somatostatin analogues to somatostatin receptor (SSTR) expressing cells and has in a number of studies been used to image DTC.6 Most studies have been performed with the somatostatin analogue 111In-octreotide, and the results have in most cases been favourable with a sensitivity from 50-100%,6 although there are reports of much lower SRS positivity.7,8 The observed differences in uptake during SRS probably depends on the protocol used, the anatomical localization of the tumours, and differences in the expression of the SSTR subtypes (sst1-sst5), in particular the sst2 subtype.9
By virtue of its superior diagnostic accuracy as compared to SRS, Gallium-68 somatostatin receptor positron emission tomography/computed tomography (Ga-68 SR-PET/CT) could replace SRS to evaluate the expression of somatostatin receptors in endocrine tumours, Ga-68 SR-PET/CT being one of the newly emerging methods holding potential for the near future. Recently, 68Ga-DOTANOC has been increasingly used in several centres and it was reported to have a more favourable dosimetry and to be more accurate for NET lesion detection than conventional imaging or SRS.10,11 68Ga-DOTATOC binds to sst2 with high affinity and to sst5 with reasonable affinity, while 68Ga-DOTA-NOC has high affinity to sst2, sst3, and sst5.10
There are several reports on the expression of SSTRs in thyroid tumours,12,13 most of which have employed mRNA analysis by e.g. RT-PCR. Klagge and collaborators12 investigated the mRNA expression of sst1-5 in benign and malignant epithelial thyroid tumours and detected a predominant expression of sst2 and sst5, a weak expression of sst1 and sst3, and expression of sst4 in only a few papillary thyroid carcinomas.
Immunohistochemistry (IHC), which in contrast to RT-PCR can reveal the cellular and subcellular pattern of expression, has been used in only a few instances to investigate SSTR expression in thyroid cancers. Sancak and coworkers14 have determined the expression of sst2, and Papotti15 analyzed the localization of all subtypes in medullary carcinomas and found a rich but heterogenous expression of all subtypes with the exception of sst4.
We have characterized the expression of SSTR subtypes in non-medullary thyroid carcinomas aiming to provide a basis for future development of imaging and therapy with somatostatin analogues for those patients with thyroid cancer who fail to respond to treatment by conventional therapies.
MATERIALS AND METHODS
Tissue samples
Paraffin block samples from 47 patients (15 males, 32 females) with thyroid carcinomas were selected blindly from archive material at the Pathology Department, Medical School, Aristotle University of Thessaloniki (AUTH), Greece. The samples included cases with known follicular cell thyroid cancer. Patients with medullary thyroid carcinoma were excluded from the present study. Thirty-three of the patients had a history of total thyroidectomy and 14 had a total thyroidectomy and cervical lymph node dissection.
Clinical and pathological data
The samples encompassed 38 papillary thyroid carcinomas (25 classical type, 13 follicular variant), four follicular, two anaplastic and three Hürthle cell thyroid carcinomas. The mean age of the patients was 49 years (range 8-85) and the male/female ratio was approximately 1:2. Eleven of the tumours measured ≤1cm at their greatest diameter, and the remaining 36 measured >1cm. Fifteen out of 47 patients presented lymph node metastases (six men and nine women) and twelve showed invasion of the thyroid parenchyma or extrathyroid soft tissue invasion. The 46 samples from the non-neoplastic thyroid tissue, obtained from areas adjacent to the carcinomas, consisted of 39 nodular goiters, six Hashimoto / lymphocytic thyroiditis, and two normal thyroid tissue.
Tissue microarray (TMA) construction
Hematoxylin and eosin-stained sections from the 47 specimens were examined and areas with adequate material of neoplasms were marked. TMA construction was performed with a tissue arraying instrument (Beecher Instruments, Silver Springs, MD) at the Department of Pathology of the AUTH. Tissue cylinders with a diameter of 0.6 mm were punched from the marked areas of each “donor” paraffin block and transferred into the recipient block. Each case was represented by 7 cores from the thyroid carcinoma (TC) and 5 cores from non-neoplastic thyroid tissue (NNTT) adjacent to the carcinoma. At the two edges of the recipient block, 8 cores from skin tissue and 5 cores from intestinal carcinoma were inserted for orientation of the block. The TMA was subsequently cut into three μm sections.
Immunohistochemistry (IHC)
The immunohistochemical staining was performed at the Department of Endocrine Oncology, Uppsala University Hospital, Uppsala, Sweden. Sections were deparaffinized in xylene and rehydrated using decreasing concentrations of ethanol. Before immunostaining, the sections were treated in a microwave oven at 700 W for 10 minutes and 350 W for 15 minutes in 50 mM Tris buffer (pH 8.0). Endogenous peroxidase was blocked with 1% hydrogen peroxidase in PBS for 30 minutes. Avidin-binding protein was blocked by incubating the sections sequentially with avidin and biotin in Blocking Kit (Vector Laboratories, Burlingame, CA, USA). Between incubations, the sections were washed in PBS and excess liquid was carefully wiped away from around the specimen. To avoid non-specific background staining, the sections were incubated with normal goat serum in a dilution 1:5 in PBS for 30 minutes before applying the primary antibody. The production and specificity of the five different polyclonal SSTR specific antibodies have been described before.16 The primary antibodies were used in the following dilutions; sst1 1:3500, sst2 1:3500, sst3 1:2500, sst4 1:3000, and sst5 1:5000. Anti-rabbit serum diluted 1:200 was used as biotinylated secondary antibody. The immunoreaction was visualized with an Elite kit (Vector Laboratories). Diaminobenzidine tetrahydrochloride (Vector Laboratories) was used as chromogen. The sections were counterstained for 30 seconds with Mayer’s hematoxylin (Apoteksbolaget, Sweden).
IHC evaluation
A scale involving percentage of positive cells for both membranous and cytoplasmic localization of stains was applied. We considered a case as negative when the percentage of positive cells was <5% of the total number of cells, focal positive when it was >5-20% and diffuse for these cases having >20% positive cells. Immunostains were evaluated independently by two persons (ETJ, GK).
Statistical methods
Variables analyzed included age, sex, tumour size, invasion of thyroid parenchyma or extrathyroidal extension, lymph node metastases, and SSTRs expression. Calculations were carried out using SPSS v14.For comparisons between different categories of immunohistochemistry results, the Pearson’s chi square (x2) test was used. Results were considered statistically significant when p value was less than 0.05.
RESULTS
The immunohistochemical analysis showed that SSTR subtypes were commonly expressed in non-medullary thyroid carcinoma cells (Table 1). All analyzed samples displayed positive immunostaining for both sst2 and sst3 and, although the staining was heterogenous, nearly all samples were scored as “diffuse”, i.e. >20% of the cells showed immunoreactivity. In addition, sst1 and sst5 were detected in the majority of the samples, whereas sst4 appeared to be infrequently expressed. Co-expression of several receptor subtypes was very common in the analyzed carcinomas and expression of three or more subtypes was detected in 36 of the 47 cases (Figure 1A-E). In the non-neoplastic thyroid tissues the expression of SSTR was quite low. No expression at all of sst1 or sst4 could be detected, whereas sst2 was expressed in 13%, sst3 in 24%, and sst5 in only 2% of the 46 samples. Thus, all SSTR subtypes showed a higher expression in non-medullary thyroid carcinomas than in the normal thyroid tissue (Figure 2).
Figure 1. A case of PTC expressing four out of five receptors. Cytoplasmic and
membranous localization of immunostains are observed for SST1 (A), SST2 (B), SST3 (C), and SST5 (E), whereas SST4 (D) is negative (SST1-5 X400).
Figure 2. Overexpression of SST3 in thyroid carcinoma compared to adjacent normal tissue (SST3 X400).
Cellular localization of somatostatin receptors
SSTRs expression was observed mainly in the cytoplasm of neoplastic cells (Figure 3A-B), although membranous localization was apparent for all subtypes in the thyroid carcinomas (Figure 3C-D). Among the different subtypes, sst2 and sst5 were most frequently localized to the plasma membrane (Table 2). In the non-neoplastic tissues no membranous localization of any SSTR could be detected.
Figure 3. Focal (SST4, A) and diffuse (SST3, B) cytoplasmic expression (IHC,
×400) of SSTRs. Membranous localization of immunostains is seen as well (C:SST1 ×400, D: SST2 ×400).
Correlation of SSTR expression to clinicopathological parameters
Statistical analysis was meaningful to perform only on the total collection of samples and the PTC subgroup, but no significant correlation was found between SSTR subtype expression and tumour size or any other clinicopathological parameter. Furthermore, there was no correlation between sst2, sst3, and sst5 cytoplasmic or membranous expression in thyroid carcinomas and the expression in the corresponding non-neoplastic tissue (x2, sst2 cytoplasmic: p= 0.506, sst2 membranous: p= 0.228, sst3 cytoplasmic: p= 0.656, sst3 membranous: p= 0.581, sst5 cytoplasmic: p= 0.244, sst5 membranous: p= 0.140). Although the number of Hürthle cell carcinomas was low, one may note that all three cases express at least four of the five SSTR subtypes.
DISCUSSION
In this study, we demonstrated that there is a high expression of all types of SSTRs in human non-medullary thyroid carcinoma tissue, while the expression of SSTRs was low in the non-neoplastic thyroid tissue obtained from the same operation material. The sst2 and sst3 subtypes appeared to be the most abundantly expressed, whereas sst4 was expressed in only a small number of tumours. In normal thyroid tissue the expression of SSTR subtypes was restricted to sst2, sst3, and sst5, which is in complete agreement with results from a study on the distribution of SSTRs in humans.17,18 The SSTR expression was located both in the cytoplasm and at the plasma membrane of the thyroid cells, also in agreement with earlier findings.17,18
The results from the immunohistochemical analysis of thyroid tumours obtained here are mainly in line with results obtained by Klagge et al. using RT-PCR.12 This group reported the predominant expression of sst2 and sst5 mRNA, a weaker expression of sst1 and sst3, and sst4 expression only in a few PCTs. The similarities between our results on non-medullary thyroid carcinomas and the results from Papotti et al15 on medullary thyroid carcinomas are also substantial. In their study, approximately 50% of the medullary thyroid carcinomas showed immunoreactivity for one or several of the subtypes sst1, sst2, sst3 or sst5, and only 4% were stained positive with antibodies to sst4. In agreement with our results, Papotti et al. did not find any significant correlation between SSTR expression and sex, age, tumour size or tumour stage. Taken together, thyroid carcinomas, of both medullary and non-medullary types, commonly express one or several of the SSTR subtypes (sst1, sst2, sst3, and sst5).19
The apparent prominent expression of SSTRs in Hürthle cell thyroid carcinomas is in agreement with positive imaging results with SRS in non-medullary thyroid cancer.20 Pathological uptake was observed in Hürthle cell carcinomas with 95% positive examinations in patients with thyroglobulin levels exceeding 10 ng/ml. The analogue 90Y-DOTATOC was, however, ineffective in the treatment of three cases of metastasizing Hürthle cell carcinomas.
SRS is not commonly used in the diagnostic work-up of patients with non-medullary thyroid carcinoma. Our results indicate that treatment with PRRT might be explored as a possible option for patients with metastatic non-medullary thyroid carcinomas refractory to conventional treatment. The development of new somatostatin analogues and new radionuclides used for treatment may provide new agents for this. However, further studies are needed to determine if PRRT can be used as an alternative for this group of patients.
ACKNOWLEDGEMENT
This work was supported by the Swedish Cancer Society and the Lions Foundation for Cancer Research at Uppsala University Hospital.
REFERENCES
1.Hundahl SA, Fleming ID, Fremgen AM, Menck HR, 1998 A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985-1995. Cancer 15: 2638-2648.
2.Pacini F, Schlumberger M, Dralle H, Elisei R, Smit JW, Wiersinga W, 2006 European consensus for the management of patients with differentiated thyroid carcinoma of the follicular epithelium and the European Thyroid Cancer Taskforce. Eur J Endocrinol 154: 787-803.
3.Sherman SI, 2003 Thyroid carcinoma. Lancet 8: 501-511.
4.Maxon HR 3rd, Smith HS, 1990 Radioiodine-131 in the diagnosis and treatment of metastatic well differentiated thyroid cancer. Endocrinol Metab Clin North Am 19: 685-718.
5.Janson ET, 2006 Treatment of neuroendocrine tumors with somatostatin analogs. Pituitary 9: 249-256.
6.Teunissen JJ, Kwekkeboom DJ, Krenning EP 2006 Staging and treatment of differentiated thyroid carcinoma with radiolabeled somatostatin analogs. Trends Endocrinol Metab 17: 19-25.
7.Garin E, Devillers A, Le Cloirec J, et al, 1998 Use of indium-111 pentetreotide somatostatin receptor scintigraphy to detect recurrent thyroid carcinoma in patients without detectable iodine uptake. Eur J Nucl Med 25: 687-694.
8.Valli N, Catargi B, Ronci N, et al, 1999 Evaluation of indium-111 pentetreotide somatostatin receptor scintigraphy to detect recurrent thyroid carcinoma in patients with negative radioiodine scintigraphy. Thyroid 9: 583-589.
9.John M, Meyerhof W, Richter D, et al, 1996 Positive somatostatin receptor scintigraphy correlates with the presence of somatostatin receptor subtype 2. Gut 38: 33-39.
10.Ambrosini V, Fani M, Fanti S, Forrer F, Maecke HR, 2011 Radiopeptide imaging and therapy in Europe. J Nucl Med 2: 42S-55S.
11.Treglia G, Castaldi P, Rindi G, Giordano A, Rufini V, 2012 Diagnostic performance of Gallium-68 somatostatin receptor PET and PET/CT in patients with thoracic and gastroenteropancreatic neuroendocrine tumours: a meta-analysis. Endocrine :DOI 10.1007/s12020-012-9631-1.
12.Klagge A, Krause K, Schierle K, Steinert F, Dralle H, Fuhrer D, 2010 Somatostatin receptor subtype expression in human thyroid tumours. Horm Metab Res 42: 237-240.
13.Pisarek H, Stepien T, Kubiak R, Borkowska E, Pawlikowski M, 2009 Expression of somatostatin receptor subtypes in human thyroid tumors: the immunohistochemical and molecular biology (RT-PCR) investigation. Thyroid Research 2: 1-8.
14.Sancak S, Hardt A, Singer J, et al, 2010 Somatostatin receptor 2 expression determined by immunohistochemistry in cold thyroid nodules exceeds that of hot thyroid nodules, papillary thyroid carcinoma, and Graves’ disease. Thyroid 20: 505-511.
15.Papotti M, Kumar U, Volante M, Pecchioni C, Patel YC, 2001 Immunohistochemical detection of somatostatin receptor types 1-5 in medullary carcinoma of the thyroid. Clin Endocrinol (Oxf) 54: 641-649.
16.Ludvigsen E, Olsson R, Stridsberg M, Janson ET, Sandler S, 2004 Expression and distribution of somatostatin receptor subtypes in the pancreatic islets of mice and rats. J Histochem Cytochem 52: 391-400.
17.Taniyama Y, Suzuki T, Mikami Y, Moriya T, Satomi S, Sasano H, 2005 Systemic distribution of somatostatin receptor subtypes in human: an immunohistochemical study. Endocr J 52: 605-611.
18.Diakatou E, Kaltsas G, Tzivras M, Kanakis G, Papaliodi E, Kontogeorgos G, 2011 Somatostatin and dopamine receptor profile of gastroenteropancreatic neuroendocrine tumors: an immunohistochemical study. Endocr Pathol 22: 24-30.
19.Pisarek H, Pawlikowski M, Kunert-Radek J, Kubiak R, Winczyk K, 2010 SSTR1 and SSTR5 subtypes are the dominant forms of somatostatin receptor in neuroendocrine tumors. Folia Histochem Cytobiol 1: 142-147.
20.Gorges R, Kahaly G, Muller-Brand J, Macke H, Roser HW, Bockisch A, 2001 Radionuclide-labeled somatostatin analogues for diagnostic and therapeutic purposes in nonmedullary thyroid cancer. Thyroid 11: 647-659.
Address for correspondence:
Georgia Karayannopoulou, MD, Department of Pathology, Medical School, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece,
FAX: +30 2310 999229, e-mail: karayan@med.auth.gr
Received 31-01-12, Revised 25-03-12, Accepted 10-04-12