Overexpression of wild-type c-RET and zero prevalence of RET/PTC rearrangements are associated with papillary thyroid cancer (PTC) in Kuwait

Background

Papillary thyroid carcinoma (PTC) is common in Kuwait. The activation of the RET oncogene by DNA rearrangement (RET/PTC) is known to have an important role in PTC carcinogenesis. However, the real frequency of the RET/PTC expression in PTC is variable between different studies. This study seeks to determine the prevalence of RET/PTC and to analyze the RET oncogene expression associated with PTC in Kuwait.

Methods

RET expression and DNA rearrangements (RET/PTC 1, RET/PTC 2 and RET/PTC 3) were studied by RT–PCR in different thyroid diseases. Results were confirmed by the Southern blot and by immunohistochemistry. Quantitative real-time PCR was used to determine the level of RET mRNA expression in PTCs.

Results

Wild-type (nonrearranged) c-RET oncogene was overexpressed in 60% of PTC cases and absent in follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma (ATC), follicular adenomas (FA) or normal thyroid. No RET/PTC rearrangement was detected in any sample. The c-RET expression in Hashimoto's thyroiditis and multinodular goiter was limited to follicular cells with PTC-like nuclear changes.

Conclusions

The overexpression of wild-type c-RET is a characteristic molecular event of PTCs in Kuwait. The prevalence of RET/PTC is zero and among the lowest recorded in the world.     

Survey of 548 oncogenic fusion transcripts in thyroid tumors supports the importance of the already established thyroid fusions genes

Neoplasms frequently present structural chromosomal aberrations that can alter the level of expression of a protein or to the expression of an aberrant chimeric protein. In the thyroid, the PAX8‐PPARG fusion is present in the neoplastic lesions that have a follicular architecture—follicular thyroid carcinoma (FTC) and follicular variant of papillary thyroid carcinoma (FVPTC), and less frequently in follicular thyroid adenoma (FTA), while the presence of RET/PTC fusions are largely restricted to papillary thyroid carcinoma (PTC). The ability to detect fusion genes is relevant for a correct diagnosis and for therapy. We have developed a new fusion gene microarray‐based approach for simultaneous analysis of all known and predicted fusion gene variants. We did a comprehensive screen for 548 known and putative fusion genes in 27 samples of thyroid tumors and three positive controls—one thyroid cancer cell line (TPC‐1) and two PTCs with known CCDC6‐RET (alias RET/PTC1) fusion gene, using this microarray. Within the thyroid tumors tested, only well known, previously reported fusion genes in thyroid oncology were identified. Our results reinforce the pathogenic role played by RET/PTC1, RET/PTC3, and PAX8‐PPARG fusion genes in thyroid tumorigenesis. © 2012 Wiley Periodicals, Inc.     

A subset of the follicular variant of papillary thyroid carcinoma harbors the PAX8-PPARgamma translocation

The occurrence of the PAX8-PPARgamma fusion gene is thought to be restricted to follicular tumors (adenomas and carcinomas) of the thyroid (FTA and FTC). Using interphase fluorescent in situ hybridization (FISH), together with recombinant tissue-type polymerase chain reaction (RT-PCR) and immunohistochemistry, we detected the PAX8-PPARgamma translocation in 4 of 8 cases of the follicular variant of papillary thyroid carcinoma (FVPTC) exclusively or almost exclusively (>95%) composed of follicles. The 4 tumors with the translocation were larger and apparently more invasive than the remaining tumors, but the series is too small to allow a statistically meaningful comparison of the data. Our findings show that follicular thyroid carcinoma (PTC) may also harbor the PAX8-PPARgamma fusion gene and indicate that a subset of FVPTC shares some molecular features of FTA and FTC.     

野生型RET和RET/PTC融合基因在成人散发性甲状腺乳头状癌中的表达及其意义

目的探讨野生型BET（WT-RET）及RET／PTC1、3融合基因在成人散发性甲状腺乳头状癌（PTC）中的表达及其与临床病理学指标的关系和意义。方法用逆转录-聚合酶链反应（RT-PCR）检测102例石蜡与新鲜（43例）甲状腺病变组织（PTC66例,对照组各种良恶性肿瘤及良性病变共36例）中WT-RET和RET／PTC1、3融合基因的表达并结合临床资料进行分析。结果（1）62％（41／66）PTC患者≥40岁。38％（25／66）PTC伴淋巴细胞性甲状腺炎,59％（39／66）伴淋巴结转移,5例（7.6％）有远处转移。（2）RET原癌基因的酪氨酸激酶区（BET-TK）检出率为68．1％（45／66）。BET原癌基因断裂点（BP）与TK的同时检出率在PTC中28．8％（19／66）,腺瘤中12．5％（1／8）,表明存在WT-BET转录物。（3）RET／PTC检出率21．2％（14／66）,其中5例BET／PTC1阳性（7．6％）,9例RET／PTC3阳性（13．6％）。6例（9％）PTC同时表达BET／PTC和WT-BET。36例对照组病例中未检测到RET／PTC融合基因。（4）统计学分析,PTC病例中WT-BET与RET／PTC1融合基因的表达与性别、年龄、肿瘤大小、多灶性、伴淋巴细胞浸润及淋巴结转移等临床病理学指标无关（P〉0．05）。结论RET／PTC融合基因在散发性成人PTC中表达率低,其诊断和判断预后的价值不大。WT-BET在甲状腺肿瘤的滤泡形成过程中起一定作用。     

Follicular variant of papillary thyroid carcinoma: genome-wide appraisal of a controversial entity

The majority of thyroid tumors are classified as papillary (papillary thyroid carcinomas; PTCs) or follicular neoplasms (follicular thyroid adenomas and carcinomas; FTA/FTC) based on nuclear features and the cellular growth pattern. However, classification of the follicular variant of papillary thyroid carcinoma (FVPTC) remains an issue of debate. These tumors contain a predominantly follicular growth pattern but display nuclear features and overall clinical behavior consistent with PTC. In this study, we used comparative genomic hybridization (CGH) to compare the global chromosomal aberrations in FVPTC to the PTC of classical variant (classical PTC) and FTA/FTC. In addition, we assessed the presence of peroxisome proliferator-activated receptor-gamma (PPARG) alteration, a genetic event specific to FTA/FTC, using Southern blot and immunohistochemistry analyses. In sharp contrast to the findings in classical PTC (4% of cases), CGH analysis demonstrated that both FVPTC (59% of cases) and FTA/FTC (36% of cases) were commonly characterized by aneuploidy (P = 0.0002). Moreover, the pattern of chromosomal aberrations (gains at chromosome arms 2q, 4q, 5q, 6q, 8q, and 13q and deletions at 1p, 9q, 16q, 17q, 19q, and 22q) in the follicular variant of PTC closely resembled that of FTA/FTC. Aberrations in PPARG were uniquely detected in FVPTC and FTA/FTC. Our findings suggest a stronger relationship between the FVPTC and FTA/FTC than previously appreciated and support further consideration of the current classification of thyroid neoplasms.     

Caveolin-1 Expression in Thyroid Neoplasia Spectrum: Comparison of Two Commercial Antibodies

We evaluated caveolin-1 expression in the human thyroid neoplasia spectrum with the aim of examining differences in expression as detected by two anti-caveolin-1 antibodies, and secondly, to investigate the association of caveolin-1 expression levels with aggressive papillary thyroid carcinoma (PTC). Immunohistochemical staining using sc894 or AV09019 antibodies revealed that caveolin-1 was generally overexpressed in the PTC group as a whole (classical and follicular variant) when compared to peritumoral tissue (PT), while it was not detected in about half of follicular thyroid carcinoma (FTC) and majority of follicular adenomas (FTA). Caveolin-1 expression decreased in the following order: clPTC, fvPTC, FTC, PT and FTA. The diagnostic accuracy of AV09019 was better than that of sc894 for discriminating: FTA from FTC, FTA or FTC from the follicular variant of PTC, total PTC from nonmalignant tissue, and malignant tumors from nonmalignant tissue. Spearman''s analysis revealed positive correlations of caveolin-1 expression and extrathyroidal invasion (p < 0.05) in PTC for both antibodies. Additionally, AV09019 antibody correlated caveolin-1 upregulation with pathological T status.To conclude, as an immunohistochemical marker AV09019 antibody performed better than sc894 in distinguishing certain histotypes of thyroid tumors. In addition, increased expression of caveolin-1 may be considered as an indicator of papillary carcinoma progression.     

Loss of heterozygosity in follicular and papillary thyroid carcinomas

In this study we aimed at investigating the incidence and the role of 3p deletions, particularly at the 3p25 approximately pter region, in follicle cell-derived thyroid neoplasms, by using loss of heterozygosity (LOH) analysis. We analyzed 12 follicular adenomas (FA), 13 follicular thyroid carcinomas (FTC), and 15 papillary thyroid carcinomas (PTC) with 11 microsatellite markers for chromosome 3. One additional marker on 3q25.2 was also investigated for assessment of deletion extent on 3q. Microsatellite instability was detected at one locus in 1 of 15 PTC (7%) and at four loci in 1 of 13 FTC (8%). Loss of heterozygosity was found in 8 of 12 cases of FTC (67%), in 6 of 15 cases of PTC (40%), and in 2 of 12 FA (17%). We identified three minimal common deleted regions (CDR) involving significant sites of LOH: two in FTC (a new terminal region, of approximately 8 cM distal to D3S1620 at 3p25.3 approximately pter and the D3S1573-D3S1595 region at 3p21.2 approximately p12) and one in PTC (D3S1304-D3S1263 region at 3p25.3 approximately p24.2). The newly identified 3p25.3 approximately pter CDR seems to be specific for FTC. Our results suggest the existence of at least three distinct regions on 3p that might harbor tumor suppressor genes involved in the carcinogenesis processes of FTC and PTC.     

Three-dimensional reconstruction of vessel distribution in benign and malignant lesions of thyroid

In order to better understand the spatial distribution of thyroid vessels, a series of benign and malignant thyroid lesions were studied with three-dimensional (3D) histological stereomicroscopic reconstruction. Cases consisted of normal autoptic thyroids (n=6), colloid goitres (n=6), Basedows disease (n=2), follicular adenoma (FA) (n=4) one of which with Hurthle cells (HC), minimally invasive, well-differentiated follicular carcinoma (FTC) (n=1), well-differentiated FTC with HC (n=1), poorly differentiated FTC (n=13) with extensive angioinvasion, papillary carcinoma (PTC) (n=8) and medullary carcinoma (MTC) (n=1). From each selected nodule, parallel sections were obtained for 3D reconstruction and for histological and immunohistochemical studies. In normal thyroid, large vessels were located at the periphery of the gland with smaller branches present within the thyroid parenchyma that encircled follicles. The same pattern of vascularisation is maintained in lesions showing a follicular architecture as colloid goitre, Basedows disease, FA, well-differentiated FTC and the follicular variant of PTC. Neoplastic lesions, at variance with non-neoplastic lesions, contained rare anastomoses. Poorly differentiated FTC and MTC contained large intratumoural vessels surrounding avascular areas corresponding to solid neoplastic cellular sheets with necrosis. PTC were more vascularised and contained numerous vascular anastomoses. In conclusion, the present data indicate that the vascular distribution is related to the follicular, papillary or solid type of growth. Vascular anastomoses and intratumoural vessels surrounding solid avascular areas are signs of malignancy.     

Clinicopathological and Molecular Histochemical Review of Skull Base Metastasis from Differentiated Thyroid Carcinoma

Skull base metastasis from differentiated thyroid carcinoma including follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC) is a rare clinical entity. Eighteen FTC cases and 10 PTC cases showing skull base metastasis have been reported. The most common symptom of skull base metastasis from FTC and PTC is cranial nerve dysfunction. Bone destruction and local invasion to the surrounding soft tissues are common on radiological imaging. Skull base metastases can be the initial clinical presentation of FTC and PTC in the presence of silent primary sites. The possibility of skull base metastasis from FTC and PTC should be considered in patients with the clinical symptoms of cranial nerve dysfunction and radiological findings of bone destruction. A variety of genetic alterations in thyroid tumors have been identified to have a fundamental role in their tumorigenesis. Molecular histochemical studies are useful for elucidating the histopathological features of thyroid carcinoma. Recent molecular findings may provide novel molecular-based treatment strategies for thyroid carcinoma.     

Synchronous papillary thyroid carcinoma and follicular thyroid carcinoma: case report and review of literature

Collision tumor is a term denoting two histologically distinct tumor types occuring at the same anatomic site, which is a rare clinical entity. In the thyroid gland, collision tumors are rare. Here we report a case of the synchronous occurrence of follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC). The current case report describes a 40-year-old woman with synchronous FTC and PTC. Pathologists and surgeons should be aware of collision tumors to avoid possible misdiagnosis.     

Cyclooxygenase-2 and inducible nitric oxide synthase expression in thyroid neoplasms and their clinicopathological correlation

To evaluate the expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in thyroid neoplasms in a Korean population, we studied a total of 154 cases: papillary carcinoma of classical type (PTC), 86; follicular adenoma (FA), 21; follicular carcinoma (FC), 35; medullary carcinoma (MC), 3; undifferentiated carcinoma (UC), 5; and Hurthle cell neoplasm (HN), 4. Using immunohistochemical staining, COX-2 expression was detected in 62 (72.1%) PTC specimens, 5 (23.8%) FA specimens, 10 (28.6%) FC specimens, 0 (0.0%) MC specimens, 1 (20.0%) UC specimen, and 3 (75%) HN specimens. iNOS expression was detected in 66 (76.7%) PTC specimens, 4 (19.0%) FA specimens, 13 (37.1%) FC specimens, 0 (0.0%) MC specimens, 3 (60.0%) UC specimens, and 4 (100%) HN specimens. The results showed that COX-2 and iNOS were frequently expressed in the PTC and HN specimens, and iNOS was more frequently overexpressed in the FC specimens than in the FA specimens. In PTC, COX-2 and iNOS were significantly overexpressed in patients over 45 yr of age (p=0.029, p=0.041), and iNOS expression was increased in patients with a large primary tumor (p=0.028). These results suggest that the upregulation of COX-2 and iNOS may contribute to the tumor progression of thyroid gland, particularly in PTC and HN, and iNOS may play an adjuvant role during the tumor progression of FC.     

RET oncogene activation in papillary thyroid carcinoma.

The RET proto-oncogene encodes a cell membrane tyrosine-kinase receptor protein whose ligands belong to the glial cell line-derived neurotrophic factor. RET functions as a multicompetent receptor complex that includes alphaGFRs and RET. Somatic rearrangements of RET designated as RET/PTC (from papillary thyroid carcinoma) were identified in papillary thyroid carcinoma before RET was recognized as the susceptibility gene for MEN2. There are now at least at least 15 types of RET/PTC rearrangements involving RET and 10 different genes. RET/PTC1 and RET/PTC3 are by far the most common rearrangements. All of the rearrangements are due to DNA damage and result in the fusion of the RET tyrosine-kinase (RET-TK) domain to the 5'-terminal region of heterologous genes. RET/PTC rearrangements are very common in radiation-induced tumors but have been detected in variable proportions of sporadic (i.e., non-radiation associated) papillary carcinomas. It is estimated that up to approximately half the papillary thyroid carcinomas in the United States and Canada harbor RET/PTC rearrangements, most commonly RET/PTC-1, followed by RET/PTC-3 and occasionally RET/PTC-2. The cause of these rearrangements in sporadic papillary carcinomas is not known, but the close association between their presence and the papillary carcinoma phenotype indicates that they play a causative role in tumor development. The proposed mechanisms of RET/PTC-induced tumorigenesis and the clinical and pathologic implications of RET/PTC activation are discussed.     

Familiäre Karzinome der Schilddrüse

Approximately 5% of differentiated thyroid carcinomas with follicular cell differentiation, papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) and 25–30% of medullary thyroid carcinoma (MTC) are hereditary. They occur either as part of a defined syndrome or are confined to the thyroid gland. Compared to their sporadic non-hereditary counterparts hereditary thyroid carcinomas generally develop earlier and regularly show multifocal tumour growth. With the exception of familial MTC, which is preceded by neoplastic C cell hyperplasia, no precursor lesions of hereditary thyroid carcinoma are known. In strong correlation with the localisation of the germline mutation of the RET protooncogene, familial MTC shows a distinct clinical course which allows precise clinical decision-making for prophylactic thyroidectomy to prevent invasive MTC. According to current knowledge prophylactic thyroidectomy of all other types of hereditary thyroid carcinoma is not justified.     

A score based on microscopic criteria proposed for analysis of papillary carcinoma of the thyroid

Morphologic examination is the gold standard for diagnosing papillary thyroid carcinoma (PTC). This diagnosis, especially for the follicular variant, may be variable even among experts. Our objective was to analyze the frequency of PTC microscopic criteria in a series of thyroid tumors including follicular adenoma (FA) and PTC to build a score which could constitute a novel way for microscopic analysis of such thyroid tumors. Sixty-six PTC and 66 FA were matched for age, sex, and thyroiditis. Two independent observers delineated the classical PTC microscopic criteria. PTC microscopic criteria were variably expressed in individual tumors. Under univariate analysis, all criteria were significantly more frequent in PTC than in FA (p<0.05). Under multivariate analysis, the most important criteria were: dark staining colloid >25%, nuclear grooves, enlarged nuclei, ovoid nuclei >5% of tumor cells, and lack of polarization. Using linear discriminant analysis, these five criteria allowed us to build a scaled score. The score proposed did not allow the making of a definite distinction between FA and PTC in routine practice. It represents the variety of microscopic presentations of FA and PTC on the basis of a statistical analysis and could help in further evaluation of immunohistochemical or molecular markers.     

甲状腺滤泡癌和乳头状癌细胞黏附分子与转移抑制基因nm23-H1蛋白表达的关系

目的 探讨黏附分子CD44、上皮性钙黏附蛋白（E-cad )和转移抑制基因nm23-H1与甲状腺滤泡源性癌分化、浸润转移的关系。方法 采用免疫组织化学SP法和EnVision法检测42例滤泡癌和54例乳头状癌中CD44、E-cad和nm23-h1的表达。结果 CD44主要表达于癌细胞及浸润淋巴细胞膜,低分化滤泡癌和有转移乳头状癌CD44表达分别高于高分化滤泡癌和无转移乳头状癌,E-cad阳性物质和nm23-H1阳性率高于滤泡癌,转移癌的阳性率和表达强度低于原发灶。甲状腺滤泡原性癌CD44检测阳性率高于E-cad和nm23-H1。在滤泡癌中E-cad与nm23-H1的表达之间呈正相关关系,而在乳头状癌中呈正相关趋势。CD44与E-cad的表达、CD44与nm23-H1的表达之间在滤泡癌和乳头状癌均呈负相关趋势。结论 综合检测CD44、E-cad和nm23对甲状腺滤泡源性癌的诊断、预后评估具有一定参考价值。     

Potent mitogenicity of the RET/PTC3 oncogene correlates with its prevalence in tall-cell variant of papillary thyroid carcinoma.

The tall-cell variant (TCV) of papillary thyroid carcinoma (PTC), characterized by tall cells bearing an oxyphilic cytoplasm, is more clinically aggressive than conventional PTC. RET tyrosine kinase rearrangements, which represent the most frequent genetic alteration in PTC, lead to the recombination of RET with heterologous genes to generate chimeric RET/PTC oncogenes. RET/PTC1 and RET/PTC3 are the most prevalent variants. We have found RET rearrangements in 35.8% of TCV (14 of 39 cases). Whereas the prevalences of RET/PTC1 and RET/PTC3 were almost equal in classic and follicular PTC, all of the TCV-positive cases expressed the RET/PTC3 rearrangement. These findings prompted us to compare RET/PTC3 and RET/PTC1 in an in vitro thyroid model system. We have expressed the two oncogenes in PC Cl 3 rat thyroid epithelial cells and found that RET/PTC3 is endowed with a strikingly more potent mitogenic effect than RET/PTC1. Mechanistically, this difference correlated with an increased signaling activity of RET/PTC3. In conclusion, we postulate that the correlation between the RET/PTC rearrangement type and the aggressiveness of human PTC is related to the efficiency with which the oncogene subtype delivers mitogenic signals to thyroid cells.