Real-time quantitative RT-PCR identifies distinct c-RET, RET/PTC1 and RET/PTC3 expression patterns in papillary thyroid carcinoma

RET/PTC1 and RET/PTC3 are the markers for papillary thyroid carcinoma. Their reported prevalence varies broadly. Nonrearranged c-RET has also been detected in a variable proportion of papillary carcinomas. The published data suggest that a wide range in expression levels may contribute to the different frequency of c-RET and, particularly, of RET/PTC detection. However, quantitative expression analysis has never been systematically carried out. We have analyzed by real-time RT-PCR 25 papillary carcinoma and 12 normal thyroid samples for RET/PTC1, RET/PTC3 and for RET exons 10-11 and 12-13, which are adjacent to the rearrangement site. The variability in mRNA levels was marked and four carcinoma groups were identified: one lacking RET/PTC rearrangement with balanced RET exon levels similar to those of the normal samples (7/25 cases, 28%), the second (6/25 cases, 24%) with balanced RET expression and very low levels of RET/PTC1, the third with unbalanced RET exons 10-11 and 12-13 expression, high RET/PTC1 levels but no RET/PTC3 (7/25 cases, 28%), and the fourth with unbalanced RET expression, high RET/PTC1 levels and low levels of RET/PTC3 (5/25 cases, 20%). Papillary carcinomas with high RET/PTC1 expression showed an association trend for large tumor size (P=0.063). Our results indicate that the variability in c-RET and RET/PTC mRNA levels contributes to the apparent inconsistencies in their reported detection rates and should be taken into account not only for diagnostic purposes but also to better understand the role of c-RET activation in thyroid tumorigenesis.     

Hyalinizing Trabecular Tumor of the Thyroid: An Update

Hyalinizing trabecular tumor (HTT) is a rare thyroid tumor of follicular cell origin with a trabecular pattern of growth and marked intratrabecular hyalinization. This tumor is known to share morphological and architectural similarities with paraganglioma and medullary thyroid carcinoma, as well as the nuclear features and RET/PTC1 translocations of papillary thyroid carcinoma. These tumors are not associated with RAS or BRAF mutations. Whether the presence of RET alterations in HTT are sufficient molecular proof of its relationship with papillary thyroid carcinoma (PTC) is still to be defined. Of great interest is the characteristic strong peripheral cytoplasmic and membranous staining of the tumor cells with MIB1 immunostain, not seen in any other thyroid neoplasm. Although cases of malignant HTT have been recorded, HTT should be considered a benign neoplasm or, at most, a neoplasm of extremely low malignant potential.     

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.     

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.     

Diffuse sclerosing variant of papillary thyroid carcinoma: lack of BRAF mutation but occurrence of RET/PTC rearrangements.

Diffuse sclerosing variant of papillary thyroid carcinoma (PTC) is a rare tumour with a characteristic morphology as well as a strong preponderance for younger female patients. The T1799A missense mutation in exon 15 of the BRAF gene and RET/PTC rearrangement have been identified as the dominant genetic tumour initiation events in the pathogenesis of PTC leading to a constitutive activation of the RAS-RAF-MAPK pathway. In order to elucidate the pathogenesis of diffuse sclerosing variant of PTC, the prevalence of BRAF mutation and RET/PTC were determined by RT-polymerase chain reaction and DNA-sequence analysis in tumour samples of seven patients with this variant (all female, age range 15-61 years, mean 33.3 years) without prior radiation exposure. None of these cases showed a BRAF mutation. RET/PTC1 (two out of seven) and RET/PTC3 (one out of seven), which have been shown in large PTC series to comprise together more than 90% of RET/PTC types, were found in <50% of the cases investigated. All seven samples expressed the RET tyrosine kinase domain but lacked its extracellular domain potentially suggesting the existence of rare types of RET/PTC rearrangement in the four remained cases of diffuse sclerosing variant of PTC. Regarding this subtype, our study confirmed the paradigm of a mutual exclusivity between RET/PTC and BRAF in PTC. Additionally, this rare variant of papillary thyroid carcinoma may represent a tumour type susceptible to RET-targeted therapies.     

Unusual metastases of papillary thyroid carcinoma: report of 2 cases

We present 2 cases of papillary thyroid carcinoma (PTC) with conventional morphology that metastasized to unusual sites. The first neoplasm was a PTC whose initial clinical manifestation was a large metastasis in the pancreas which mimicked a primary neoplasm. The mediastinal location of the thyroid gland was responsible for the delay in identification of the primary tumor. Eventually, the patient, a 72-year-old man, developed brain and vertebral metastases. The second case was that of a 58-year-old woman with a PTC with initial metastases in cervical lymph nodes; subsequently, the tumor spread to axillary lymph nodes and finally to the breast. Transformation to anaplastic spindle and giant cell carcinoma within the breast metastasis occurred 20 years after the primary thyroid tumor had been diagnosed and surgically treated. The metastatic anaplastic spindle and giant cell carcinoma contained rhabdoid inclusions further complicating identification. To the best of our knowledge, only 3 cases of PTC metastatic to the breast have been reported, none of them with anaplastic transformation. On the other hand, only 3 cases of PTC metastatic to the pancreas have been published, 2 of them of the tall cell variant, and in none of these cases were the first symptoms attributable to the metastasis. Brief comments about the differential diagnosis are included.     

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.     

The oncogenic activity of RET point mutants for follicular thyroid cells may account for the occurrence of papillary thyroid carcinoma in patients affected by familial medullary thyroid carcinoma

Activating germ-line point mutations in the RET receptor are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinoma (MTC), whereas somatic RET rearrangements are prevalent in papillary thyroid carcinomas (PTCs). Some rare kindreds, carrying point mutations in RET, are affected by both cancer types, suggesting that, under specific circumstances, point mutations in RET can drive the generation of PTC. Here we describe a family whose siblings, affected by both PTC and MTC, carried a germ-line point mutation in the RET extracellular domain, converting cysteine 634 into serine. We tested on thyroid follicular cells the transforming activity of RET(C634S), RET(K603Q), another mutant identified in a kindred with both PTC and MTC, RET(C634R) a commonly isolated allele in MEN2A, RET(M918T) responsible for MEN2B and also identified in kindreds with both PTC and MTC, and RET/PTC1 the rearranged oncogene that characterizes bona fide PTC in patients without MTC. We show that the various RET point mutants, but not wild-type RET, scored constitutive kinase activity and exerted mitogenic effects for thyroid PC Cl 3 cells, albeit at significantly lower levels compared to RET/PTC1. The low mitogenic activity of RET point mutants paralleled their reduced kinase activity compared to RET/PTC. Furthermore, RET point mutants maintained a protein domain, the intracellular juxtamembrane domain, that exerted negative effects on the mitogenic activity. In conclusion, RET point mutants can behave as dominant oncogenes for thyroid follicular cells. Their transforming activity, however, is rather modest, providing a possible explanation for the rare association of MTC with PTC.     

RET/PTC and CK19 expression in papillary thyroid carcinoma and its clinicopathologic correlation

Recently, the rearrangement of RET proto-oncogene has been reported to be the most common genetic change in papillary thyroid carcinoma (PTC). However, its prevalence has been reported variably and its relation to clinical outcome has been controversial. The characteristic nuclear features of PTC usually render the diagnosis, but problem arises with equivocal cytologic features that are present focally. Although there remains some controversy, CK19 has been reported to be a useful ancillary tool for diagnosis of PTC. To evaluate the expression rate of RET/PTC rearrangement and CK19 in PTCs in a Korean population, we studied 115 papillary thyroid carcinomas in 3 mm-core tissue microarray based immunohistochemical analysis. The prevalence of Ret protein expression was 62.6% and the CK19 immunoreactivity was 80.9%. There was no statistically significant association between the Ret positivity and CK19 immunoreactivity, although the percent agreement of the two was relatively high. The clinicopathological variables did not correlate with the expression of Ret. In conclusion, the prevalence of Ret protein expression and its clinicopathological implications in a Korean population are not much different from those reported in previous studies. However, its detection via immunohistochemistry can be a useful diagnostic tool for diagnosing papillary thyroid carcinoma in conjunction with CK19.     

Papillary thyroid carcinoma-like tumor of the kidney: a case report

Thyroid carcinoma-like tumor of the kidney is an extremely rare variant of renal cell carcinoma. Most previously reported cases were incidental finding; and none of them showed papillary thyroid carcinoma (PTC) nuclear features. This study reports the first case of PTC (follicular variant)-like tumor of the kidney in which a female patient presented with hematuria, weight loss, and flank pain. Imaging studies revealed a left renal mass with enlarged hilar lymph nodes. Histologically, the renal tumor had a striking resemblance to follicular variant of PTC. However, no radiological abnormalities were found in the thyroid, mediastinum, or pelvis. Tumor cells were negative for thyroid markers (thyroglobulin and TTF1). According to the authors, this is the first case of PTC (follicular variant)-like tumor of the kidney.     

Hashimoto's thyroiditis shares features with early papillary thyroid carcinoma

Neoplastic transformation is a multistep process that results in a continuous spectrum from the normal (physiological) state to a fully established neoplasm. The gold standard for diagnosis of papillary thyroid carcinoma is conventional histology, the essential element being the characteristic nuclear features, regardless of whether papillary structures are present or not. However, other criteria are being used increasingly in the diagnosis of neoplasms, including immunohistochemical staining and molecular profile. The RET/PTC gene rearrangement is highly specific for papillary thyroid carcinoma and is associated with the characteristic nuclear features seen in papillary thyroid carcinoma. There is an overlap in the morphological features, immunohistochemical staining pattern, and most importantly, molecular profile between papillary thyroid carcinoma and Hashimoto's thyroiditis. Although considered a 'benign' condition, Hashimoto's thyroiditis almost always harbours a genetic rearrangement that is strongly associated with and is highly specific for papillary thyroid carcinoma. Submicroscopic foci of papillary thyroid carcinoma must be present in Hashimoto's thyroiditis, although the clinical behaviour is still benign. Further studies are required to predict which foci will progress to papillary thyroid carcinoma.     

Papillary Thyroid Carcinoma Oncogene (RET/PTC) Alters the Nuclear Envelope and Chromatin Structure

Current evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated (p 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists (p 0.0001). In addition, RET/PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway.     

Primary thyroid spindle cell tumors: spindle cell variant of papillary thyroid carcinoma?

Primary thyroid spindle cell tumors or spindle cell component in the thyroid tumors are very rare. The spindle tumor cells were positive for thyroid papillary carcinoma markers. So these tumors were diagnosed as spindle cell variant of papillary thyroid carcinoma (PTC). To further delineate clinico-pathological features of primary thyroid spindle cell tumors and discuss differential diagnosis, we reported a 67-year-old man with a mass in the right thyroid without clinical symptom. Microscopy revealed that an encapsulated tumor with lot criss spindle cells arranged in bundles. Nuclear grooves were easy to see and rare displayed pseudoinclusions. Immunohistochemical studied showed that the spindle cells were all strong positive for TTF-1, Pax-8, thyroglobulin. Rare follicular were seen in the periphery of the tumor near the thyroid tissue. The cells formed follicular but the spindle tumor cells were positive for pan-keratins. The pathological diagnosis was primary thyroid spindle cell tumors, suspected spindle cell variant of PTC. Primary thyroid spindle cell tumors were presence and without the unified name. The further reports and more discussion were need about these tumors.     

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.     

Anaplastic thyroid carcinoma with rhabdoid features

Anaplastic thyroid carcinoma (ATC) is a rare, highly aggressive neoplasm, characterized by complete or partial composition by undifferentiated cells. We report a case of ATC with rhabdoid features in a 68‐year‐old male, who presented with a rapidly enlarging neck mass. Fine‐needle aspiration (FNA) of the thyroid mass showed discohesive, pleomorphic round to polygonal rhabdoid cells with one to multiple eccentric, large, rounded nuclei with a prominent nucleolus, moderate to abundant, globoid cytoplasm which oftentimes harbor a pale para‐nuclear inclusion. The cytoplasm of some cells contained variously sized, eosinophilic granules. Rare cells contained neutrophils in their cytoplasm. Mitoses including atypical mitotic figures and necrosis were readily seen. Histologic examination of needle core biopsy (NCB) revealed individual dispersed and sheets of pleomorphic neoplastic cells with similar cytomorphologic features as described above. The tumor extensively infiltrated a myxocollagenous stroma containing lymphocytes and neutrophils, and demonstrated foci of necrosis. Tumor cells were immunoreactive for keratins AE1/AE3, CAM5.2, and CK19; PAX‐8, and p63, but negative for S‐100, HMB‐45, calcitonin, TTF‐1, thyroglobulin, CD56, HBME‐1, glypican‐3, PAX‐5, myogenin, CD31, and INI‐1. The differential diagnosis of this malignant rhabdoid tumor is discussed. Diagn. Cytopathol. 2015;43:416–420. © 2015 Wiley Periodicals, Inc.     

Warthin-like tumour of the thyroid gland: RET/PTC expression indicates it is a variant of papillary carcinoma

AIMS: Three cases with features of so-called 'Warthin-like tumour' of the thyroid (WaLTT) are described, in order to evaluate its relationship with papillary carcinoma (PC). METHODS AND RESULTS: We performed an histological and immunohistochemical study with emphasis on RET/PTC expression. The most striking features are represented by marked lymphocytic infiltration in the stalks of papillae and by oxyphilic metaplasia of epithelium, resembling Warthin tumour of the salivary gland. In all cases, we found nuclear features reminiscent of PC. The neoplastic cells were strongly positive for Leu M1 and epithelial membrane antigen (EMA), less for thyroglobulin and negative for calcitonin. The lymphocytic infiltrate was composed of a mixed population of B and T-cells with sparse S100-positive Langerhans cells. An interesting finding was the strong positivity with the antibody against RET/PTC. CONCLUSION: All clinicopathological data along with the presence of the extensive lymphocytic infiltrate could imply a more favourable prognosis. The expression of RET/PTC fusion gene adds support to the hypothesis that this tumour is a variant of PC, probably related to the oncocytic variant of PC.     

Renal cell carcinoma with rhabdoid features: an aggressive neoplasm

AIMS: Only a few reports on renal cell carcinoma with rhabdoid features have been published. This study was performed to investigate the clinicopathological characteristics of renal cell carcinomas with rhabdoid features. METHODS AND RESULTS: Among 253 cases of renal cell carcinoma in adults, eight cases with rhabdoid features were detected. Rhabdoid areas ranged from 10% to 90% of each of the cases. Seven of the eight cases were TNM stage III or IV, and four of the eight cases died within 8 months of surgery. Immunohistochemically, the rhabdoid areas were positive for CAM 5.2 (4/8), AE1/AE3 (6/8), epithelial membrane antigen (6/8) and vimentin (8/8), and negative for myogenetic markers (0/8). The mean MIB-1 labelling index in the rhabdoid areas was higher than that in the definite carcinomatous areas. Ultrastructurally, perinuclear whorls of intermediate filaments were demonstrated in three of the eight cases using paraffin-embedded blocks. CONCLUSIONS: The rhabdoid areas in renal cell carcinoma have histological, immunohistochemical and ultrastructural similarities to malignant rhabdoid tumours. Renal cell carcinoma with rhabdoid features is a highly aggressive neoplasm and its malignant behaviour may be due to the high cell-proliferative activity of the rhabdoid areas. Rhabdoid features in renal cell carcinoma may represent the endpoint of clonal evolution of renal cell carcinoma (especially in clear cell type cases).