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.     

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.     

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.     

Pathologic features of Hashimoto's-associated papillary thyroid carcinomas

Some investigators have found an increased incidence of papillary carcinoma (PC) of the thyroid in patients with Hashimoto's (autoimmune) thyroiditis (HT), which raises the possibility that there may be more than an incidental association between these 2 diseases. In this study, we analyzed the pathology of Hashimoto's-associated thyroid carcinomas to see if these tumors showed any distinctive features. The possible significance of solid cellular nodules as preneoplastic lesions in patients with HT was investigated. A review of all the cases of HT during a 16-year period yielded 30 PC and 3 follicular carcinomas (FC). Within the PC there were 7 (23%) follicular variants. Twenty (67%) of the PC showed various degree of intratumoral fibrosis, ranging from thick fibrous septa separating tumor nodules to almost complete obliteration of the tumor by the fibrosis, with only microscopic residual tumor nests. In most of the cases, the desmoplastic response within the tumors was of the fibromatosis-like type with dense hyalinized collagen and bland-appearing spindle cells. All the tumors, independently of the degree of fibrosis, showed the nuclear features of PC. No correlation was found between the degree of fibrosis in the tumors and the thyroid gland outside the tumors. There were tumors with marked fibrosis without fibrosis outside the tumors. Four cases of PC (13%) showed a growth pattern characterized by cystic spaces with thick hyalinized walls and focal papillary hyperplasia lined by flat and cuboidal epithelium, reminiscent of a vascular neoplasm. There were 4 atypical solid microscopic nodules with confluent cellularity; 2 of them associated with a PC and the other 2 with diffuse HT without PC. These nodules were composed of cells with clear nuclei and occasional grooves without nuclear pseudoinclusions. By immunohistochemistry, 2 of 3 nodules showed cytoplasmic reactivity for cytokeratin 19, and 2 of 3 nodules were positive for the RET/PTC (rearranged during transfection, papillary thyroid carcinoma) antibody. In summary, HT-associated PC may frequently display prominent stromal desmoplasia and a pseudovascular pattern, both of which can present diagnostic difficulties if the cytologic features of PC are not recognized because of the marked obliteration of the tumor by the fibrosis. Atypical nodules may represent a precursor lesion of PC in patients with HT.     

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。     

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.     

Prevalence of RET/PTC rearrangements in Hashimoto's thyroiditis and papillary thyroid carcinomas

The relationship between Hashimoto's thyroiditis (HT) and follicular cell-derived thyroid cancer remains unclear. Recently, 2 studies reported a 95% prevalence of RET/PTC rearrangements in histologically benign tissue affected by HT, suggesting that multiple occult tumors exist in HT patients with high frequency. We tested the prevalence of RET/PTC rearrangements in 26 HT, in 6 papillary carcinomas arising in the background of HT, and in 27 papillary carcinomas not associated with HT. We detected no RET/PTC rearrangements in HT or papillary carcinomas arising in the background of HT, in contrast to a 33% prevalence among papillary carcinomas not associated with HT. However, the expression of wild-type RET was found in more than half of papillary carcinomas. These results suggest that, if the association between HT and thyroid cancer exists, its molecular basis is different from RET/PTC rearrangement.     

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.     

Clinical significance of RET/PTC and p53 protein expression in sporadic papillary thyroid carcinoma

AIMS: Rearranged during Transfection (RET)/papillary thyroid carcinoma (PTC) and p53 are two genes involved in the pathogenesis of PTC. It has been suggested that RET/PTC expression is associated with higher rates of local extension and lymph node involvement, whereas p53 mutations are more frequent in poorly differentiated and anaplastic carcinomas. In addition, experimental studies have shown that p53 activity can modify the behaviour of PTC carrying RET/PTC. The aim of this study was to investigate the expression of both RET/PTC and p53 in order to evaluate their usefulness as prognostic factors. METHODS AND RESULTS: Resected specimens of 61 cases of PTC were studied immunohistochemically using a polyclonal antibody to RET and a monoclonal antibody to p53 protein. RET/PTC expression was associated with extrathyroid extension of PTC, at diagnosis (P < 0.05). In contrast, no relationship between p53 immunoreactivity and clinical status was found. In addition, p53 expression was more prevalent among RET/PTC+ patients, and significantly influenced the relationship observed between RET/PTC and extrathyroid extension of the disease. CONCLUSION: Our results suggest that immunohistochemistry for both PTC/RET and p53 could be useful in the clinical evaluation of patients with PTC.     

The utility of aspiration cytology for the distinction of aggressive and non-aggressive papillary carcinoma of the thyroid

Preoperative identification of the aggressive variants of papillary thyroid carcinoma (PTC) by fine needle aspiration (FNAC) has been suggested and different systems for distinguishing them from classical variant of PTC have been employed, including a point-based cytology grading system as suggested by Damiani et al., that we currently use in our Institution. The aim of this paper is to verify if distinction of PTC in aggressive or nonaggressive variants impacts on surgical treatment. In 7 years, from 1998 to 2005, 13586 cases of FNAC of thyroid have been performed; among them 156 PTC. The cytological material of thirty complete thyroidectomies with histology proven papillary thyroid carcinoma were reviewed. 11/30 cases were correctly stratified into the appropriate low or high-grade category. The cytological grading system was discordant with the final histopathological diagnosis in 9/30 cases while in 10 cases the grade was not assessed. Eight cases were downgraded and therefore the low grade papillary carcinoma diagnosed at cytological level turned to be high grade at histology. When those cases were reviewed, the histology of seven cases diagnosed as tall cell variant of PTC failed to show a percentage of tumour cells higher than 50% and therefore it would be better to diagnose them as classical variant of PTC. One case was under-graded at FNAC. The case that was cytologically upgraded was a follicular variant of PTC in Hashimoto thyroiditis with a focus of tall cells. All patients underwent thyroidectomy or thyroidectomy plus lymphadenectomy and from our results the pre-operative diagnosis did not effect the surgical treatment. No patient died of the disease.     

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.     

Immunohistochemical study of papillary thyroid carcinoma and possible papillary thyroid carcinoma-related benign thyroid nodules

Recent immunohistochemical studies have identified different antisera that have various degrees of sensitivity and specificity for papillary thyroid carcinoma (PTC). In this study, we performed immunostaining for CK, EMA, HBME, CD57 and CD15 in PTC, and benign thyroid nodular lesions to compare the sensitivity and the specificity of these antisera for PTC. In addition, we studied the patterns of immunostaining of these antisera in benign nodular thyroid lesions displaying a fine chromatin pattern, foci of cells with nuclear grooves, and optically clear nuclei. Fifty-five PTC (composed of 30 papillary variants and 25 follicular variants), 5 follicular carcinomas, 30 follicular adenomas, and 20 thyroid nodular lesions (5 papillary variants and 15 follicular variants) were submitted for immunostaining with CK, EMA, HBME, CD57, and CD15. CK and HBME showed the highest sensitivity and specificity for PTC when an arbitrary cutoff of more than 10% positive cells was considered as positive diagnostic immunostaining for these sera. The other antisera were less sensitive and less specific. One case of PTC showed negative HBME but positive CD15, whereas three papillary variants and two follicular variants of benign thyroid nodules revealed a positive diagnostic HBME immunostaining for PTC and negative CK immunostaining. Any combination of positive diagnostic immunostaining with CK+ HBME, CK+ CD57 or CK+ CD15 has a sensitivity of 95% and specificity of 90% for PTC. Thyroid nodules with a diffuse or focal fine chromatin pattern and focal areas with nuclear grooves or optically clear nuclei displayed immunoreactivity ranging from 0% to 50% of cells. Three of five follicular carcinomas showed negative reactivity for HBME, CD57, and CD15. A combination of immunostaining with CK, HBME and CD57 (or CD15) is a sensitive and specific test for PTC. This panel can be used to rule out thyroid nodules posing a diagnostic problem with PTC. Follicular adenoma and nodules of the thyroid, with a fine chromatin pattern and focal nuclear grooves or optically clear nuclei, displayed an intermediate range of reactivity between reactive thyroid tissue and PTC.     

甲状腺乳头状癌中BRAFV599E点突变与RET/PTC融合基因的检测

目的检测甲状腺乳头状癌(PTC)及其他类型甲状腺良恶性肿瘤中BRAFV599E的点突变及 RET/PTC1、3融合基因的表达状况,探讨二者与PTC临床病理学特征的关系.方法用聚合酶链反应(PCR)及逆转录(RT)-PCR分别检测95例石蜡与新鲜甲状腺病变组织中BRAFV599E点突变和RET/PTC1、3融合基因.结果 (1)仅在PTC中检测到BRAFV599E的突变,突变率56%(37/66),在经典型PTC和高细胞型PTC中突变率分别为70%(29/41)和2/3,在滤泡型PTC及其他类型甲状腺病变中未检测到BRAFV599E的突变.统计学分析BRAF突变与性别、年龄、伴慢性淋巴细胞浸润及淋巴结转移无明显关系(P>0.05).(2)PTC中RET/PTC检出率21.2%(14/66),其中5例RET/PTC1阳性(7.6%),9例RET/PTC3阳性(13.6%).RET/PTC融合基因阳性的14例PTC中未检测到BRAFV599E突变.其余29例良恶性病例中未检测到RET/PTC融合基因.RET/PTC融合基因的表达与PTC的临床病理学特征无明显关系(P>0.05).结论 (1)BRAFV599E突变和RET/PTC融合基因是PTC较特征性的遗传学改变,可作为PTC诊断和鉴别诊断提供分子学的依据,BRAFV599E突变可能是甲状腺乳头状癌表型的重要决定因素之一;(2)BRAFV599E突变与PTC的经典型和高细胞型两种主要亚型密切相关;(3)BRAFV599E突变与RET/PTC融合基因可能在PTC中是独立事件.     

Multicentric encapsulated papillary oncocytic neoplasm of the thyroid: A case diagnosed by a combined cytological, histological, immunohistochemical, and molecular approach

Fine-needle aspiration (FNA) diagnosis of oncocytic lesions is challenging. In fact, oncocytic changes occur in inflammatory, hyperplastic, and neoplastic settings, including both benign and malignant tumors. The rare oncocytic variant of papillary thyroid carcinoma (PTC), shows papillae composed by cells with large oncocytic granular cytoplasm featuring clear PTC nuclear features. A morphological similar, but biologically distinct lesion, is the encapsulated papillary oncocytic neoplasia. Here, we first report on FNA, its cytological features together with histological, immunohistochemical, and molecular correlates.     

Follicular epithelial dysplasia of the thyroid: morphological and immunohistochemical characterization of a putative preneoplastic lesion to papillary thyroid carcinoma in chronic lymphocytic thyroiditis

In chronic lymphocytic thyroiditis (CLT), the follicular epithelial cells display cytological atypia resembling papillary thyroid carcinoma (PTC), and epidemiological studies have suggested an increased risk of PTC in patients with this condition. While reactive atypia is observed diffusely in CLT-affected thyroid parenchyma, it is not unusual to find microscopic foci morphologically distinct from the surrounding parenchyma, exhibiting more pronounced cytological and architectural atypia. These small atypical lesions, which we term “follicular epithelial dysplasia” (FED), are particularly prominent in cases of severe CLT, yet lack invasive growth, papillary architecture, or intranuclear pseudoinclusions. To gain further insight into their biological significance, we constructed a tissue microarray of 70 cases of CLT, comprised of morphologically normal thyroid, thyroid with reactive atypia, FED, follicular nodular disease (nodular hyperplasia or follicular adenoma), and PTC. Immunohistochemical staining was performed for a marker panel including PTC (HBME-1, cytokeratin 19, galectin-3, and cyclin-D1) as well as TTF-1, thyroglobulin, and p63. Slides were digitally scanned and immunohistochemical staining evaluated using automated image analysis software. FED lesions were positive for TTF-1 and thyroglobulin (50/50, 100 %), though some (13/50, 26 %) also expressed p63. Similar to PTC, strong diffuse staining was observed for HBME-1 (43/50, 86 %), cytokeratin 19 (48/50, 96 %), galectin-3 (20/50, 40 %) and cyclin-D1 (38/50, 76 %). In contrast, normal thyroid, reactive atypia, and follicular nodular disease were negative, or at most, exhibited focal weak staining for HBME-1, cytokeratin 19, and galectin-3. The results of this study demonstrate the presence of atypical microscopic lesions in CLT with an immunohistochemical profile similar to PTC, supporting the concept of a premalignant lesion preceding PTC, arising in the context of severe chronic inflammation.     

甲状腺乳头状癌RET、CK19、TG、Ki-67的表达

目的 研究甲状腺乳头状癌RET、CK19、TG、Ki-67蛋白表达特点及其临床意义。方法 应用免疫组织化学SP法检测RET、CK19、TG、Ki-67蛋白在30例甲状腺乳头状癌、10例结节性甲状腺肿和18例癌旁正常甲状腺中的表达。结果 RET、CK19在乳头状癌的阳性率（66．7％、83．3％）明显高于结节性甲状腺肿和正常甲状腺阳性率（7．1％、25．0％）,两者差异有显著性（P〈0．01）。乳头状癌组及良性病例组TG表达阳性率差异无显著性（P〉0．05）。96．7％的乳头状癌Ki-67阳性细胞数小于10％。结论 RET及CK19在甲状腺乳头状癌表达增加,具有一定的病理诊断价值。