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 oncogene amplification in thyroid cancer: correlations with radiation-associated and high-grade malignancy

A radiation etiology is well known in thyroid carcinogenesis. RET oncogene rearrangement is the most common oncogenic alteration in Chernobyl-related papillary thyroid cancer (PTC). To find the characteristic alteration associated with RET rearrangements in radiation-induced thyroid cancers, we analyzed the RET oncogene by fluorescence in situ hybridization. The fluorescence in situ hybridization technique has the possibility of detecting RET rearrangements at a single-cell level regardless of the specific fusion partner involved and directly reveals RET copy number on a per-cell basis. Our study demonstrated RET amplification in all 3 cases of radiation-associated thyroid cancers but not in sporadic well-differentiated PTC (n = 11). Furthermore, RET amplification was observed in all 6 cases of sporadic anaplastic thyroid cancers (ATCs). The frequency of RET amplification-positive cells was higher in ATC (7.2%-24.1%) than in PTC (1.5%-2.7%). The highest frequency of RET amplification-positive cells was observed among ATC cases with a strong p53 immunoreactivity. In conclusion, we found RET amplification, which is a rare oncogenic aberration, in thyroid cancer. This report is the first one to suggest the presence of RET amplification in PTC and ATC. RET amplification was correlated with radiation-associated, high-grade malignant potency, and p53 accumulation, suggesting genomic instability. RET amplification might be induced by a high level of genomic instability in connection with progression of thyroid carcinogenesis and, subsequently, be associated with radiation-induced and/or high-grade malignant cases.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

Etiology and significance of the optically clear nucleus

Papillary thyroid carcinoma (PTC) is diagnosed in both cytology and surgical pathology specimens on the basis of distinct nuclear morphology, characterized by nuclear elongation, chromatin clearing, intranulcear grooves, and inclusions. Although these nuclear features are specific to papillary carcinoma, they can be mimicked in some benign conditions. The majority of PTC cases do not pose diagnostic problems. However, a distinct subset of cases has generated controversy among experts. These cases are follicular patterned tumors that show minimal nuclear changes in PTC. Several investigators have explored the role of immunohistochemical markers in the histologic diagnosis of PTC. Somatic rearrangements of the RET protooncogene are the most frequent genetic abnormality found in PTC. The frequency of these rearrangements has varied according to the geographic region, radiation exposure, and methodologies used and histologic variant of PTC. Recent studies have suggested that RET/PTC may be the cause of this specific nuclear change in PTC; however, the role of RET/PTC in tumor progression still needs to be defined.     

Ret/PTC3 is the most frequent form of gene rearrangement in papillary thyroid carcinomas in Japan

Rearrangements of the RET and TRK proto-oncogenes, which generate fusion oncogenes, are frequent in papillary thyroid carcinomas in Caucasian populations. To determine the spectrum of gene rearrangements in Japanese patients, we systematically examined 40 papillary thyroid carcinomas for all possible types of gene fusion events involving RET or TRK genes. RET rearrangements were found in ten tumors (25%): ret/PTC1 had occurred in two tumors, ret/PTC2 in one, ret/PTC3 in six, and a novel RET rearrangement in the remaining patient. In this last patient, the 5′ novel sequence was fused in-frame to the RET amino acid sequence; thus, the fusion gene may encode a protein with a RET kinase domain at the carboxy terminus. The RET gene was fused to 5′ donor sequences at the beginning of exon 12 in all ten tumors. No rearrangements involving the TRK gene were found in this panel of carcinomas. Our results indicated that constitutive activation of the RET by gene rearrangement is a frequent mechanism of papillary thyroid carcinogenesis in Japanese adults. Received: November 24, 1998 / Accepted: November 28, 1998     

Distinguishing tall cell variant of papillary thyroid carcinoma from usual variant of papillary thyroid carcinoma in cytologic specimens

Tall cell variant (TCV) is an aggressive form of papillary thyroid carcinoma (PTC), usually associated with higher local recurrence and distant metastasis. Some authors have suggested that TCV can be effectively diagnosed on thyroid fine-needle aspiration (FNA); this diagnosis may help clinicians plan a more effective treatment regimen. The objective of this study was to compare the FNA specimens of TCV with those of usual variant of PTC (UV-PTC) and to define a set of distinguishing cytologic features. Thirty FNA specimens of histologically proven TCV were compared with 32 FNA specimens of histologically proven UV-PTC. All specimens were evaluated for the following features: papillary groups (PG), elongated/tall cells (EL/TC), oncocytic cytoplasm (OC), distinct cell borders (DCB), prominent central nucleoli (PCN), intranuclear grooves (NG), and intranuclear inclusions (NI). These features were semiquantitatively measured on a sliding scale of 0-4 in both air-dried Diff-Quik-stained and ethanol-fixed Papanicolaou-stained preparations. TCV showed distinctive cytologic features, which can distinguish them from UV-PTC. These included EL/TC, OC, and DCB and were also found to be statistically significant (P < 0.0001). No significant differences were noted for PG and NG. The NIs in TCV cases were qualitatively different than those in UV-PTC. In TCV there were multiple inclusions within the same nucleus imparting a "soap bubble appearance" to the nucleus. This feature was seen in almost all cases of TCV and was rarely seen in usual PTC. On the basis of the above-mentioned cytologic features, TCV can be distinguished from usual PTC in FNA specimens.