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。     

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.     

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.     

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.     

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.     

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.     

LIMD2 Is Overexpressed in BRAF V600E-Positive Papillary Thyroid Carcinomas and Matched Lymph Node Metastases

We previously described that LIM domain containing 2 (LIMD2) overexpression was closely correlated with metastatic process in papillary thyroid carcinoma (PTC). We here evaluated the expression of LIMD2 in a series of non-metastatic and metastatic PTC and their matched lymph node metastases via immunohistochemistry. LIMD2 was expressed in 74 (81%) of primary PTC and 35 (95%) of lymph node metastases. Sub-analysis performed in 37 matched samples demonstrated that in four cases, LIMD2 is expressed in lymph node metastases, while it is not expressed in primary tumors. Moreover, in eight cases, the staining intensity of LIMD2 was stronger in the patient-matched lymph node metastases than in the primary tumors. Next, the expression of LIMD2 was correlated with clinical pathological parameters and BRAF V600E and RET/PTC mutational status. The expression of LIMD2 in primary tumors was correlated with the presence of BRAF V600E mutation (P =?0.0338). Western blot analysis in thyroid cell lines demonstrated that LIMD2 is expressed in two PTC cell lines, while it is not expressed in normal thyroid and follicular thyroid carcinoma cell lines. Importantly, its expression was higher in a PTC cell line that harbors BRAF V600E mutation than in a PTC cell line that harbors RET/PTC1. The available genomic profiling data generated by The Cancer Genome Atlas Research Network confirmed that LIMD2 expression is higher in BRAF-like PTC samples. Our data suggest that LIMD2 may play an important role in the metastatic process of PTC, predominantly in BRAF V600E-positive tumors.     

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.     

Comparison of metabolic ratios of urinary estrogens between benign and malignant thyroid tumors in postmenopausal women

Background

Estrogen metabolism may be associated with the pathophysiological development of papillary thyroid carcinoma (PTC).

Methods

To evaluate the differential estrogen metabolism between benign and malignant PTCs, estrogen profiling by gas chromatography–mass spectrometry was applied to urine samples from postmenopausal patients with 9 benign tumors and 18 malignant stage I and III/IV PTCs.

Results

The urinary concentration of 2-methoxyestradiol was significantly lower in the stage I malignant patients (3.5-fold; P?<?0.025) than in the benign group. The metabolic ratios of 16α-OH-estrone/estrone and estriol/estradiol, which are responsible for 16α-hydroxylase activity, were increased more than 2.5-fold in the advanced-stage malignant PTC (P?<?0.02 each). The more than 6.2-fold decrease in the urinary 2-/16α-hydroxylase ratio in stage III/IV malignant PTC was consistent with the ratio in postmenopausal patients with endocrine gland cancers. In addition, reductive 17β-hydroxysteroid dehydrogenase (17β-HSD; estradiol/estrone or estriol/16α-OH-estrone) was present at significantly higher levels in subjects with stage III/IV malignant PTCs than in benign subjects (>3.5-fold difference; P?<?0.002). In particular, the estriol/16α-OH-estrone ratio differentiated between the benign and early-stage malignant patients (P?<?0.01).

Conclusions

Increased 16α-hydroxylation and/or a decreased 2-/16α-ratio, as well increased reductive 17β-HSD, with regard to estrogen metabolism could provide potential biomarkers. The devised profiles could be useful for differentiating malignant thyroid carcinomas from benign adenomas in postmenopausal women.

     

The Flip Side of NIFTP: an Increase in Rates of Unfavorable Histologic Parameters in the Remainder of Papillary Thyroid Carcinomas

The term noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) was recently proposed to replace noninvasive follicular variant of papillary thyroid carcinoma (FVPTC) both to promote more conservative management of these tumors and spare patients the psychological burden of a cancer diagnosis. This reclassification will lower the incidence of papillary thyroid carcinoma (PTC). In addition, it could result in an increase in the rates of unfavorable histologic prognosticators for PTC overall because NIFTPs had previously accounted for many of the PTCs without these features. Our aim was to evaluate the potential impact of the reclassification of NIFTP on the rates of extrathyroidal extension, lymphovascular invasion, and lymph node metastases in PTC. We identified all PTCs clinically over 1 cm diagnosed on surgical resection between August 2010 and August 2012. The histopathologic characteristics, including PTC subtype, tumor size, presence of extrathyroidal extension and lymphovascular invasion, and surgical margin and lymph node status were all recorded. Based on these parameters, cases were classified according to the American Thyroid Association (ATA) risk stratification system for structural disease recurrence. Tumor slides for cases initially diagnosed as FVPTC were reviewed to identify tumors that would now be classified as NIFTPs. Our cohort included 348 cases of PTC, of which 94 (27%) would now be classified as NIFTPs. After excluding NIFTPs from the PTC category, there were increased rates of extrathyroidal extension (26% up from 19%, p = 0.046), lymphovascular invasion (37% up from 27%, p = 0.0099), and lymph node metastases (26% up from 19%, p = 0.045) among the remaining PTCs. Based on these changes in histologic features, 10% fewer cases were defined as ATA low risk (62% down from 72%, p = 0.0081). Our results indicate that the downgrading of some carcinomas to NIFTP will increase the rates of higher risk histologic parameters in the remaining PTCs by statistically significant margins. Although the overall survival for PTC is very high and would likely not be changed significantly by the introduction of NIFTP, additional studies evaluating the impact of the NIFTP shift are warranted.     

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。     

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.     

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.     

The RET proto-oncogene in medullary and papillary thyroid carcinoma

 The evolution of cancer is a multistep phenomenon, and multiple cellular genetic lesions are involved in the emergence of the malignant neoplasm. Several early events have been implicated in the neoplastic transformation of thyrocytes, and recent reports have described the involvement of specific genetic alterations in different types of thyroid neoplasms: ras point mutations are frequently observed in tumours with follicular histology, gsp – the mutated form of the alpha subunit of the Gs-protein – is encountered in up to 73% of papillary or follicular thyroid carcinomas, and a high prevalence of p53 point mutations has been found in anaplastic thyroid carcinomas but not in differentiated follicular tumours. More recent studies revealed that the RET proto-oncogene is involved in the oncogenesis of medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC) by activation of its tyrosine kinase either by point mutation or rearrangement. In this review the most important recently published data on alterations of the RET proto-oncogene in heritable and sporadic MTCs and in PTCs will be summarized. Emphasis will be directed to the pathophysiological mechanisms of tumour initiation, the indications and limitations of DNA testing, and the clinical implications of identified RET defects in thyroid lesions. Received: 9 January 1997 / Accepted: 17 February 1997     

Frequent <Emphasis Type="Italic">BRAF</Emphasis><Superscript><Emphasis Type="Italic">V600E</Emphasis></Superscript> and Absence of <Emphasis Type="Italic">TERT</Emphasis> Promoter Mutations Characterize Sporadic Pediatric Papillary Thyroid Carcinomas in Japan

Pediatric papillary thyroid carcinoma (PTC) has unique features but requires further genetic investigation. Moreover, there has been increasing concern about the risk for pediatric PTC in Japan after the Fukushima accident. This study aims to evaluate the frequencies of BRAF and TERT promoter mutations and to examine their significance in non-radiation-associated pediatric PTCs in Japan. We enrolled 81 pediatric PTC patients aged ≤20 years. The control group included 91 adult PTCs from patients >20 years old. BRAF and TERT mutations were analyzed by allele-specific-PCR and/or Sanger sequencing. Compared with adult PTCs, pediatric PTCs exhibited larger tumor size, more frequent lymph node metastasis, and less classical histology. The prevalence of BRAF ^V600E in pediatric PTCs was 54% and significantly lower than that in adults of 85%. In the pediatric PTCs, BRAF ^V600E was positively associated with older age, classical histology, and the lymph node metastasis but independent from other clinicopathological factors. TERT mutations were identified in 13% of adults and in none of the pediatric PTCs. In conclusion, pediatric PTCs are characterized by more advanced clinicopathological features, lower BRAF ^V600E frequency, and absence of TERT mutation. The BRAF ^V600E frequency in this study is similar to the reported BRAF ^V600E frequency in the ultrasonographically screened pediatric PTCs in Fukushima.