Molecular genetic study comparing follicular variant versus classic papillary thyroid carcinomas: association of N-ras mutation in codon 61 with follicular variant

Although the follicular variant of papillary thyroid carcinoma (FVPTC) has been classified as a papillary cancer based on nuclear features, its follicular growth pattern and potential for hematogenous spread are more characteristic of follicular carcinoma. To gain insight into the biologic nature of FVPTC, we compared genetic alterations characteristic of papillary and follicular thyroid carcinomas in 24 FVPTCs and 26 classic PTC (CPTCs). In FVPTCs, we observed ras mutation in 6 of 24 cases (25%), BRAF mutation in 1 of 13 cases (7.6%), and ret rearrangement in 5 of 12 cases (41.7%). In CPTCs, we found ras mutation in no case, BRAF mutation in 3 of 10 cases (30%), and ret rearrangement in 5 of 11 cases (45%). One FVPTC exhibited simultaneous ras mutation and ret/PTC1 rearrangement, and one CPTC harbored simultaneous BRAF mutation and ret/PTC3 rearrangement. Based on these findings, we concluded that ras mutation correlates with follicular differentiation of thyroid tumors whereas ret activation is associated with papillary nuclei but not with papillary architecture. ret activation is not exclusive of ras or BRAF mutation, whereas ras and BRAF mutations are mutually exclusive. The implications of these results for follicular and papillary carcinogenesis are discussed.     

BRAF mutations are associated with some histological types of papillary thyroid carcinoma

Mutations in the BRAF gene have recently been detected in a wide range of neoplastic lesions with a particularly high prevalence in melanoma and papillary thyroid carcinoma (PTC). The hot-spot mutation BRAF(V599E) is frequently detected in PTC (36-69%), in contrast to its absence in other benign or malignant thyroid lesions. In order to unravel whether there is any association between the occurrence of the BRAF mutation and the histological pattern of PTC, in this study a previous series of 50 PTCs was extended to 134 cases, including ten cases of PTC-related entities-hyalinizing trabecular tumour (HTT) and mucoepidermoid carcinoma (MEC). Using PCR/SSCP and sequencing, the BRAF(V599E) mutation was detected in 45 of the 124 PTCs (36%). No mutations were detected in any case of HTT and MEC. BRAF(V599E) was present in 75% of Warthin-like PTCs and 53% of conventional PTCs, whereas no BRAF(V599E) mutations were detected in any of the 32 cases of the follicular variant of PTC. BRAF(V599E) was also detected in 6 of 11 cases of the oncocytic variant of PTC that displayed a papillary or mixed follicular-papillary growth pattern and in none of the four oncocytic PTCs with a follicular growth pattern. A distinct mutation in BRAF (codon K600E) was detected in three cases of the follicular variant of PTC. This study has confirmed the high prevalence of BRAF(V599E) in PTC and has shown that the mutation is almost exclusively seen in PTC with a papillary or mixed follicular-papillary growth pattern, regardless of the cytological features of the neoplastic cells. The results support the existence of an oncocytic variant of PTC that should be separated from the oncocytic variant of follicular carcinoma and suggest that the follicular variant of PTC may be genetically different from conventional PTC.     

Diffuse (or multinodular) follicular variant of papillary thyroid carcinoma: a clinicopathologic and immunohistochemical analysis of ten cases of an aggressive form of differentiated thyroid carcinoma

In an attempt to advance and improve the characterization of the so-called diffuse follicular variant of papillary thyroid carcinoma (diffuse FVPTC) we studied a series of 59 thyroid carcinomas consecutively treated in a specialized center. The clinicopathologic and some of the immunohistochemical characteristics (uPA-R, Lewis X, Sialyl Lewis X, and MIB-1) of ten cases of FVPTC displaying a multinodular or diffuse pattern of growth, and histologic features similar to those previously described in diffuse FVPTC, were compared with those of common papillary thyroid carcinoma (PTC, 25 cases) and common FVPTC (8 cases). Cases of diffuse FVPTC differed significantly from common PTC and FVPTC in targeting younger patients and in exhibiting a prevalence of multicentricity, extrathyroid extension, nodal metastasis, and vascular invasion. Immunohistochemically, diffuse FVPTC cases were characterized by the overexpression of uPAR and sialyl Lewis X. No differences were observed regarding MIB 1 immunoreactivity. Regardless of the term used to designate the multicentric, invasive form of FVPTC (diffuse or multinodular FVPTC) it is crucial to acknowledge the existence of cases of FVPTC with a guarded prognosis that should be distinguished from the classic, uninodular form of FVPTC.     

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.     

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     

甲状腺乳头状癌中ret癌基因的表达

目的：观察人甲状腺乳头状癌中ret癌基因的表达。方法：应用RT－PCR技术研究甲状腺乳头状癌新鲜组织中ret癌基因活化形式PTC基因的表达。结果：25例甲状腺乳头状癌中有6例（24％）PTC基因表达阳性,主要分布于Ⅱ级以上肿瘤中;而甲状腺滤泡型癌、甲状腺瘤和正常甲状腺组织吕PTC均为阴性。结论：PTC基因是ret癌基因新的活化形式,ret基因的活化仅限于甲状腺头状癌类型中,可能是乳头状癌的特殊遗传事件,而无PTC基因表达的乳头状癌,其分子发生机制可能与其他基因的遗传性改变有关。因此检测PTC基因可作为判断甲状腺乳头状癌生物学行为的有效参考指标。     

<Emphasis Type="Italic">BRAF</Emphasis> mutations typical of papillary thyroid carcinoma are more frequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas

Somatic mutations of the BRAF gene (BRAF^V599E and BRAF^K600E) were found to be closely associated with different histotypes of papillary thyroid carcinoma (PTC). The V599E mutation is highly prevalent in PTC with a papillary or mixed papillary follicular growth pattern, and the K600E mutation is apparently restricted to the follicular variant of PTC. It is usually accepted that thyroid malignancies may follow a progression path from well-differentiated to poorly differentiated (PDC) and undifferentiated (UC) carcinomas. One would expect that at least some of the less differentiated carcinomas would harbour the genetic alterations of pre-existing well-differentiated tumours. In order to find the prevalence of BRAF mutations in PDC and UC, we screened a series of 19 PDCs and 17 UCs, as well as 3 UC-derived cell lines, for both mutation types. The group of PDCs was restricted to the so-called insular and insular-like PDCs, thus excluding PTCs with solid, insular or trabecular foci of growth and PDCs displaying typical PTC nuclei. No BRAF mutations were detected in any of the 19 cases of PDC, whereas 6 of the UCs (35%) and one UC-derived cell line presented the BRAF^V599E mutation. The BRAF^K600E mutation was not detected in any case. We conclude that UC may progress from BRAF^V599E-mutated PTC. The absence of BRAF mutations in our series of PDC supports the assumption that pure insular and insular-like PDCs are more closely related to follicular carcinoma than to PTC.Paula Soares and Vítor Trovisco contributed equally to this work.     

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.     

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.     

Molecular pathobiology of thyroid neoplasms

Tumors of thyroid follicular cells provide a very interesting model to understand the development of human cancer. It is becoming apparent that distinct molecular events are associated with specific stages in a multistep tumorigenic process with good genotype/phenotype correlation. For instance, mutations of the gsp and thyroid-stimulating hormone receptor genes are associated with benign hyperfunctioning thyroid nodules and adenomas while alterations of other specific genes, such as oncogenic tyrosine kinase alterations (RET/PTC, TRK) in papillary carcinoma and the newly discovered PAX8/peroxisome proliferator-activated receptor γ rearrangement, are distinctive features of cancer. Although activating RAS mutations occur at all stages of thyroid tumorigenesis, evidence is accumulating that they may also play an important role in tumor progression, a role that is well documented for p53. Environmental factors (iodine deficiency, ionizing radiations) have been shown to play a crucial role in promoting the development of thyroid cancer, influencing both its genotypic and phenotypic features. It is possible that the follicular thyroid cell has unique ways to respond to DNA damage. Similarly to leukemia or sarcomas (and unlike most epithelial cancers), numerous specific rearrangements are being discovered in thyroid cancer suggesting preferential activation of DNA repair instead of cell death programs after environmentally induced genetic alterations.     

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.     

Familial Follicular Cell Tumors: Classification and Morphological Characteristics

Familial follicular cell-derived well-differentiated thyroid cancer, papillary (PTC), and follicular thyroid carcinomas (FTC), accounts for 95% of thyroid malignancies. The majority of are sporadic, and at least 5% of these patients will have familial disease. Familial thyroid syndromes are classified into familial medullary thyroid carcinoma (FMTC), derived from calcitonin-producing C cells, and familial follicular cell tumors or non-medullary thyroid carcinoma (FNMTC), derived from follicular cells. Twenty-five percent of patients with medullary thyroid cancer (MTC) have a familial form; however, this accounts for only 1% of all patients with thyroid cancer. The familial follicular cell-derived lesions or familial non-medullary thyroid cancer can be divided into two clinical-pathological groups. The first group includes familial syndromes characterized by a predominance of non-thyroidal tumors, such as familial adenomatous polyposis (FAP), PTEN-hamartoma tumor syndrome (Cowden disease; PHTS), Carney complex, Werner syndrome, and Pendred syndrome. The second group includes familial syndromes characterized by predominance of papillary thyroid carcinoma (PTC), such as pure fPTC, fPTC associated with papillary renal cell carcinoma, and fPTC with multinodular goiter. Most of the progress in the genetics of familial thyroid cancer has been in patients with MTC. This is usually a component of multiple endocrine neoplasias IIA or IIB, or as pure familial medullary thyroid carcinoma syndrome. The genetic events in the familial C-cell-derived tumors are known and genotype-phenotype correlations are well established. The mutations in patients with isolated NMFTC have not been as well defined as in MTC. In many cases, patients have a known familial syndrome that has defined risk for thyroid cancer. The clinician must be knowledgeable in recognizing the possibility of an underlying familial syndrome when a patient presents with thyroid cancer. Some characteristic thyroid morphologic findings should alert the pathologist of a possible familial cancer syndrome, which may lead to further molecular genetics evaluation.     

Immunohistochemical diagnosis of papillary thyroid carcinoma.

In thyroid, the diagnosis of papillary carcinoma (PC) is based on nuclear features; however, identification of these features is inconsistent and controversial. Proposed markers of PC include HBME-1, specific cytokeratins (CK) such as CK19, and ret, the latter reflecting a ret/PTC rearrangement. We applied immunohistochemical stains to determine the diagnostic accuracy of these three markers. Formalin-fixed, paraffin-embedded tissue from 232 surgically resected thyroid nodules included 40 hyperplastic nodules (NH), 35 follicular adenomas (FA), 138 papillary carcinomas (PC; 54 classical papillary tumors and 84 follicular variant papillary carcinomas [FVPC]), 4 follicular carcinomas (FC), 6 insular carcinomas (IC), 7 Hürthle cell carcinomas (HCC), and 2 anaplastic carcinomas (AC). HBME-1 and ret were negative in all NH and FA; some of these exhibited focal CK19 reactivity in areas of degeneration. Half of the FC and AC exhibited HBME-1 staining but no positivity for CK19 or ret. In PC, 20% of cases stained for all three markers. Classical PC had the highest positivity with staining for HBME-1 in 70%, CK19 in 80%, and ret in 78%. FVPC were positive for HBME-1 in 45%, for CK19 in 57%, and for ret in 63%; only 7 FVPC were negative for all three markers. The six IC exhibited 67% staining for HBME-1 and 50% positivity for CK19 and ret. The seven HCC had 29% positivity for HBME-1 and CK19, and 57% positivity for ret. This panel of three immunohistochemical markers provides a useful means of diagnosing PC. Focal CK19 staining may be found in benign lesions, but diffuse positivity is characteristic of PC. HBME-1 positivity indicates malignancy but not papillary differentiation. Only rarely are all three markers negative in PC; this panel therefore provides an objective and reproducible tool for the analysis of difficult thyroid nodules.     

Concomitant papillary thyroid carcinoma and Hashimoto's thyroiditis

An association between papillary thyroid carcinoma (PTC) and Hashimoto's thyroiditis (HT) is well recognized. Both entities may often display overlapping morphologic features. The aim of this study was to evaluate the accuracy of fine needle aspiration (FNA) of concomitant PTC and HT. Twenty nine thyroid FNAs with a diagnosis of concomitant PTC and HT on follow-up surgical material were retrospectively reviewed (11% of all HT cases diagnosed in the same period of time). The cytologic specimens were evaluated for the presence of diagnostic features of PTC and HT. In 16 of 29 cases, the diagnosis of PTC was made or suggested; however, only in 3 cases were both entities recognized on the FNA material. The review of the remaining cases (13 cases) showed diagnostic features of PTC in 2 cases (interpretation errors), some features of PTC in 8 cases (insufficient diagnostic features), features of only HT in 2 cases, and 1 case was acellular (sampling errors). Originally, 10 cases with features of PTC were diagnosed as either follicular neoplasm or colloid nodule with or without HT. Histologically, 1 of 13 cases was a cystic variant and 7 of 13 cases were follicular variants of papillary carcinoma. It is important to be aware of the coexistence of PTC and HT. Deliberate search for evidences of PTC in every case of HT may be necessary to improve diagnostic accuracy of the FNA. However, the cytologic diagnosis of follicular variant of PTC coexisting with HT can be challenging. The sampling error may also cause false negative results.     

Real-time analysis of beta- and gamma-catenin mRNA expression in ret/PTC-1 activated and nonactivated thyroid tissues.

Our group has previously demonstrated an association between ret/PTC-1 activation and decreased E-cadherin mRNA levels in papillary thyroid carcinoma. We also observed similarities in the E-cadherin expression profiles of Hashimoto thyroiditis and ret/PTC-1-positive papillary thyroid carcinomas and have hypothesized that ret/PTC-1 activation might cause not only the structural and nuclear peculiarities of PTC but also an immune reaction to thyroid epithelium. The objective of this study was to examine the expression of E-cadherin's ligands, beta- and gamma-catenin, in various thyroid tissue types in the context of ret/PTC-1 positivity using laser capture microdissection and TaqMan (Applied Biosystems, Foster City, CA). One-Step RT-PCR. Beta-catenin mRNA levels were found to be consistently decreased in both papillary and anaplastic carcinomas when compared with a normal/follicular adenoma group. A significant difference in expression levels was observed between papillary and follicular thyroid carcinomas with the latter having elevated mRNA levels of beta-catenin. Gamma-catenin mRNA was decreased in anaplastic carcinomas compared with normal/follicular adenoma groups. A similar expression profile of gamma-catenin as beta-catenin was observed in papillary and follicular carcinomas with the latter once again having higher mRNA levels. These results therefore suggest that although beta- and gamma-catenin may play a role in the progression of thyroid cancer in general, they do not appear to be associated with ret/PTC-1-modulated pathways.     

Molecular Analysis of Thyroid Tumors

Thyroid cancer is the most common endocrine malignancy, and its incidence is rising in the USA and other countries. Papillary and follicular thyroid carcinomas are the two most common types of thyroid cancer. Non-overlapping genetic alterations, including BRAF and RAS point mutations, and RET/PTC and PAX8/PPARγ rearrangements, are found in more than 70% of papillary and follicular thyroid carcinomas. These represent the most common genetic alterations in thyroid cancer, as well as molecular markers of diagnostic and prognostic significance. The use of these and other emerging molecular markers will likely improve the diagnosis of malignancy in thyroid nodules as well as facilitate more individualized operative and postoperative management. Herein, we provide a brief overview of the common genetic alterations in papillary and follicular thyroid carcinoma and discuss the diagnostic and prognostic significance thereof.     

N-ras mutation of thyroid tumor with special reference to the follicular type

Using the method of polymerase chain reaction-single strand conformation polymorphism, the point mutations of the ras oncogenes in a total of 33 thyroid tissues, including 12 follicular adenomas, 6 follicular carcinomas, 11 papillary carcinomas, and 4 undifferentiated carcinomas, were examined. The frequency of the mutation was 3% (1/33) in codon 12,13 of Ki-ras and 18% (6/33) in codon 61 of N- ras , including 17% (2/12) in follicular adenoma, 50% (3/6) in follicular carcinoma, 0% (0/11) in papillary carcinoma and 25% (114) in undifferentiated carcinoma. In follicular adenoma, positivity was observed in microfollicular or trabecular subtypes. Furthermore, the mutation of ras was examined in histologically different parts, coexisting in the same tumor in a total of four cases. Both the undifferentiated carcinoma and coexisting follicular adenoma, and both the microfollicular adenoma and trabecular nodule growing in the tumor, had the same N-ras (61) mutation. Direct sequencing analysis showed that all mutations were CAA (Gln) to CGA (Arg) transition of codon 61, except for CAA to AAA transversion in one case of follicular carcinoma. A similar genetic abnormality of N-ras genes at codon 61 between follicular adenoma and follicular carcinoma suggests that the mutation of N- ras at codon 61 might play a part in oncogenesis in follicular tumors.