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     

The contribution of molecular pathology to the classification of thyroid tumors

Significant molecular advances have been undertaken for the past two decades in the field of thyroid follicular neoplasms, including a detailed genomic profile of papillary thyroid carcinoma (PTC) by The Cancer Genome Atlas (TCGA) project. These molecular discoveries led to a better understanding of the pathogenesis of thyroid neoplasms and resulted in reclassification of certain types of thyroid tumors. This review discusses how, 1) the molecular profiles of follicular-patterned lesions led to the reclassification of the follicular variant of PTC into non-invasive follicular thyroid neoplasm with papillary like nuclei, 2) the genotyping of Hürthle cell neoplasm provided the rationale to classify these tumors independently from follicular adenomas and carcinomas, and 3) BRAF and RAS molecular signatures have the potential of subclassifying PTC and poorly differentiated thyroid carcinoma into clinically relevant molecular subtypes.     

Alterations of the BRAF gene in thyroid tumors

BRAF belongs to the RAF family of protein kinases that are important components of the MAPK signaling pathway mediating cell growth, differentiation and survival. Activating point mutation of the BRAF gene resulting in V600E (previously designated as V599E) is a common event in thyroid papillary carcinoma, being found in approx 40% of this tumor. It has strong association with classical papillary carcinoma and tall cell and possibly Warthin-like variants. This mutation also occurs in thyroid poorly differentiated and anaplastic carcinomas, usually those containing areas of papillary carcinoma. Alterations in the BRAF gene do not overlap with RAS mutations and RET/PTC rearrangement, indicating that activation of one of the effectors of the MAPK pathway is sufficient for papillary thyroid carcinogenesis. Recently, another mechanism of BRAF activation has been identified, which involves chromosome 7q inversion that results in the AKAP9-BRAF fusion. It is rare in sporadic papillary carcinomas and is more common in tumors associated with radiation exposure. Yet another mechanism of BRAF activation may involve copy number gain, which is seen in a significant portion of thyroid follicular tumors of both conventional and oncocytic (Hürthle cell) types.     

Concurrent Pheochromocytoma,Paraganglioma, Papillary Thyroid Carcinoma,and Desmoid Tumor: A Case Report with Analyses at the Molecular Level

Reports on the association of papillary thyroid carcinoma with paraganglionic or desmoid tumors have appeared infrequently. The former setting usually affects middle-aged females; the latter is typical of familial adenomatous polyposis. We report the case of a 69-yr-old man in whom two abdominal masses had been instrumentally detected following an access of abdominal pain. Save for a moderate hypertension, he was asymptomatic and an impalpable thyroid nodule was detected by ultrasonography. A high urinary noradrenaline output and cytology of the masses raised the suspicion of pheochromocytoma. At laparotomy, an adrenal pheochromocytoma and a paracaval paraganglioma were excised. Subsequently, hemithyroidectomy was performed, and histopathology revealed papillary microcarcinoma. A nodule of desmoid tumor was also removed from the abdominal wall. An analysis ofRET, APC, andTP53 gene mutations, and ofRET andNTRK1 gene rearrangements, yielded negative results. No in vitro transforming activity was detected in the tumor DNA when assayed in transfection experiments. The lack of a consistent family history also made unlikely the possibility of identifying the putative germline defect by linkage analyses. Should this unusual aggregation of tumors represent a new entity, a number of genetic alterations have now been excluded.     

<Emphasis Type="Italic">BRAF</Emphasis><Superscript>V600E</Superscript> Mutation in Papillary Thyroid Carcinoma: Significant Association with Node Metastases and Extra Thyroidal Invasion

B-Raf (BRAF) is the strongest activator in the downstream of MAP kinase signaling. The somatic point mutation of BRAF gene (V600E) is the most common and specific event in papillary thyroid carcinoma (PTC). However, its prevalence is variable among different studies and its association with clinico-pathological features is controversial. This study tests the prevalence of BRAF ^V600E mutation in thyroid cancer patients in Indian subcontinental population. We analyzed 140 thyroid tumor specimens for BRAF gene mutation at codon 600 using mutant-allele-specific amplification, single-strand conformation polymorphism, Mutector assay, and DNA sequencing of the PCR-amplified exon 15. BRAF mutation at codon 600 was detected in 46 of 86 PTC patients (53.4%) from Indian subcontinental cohort. Frequency of mutation varied across the subtypes of PTCs. BRAF ^V600E mutation was more common in the conventional PTC (38 out of 62; 61%) than in the follicular variant of PTC (2 out of 17; 11.7%). None of the 8 follicular thyroid adenomas, 14 follicular thyroid carcinomas, 16 medullary thyroid carcinomas, and 16 benign hyperplasia patients showed any exon 15 mutation. We found significant correlation between BRAF mutation status and extra-thyroidal invasion, lymph node metastasis, and tumor stage. However no correlation was observed with gender, age, and tumor size of the patients. Thus our findings suggest that BRAF ^V600E is a prevalent genetic alteration in adult sporadic PTCs in Indian cohort and it may be responsible for the progression of classic variant of PTC to metastatic and poorly differentiated subtype and likely to have significant impact on its diagnostic and prognostic management.     

Understanding the genotype of follicular thyroid tumors

Tumors of the thyroid with a follicular growth pattern are controversial and can be diagnostically challenging for the pathologist. This group of tumors includes both follicular-derived lesions (adenomas and carcinomas) and papillary carcinoma (follicular variant of papillary carcinoma). H&E morphology has classically been the gold standard for diagnosis. In the past several decades, however, several important molecular markers have been identified that may be unique to different types of thyroid carcinomas. These include the translocations RET/PTC and PAX8-PPARγ and point mutations in the BRAF and RAS genes. Other molecular events in tumor suppressor genes may be useful for diagnosis of these tumors as well. None of the mutational markers are very sensitive, and there is some question regarding specificity for malignancy, because mutations have also been described in histologically benign tumors. However, with increasing availability of molecular testing for the general pathologist, a molecular testing panel used in conjunction with the H&E morphology and immunohistochemical stains may become useful in the clinical setting for the diagnosis of thyroid tumors.     

<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.     

A Novel Complex BRAF Mutation Detected in a Solid Variant of Papillary Thyroid Carcinoma

BRAF gene mutations are identified in about 45% of papillary thyroid carcinomas (PTC) and represent the most common genetic event in this tumor. Here, we report a case of PTC, solid variant, with a complex BRAF mutation that involves one nucleotide substitution, C1796T, and a CTT triplet insertion, 1798_1799insCTT, located on the same allele. This mutation leads to the replacement of a threonine with an isoleucine, T599I, and replacement of a valine with an alanine and a leucine, V600delinsAL. This mutation was identified both in the preoperative fine needle aspirate sample and in the surgical specimen after total thyroidectomy. Other rare BRAF mutations in PTC are reviewed.     

Molecular Detection of <Emphasis Type="Italic">PPARγ</Emphasis> Rearrangements and Thyroid Carcinoma in Preoperative Fine-Needle Aspiration Biopsies

The pathologic diagnosis of thyroid follicular tumors is difficult, particularly in preoperative fine-needle aspiration biopsies. To investigate whether the molecular diagnosis of PPARγ rearrangements can detect thyroid carcinomas in fine-needle aspiration biopsies, we performed interphase fluorescence in situ hybridization on 24 thyroid fine-needle aspiration and 17 follow-up thyroidectomy specimens. Two of the 24 fine-needle aspiration biopsies contained PPARγ rearrangements, and both were diagnosed suggestive of a thyroid follicular neoplasm by cytology. The two corresponding thyroidectomies each contained PPARγ rearrangements in all tumor cells and, both were diagnosed follicular-patterned thyroid carcinomas—one a follicular carcinoma and the other a follicular variant of papillary carcinoma, the latter by majority of expert endocrine pathologists. Our experiments demonstrate that PPARγ rearrangements can detect a subset of follicular-patterned thyroid carcinomas in preoperative thyroid fine-needle aspiration biopsies. The ultimate utility of mutations such as PPARγ rearrangements in diagnosis of thyroid carcinoma must be proven by direct correlation of mutation status with thyroid tumor biology and not just with thyroid tumor morphology, a subjective and imprecise marker of clinical behavior. The application of specific mutations to preoperative diagnosis of thyroid carcinoma is predicted to improve the accuracy and reduce the costs of treating patients with thyroid tumors. An erratum to this article can be found at     

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.     

BRAF copy number gains in thyroid tumors detected by fluorescence In Situ hybridization

Point mutation of the BRAF gene is a common genetic event in papillary thyroid carcinomas. More recently, it has been found that BRAF can also participate in chromosomal rearrangement. In this study, we explore yet another possible mechanism of BRAF alteration, which involves copy number gain. Using fluorescence in situ hybridization with BRAF specific and chromosome 7 centromeric probes, we studied 62 follicular thyroid tumors and 32 papillary carcinomas. We found that numerical changes in BRAF copy number were rare in papillary thyroid carcinomas, while they occurred in 16–45% of follicular tumors of conventional and oncocytic (Hürthle cell) types. They were due to amplification of the gene or gain of one or more copies of chromosome 7. Tetrasomy for chromosome 7 was overall the most common finding. The changes in BRAF copy number did not overlap with RAS mutations in follicular tumors. In a group of follicular carcinomas, tumors with BRAF copy number gain were significantly more often widely invasive (67%) compared to tumors with no copy number change (18%). By Western blotting, the tumors carrying four copies of the gene revealed higher expression of BRAF protein, suggesting that copy number gain may represent another mechanism of BRAF activation in thyroid tumors.     

Genetic alterations involved in the transition from well-differentiated to poorly differentiated and anaplastic thyroid carcinomas

Recent molecular studies have provided new insights into thyroid carcinogenesis. In thyroid papillary carcinomas at least three initiating events may occur, which are point mutations in the BRAF and RAS genes and RET/PTC rearrangements. Tumors harboring mutant BRAF and RAS are prone to progression to poorly differentiated and anaplastic carcinoma, but most likely require additional mutations to trigger this process. In thyroid follicular carcinomas, two known initiating events are RAS mutations and PAX8-PPARγ rearrangements, and RAS predisposes to dedifferentiation of follicular carcinomas. p53 and β-catenin mutations, found with increasing incidence in poorly differentiated and anaplastic carcinomas but not in well-differentiated tumors, may serve as a direct molecular trigger of tumor dedifferentiation. Additional evidence for progression from a preexisting well-differentiated carcinoma to poorly differentiated and anaplastic carcinoma comes from the studies of loss of heterozygosity and comparative genomic hybridization. Molecular studies, although limited by the lack of uniform histologic criteria for poorly differentiated carcinomas, revealed no genetic mutations or chromosomal abnormalities that are unique for poorly differentiated carcinoma and not present in well-differentiated or anaplastic carcinomas. This suggests that poorly differentiated carcinoma, as a group, represents a distinct step in the evolution from well-differentiated to anaplastic thyroid carcinoma, rather than an entirely separate type of thyroid malignancy.     

Familial Non-Medullary Thyroid Carcinoma: An Update

Familial thyroid cancer can arise from follicular cells (familial non-medullary thyroid carcinoma (FNMTC)) or from the calcitonin-producing C-cell (familial medullary thyroid carcinoma). This is usually a component of multiple endocrine neoplasias (MEN) 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. In contrast, the case for a familial predisposition of non-medullary thyroid carcinoma is only now beginning to emerge. Although the majority of papillary (PTC) and follicular thyroid carcinomas (FTC) are sporadic, familial tumors account for over 5% of cases. The presence of multifocal papillary carcinoma is a common feature of FNMTC. The familial follicular cell-derived tumors or non-medullary thyroid carcinomas encompass a heterogeneous group of diseases, including diverse syndromic-associated tumors and non-syndromic tumors. Based on clinico-pathologic findings, FNMTC is divided into two groups. The first includes familial syndromes characterized by a predominance of non-thyroidal tumors, such as familial adenomatous polyposis (FAP), PTEN hamartoma tumor syndrome (PHTS), Carney complex type 1, and Werner syndrome. The second group includes familial syndromes characterized by a predominance of NMTC, such as pure familial (f) PTC with or without oxyphilia, fPTC with papillary renal cell carcinoma, and fPTC with multinodular goiter. Some characteristic morphologic findings should alert the pathologist of a possible familial cancer syndrome, which may lead to further molecular genetic evaluation.     

The two genes generating RET/PTC3 are localized in chromosomal band 10q11.2

PCR analysis of DNA from a selected panel of human-rodent somatic cell hybrids and fluorescent in situ hybridization (FISH) analysis allowed us to localize the human ELEI gene. This previously uncharacterized gene is fused with the tyrosine kinase (tk) domain of the RET proto-oncogene to generate the oncogenic sequence RET/PTC3, thus providing a third example of RET oncogenic activation in papillary thyroid carcinomas. ELEI was localized to band 10q11.2, the subband where RET also maps, at a minimum distance of more than 500 kb from the proto-oncogene. The fusion event corresponding to the rearrangement reciprocal to that leading to the formation of RET/PTC3 was also identified and characterized. The karyotype of two RET/PTC3 positive tumors did not show any evidence of chromosome 10 abnormalities. The data indicate that a cytogenetically undetectable paracentric inversion within 10q11.2 generates RET/PTC3.     

Immunohistochemical separation of follicular variant of papillary thyroid carcinoma from follicular adenoma

The accurate diagnosis of differentiated thyroid tumors is very important for clinical management of patients. The histopathological distinction between some types of differentiated thyroid tumors can be very difficult even for experienced pathologists. We used immunohistochemical markers from published data obtained from DNA expression profiling, tissue microarray analysis, and immunohistochemistry to analyze a series of 157 thyroid tumors and 5 normal thyroids. These analyses showed that several antibodies were useful in distinguishing follicular adenomas from follicular variant of papillary thyroid carcinomas including HBME-1, CITED 1, galectin-3, cytokeratin 19, and S100A4 (p<0.0001). A combination of markers consisting of a panel of HBME-1, galectin-3, and CK19 or a panel of HBME-1, CITED1, and galectin-3 was usually most effective in distinguishing follicular adenoma from follicular variant of papillary thyroid carcinoma. Because individual tumors may not express some of these markers, the use of a panel of antibodies is recommended. These results indicate that some individual antibodies or a panel of antibodies combined with histopathological analysis can be useful in separating follicular adenoma (FA) from follicular variant of papillary thyroid carcinoma (FVPTC).     

A t( 10; 17) translocation creates the RET/PTC2 chimeric transforming sequence in papillary thyroid carcinoma

Activation of the RET protooncogene tyrosine kinase (tk) by fusion with other genes is a frequent finding in papillary thyroid carcinoma. The tk domain of proto-RET can be fused either with the D10S170 gene generating the RET/PTCI transforming sequence or with sequences belonging to the gene encoding the regulatory subunit R/A of c-AMP-dependent protein kinase A, thus forming the RET/PTC2 oncogene. We have previously shown that an inversion of chromosome 10, inv(10)(q11.2q21), is responsible for the generation of the RET/PTCI. Here we report that a chromosomal translocation, t(10;17)(q11.2;q23), juxtaposes the tk domain of the RET protooncogene, which resides on chromosome 10, to a 5′ portion of the R/A gene on chromosome 17, leading to the formation of the chimeric transforming gene RET/PTC2. The finding of the transforming protein in primary tumor cell extracts supports the conclusion that RET/PTC2 activation plays a role in papillary thyroid tumorigenesis. Genes Chrom Cancer 9:244-250 (1994). © 1994 Wiley-Liss, Inc.     

Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant

Mutation of the BRAF gene is common in thyroid cancer. Follicular variant of papillary thyroid carcinoma is a variant of papillary thyroid carcinoma that has created continuous diagnostic controversies among pathologists. The aims of this study are to (1) investigate whether follicular variant of papillary thyroid carcinoma has a different pattern of BRAF mutation than conventional papillary thyroid carcinoma in a large cohort of patients with typical features of follicular variant of papillary thyroid carcinoma and (2) to study the relationship of clinicopathological features of papillary thyroid carcinomas with BRAF mutation. Tissue blocks from 76 patients with diagnostic features of papillary thyroid carcinomas (40 with conventional type and 36 with follicular variant) were included in the study. From these, DNA was extracted and BRAF V600E mutations were detected by polymerase chain reaction followed by restriction enzyme digestion and sequencing of exon 15. Analysis of the data indicated that BRAF V600E mutation is significantly more common in conventional papillary thyroid carcinoma (58% versus 31%, P = .022). Furthermore, the mutation was often noted in female patients (P = .017), in high-stage cancers (P = .034), and in tumors with mild lymphocytic thyroiditis (P = .006). We concluded that follicular variant of papillary thyroid carcinoma differs from conventional papillary thyroid carcinoma in the rate of BRAF mutation. The results of this study add further information indicating that mutations in BRAF play a role in thyroid cancer development and progression.     

Global DNA methylation evaluation: potential complementary marker in differential diagnosis of thyroid neoplasia

The implications of global DNA hypomethylation were recently reported in several models of tumorigenesis. Little is known about this epigenetic event in thyroid neoplasia. The study aimed to evaluate the status of global DNA methylation in several types of thyroid tumors using a monoclonal antibody specific for 5-methylcytidine (5-mc) and to define the diagnosis potential of this marker. 5-mc immunostaining scores were calculated in 17 papillary thyroid carcinomas (PTC), 6 follicular thyroid carcinomas (FTC), 16 follicular adenomas (FA), 19 nodular goiters (NG) and ten Hürthle cells adenomas (HCA). The expression of galectin-3 was also evaluated. Computerized image analysis showed a significant lower level of 5-mc immunostaining in thyroid carcinoma when compared with benign tumors or adjacent normal thyroid parenchyma (P<0.0001). Overall, 5-mc accuracy to distinguish malign from benign thyroid tumors was similar to that of galectin-3 (89% versus 87%, P>0.05). The combination of 5-mc with galectin-3 led to an excellent accuracy level of 96%. Among follicular neoplasia 5-mc accuracy to differentiate malign tumors trends to be higher than galectin-3 one (90% versus 66%, P=0.06). These data stress the necessity of epigenetic events evaluation among thyroid nodules and propose global DNA methylation assessment as a potential diagnostic tool to combine with other valuable markers.