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.     

An evaluation and recommendation of the optimal methodologies to detect RET gene rearrangements in papillary thyroid carcinoma

To recommend a reliable and clinically realistic RET/PTC rearrangement detection assay for papillary thyroid carcinoma (PTC), we compared multiplex quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). RET/PTC rearrangement was detected using either RET break‐apart FISH followed by multicolor FISH to confirm CCDC6/RET or NCOA4/RET fusions, or by multiplex qPCR to detect 14 RET/PTC subtypes with simultaneous RET mRNA expression. RET protein expression was detected by IHC. The specificity and sensitivity of multiplex qPCR and IHC were calculated using break‐apart FISH as a reference. Among 73 PTC patients with sufficient tissue available for FISH and multiplex qPCR, 10 cases were defined as RET/PTC positive by both assays, including eight CCDC6/RET and two NCOA4/RET fusions with relatively high RET mRNA. In addition, multiplex qPCR identified another two CCDC6/RET fusion positive cases, but with low RET mRNA expression. IHC staining identified 11 RET positive cases among 39 patients with available samples. In comparison to FISH, multiplex qPCR displayed 100% sensitivity and 97% specificity to detect RET/PTC fusions, while IHC was neither sensitive nor specific. Our data reveal that both multiplex qPCR and FISH assays are equally applicable for detection of RET/PTC rearrangements. Break‐apart FISH methodology is highly recommended for the wider screening of RET rearrangements (regardless of partner genes), while multiplex qPCR is preferred to identify all known fusion types using one assay, provided mRNA expression is also measured. IHC analysis could potentially provide an additional method of fusion detection dependent on further optimization of assay conditions and scoring cutoffs. © 2014 Wiley Periodicals, Inc.     

RET/PTC rearrangement in thyroid tumors

Rearrangement of the RET gene, also known as RET/PTC rearrangement, is the most common genetic alteration identified to date in thyroid papillary carcinomas. The prevalence of RET/PTC in papillary carcinomas shows significant geographic variation and is approx 35% in North America. RET/PTC is more common in tumors in children and young adults, and in papillary carcinomas associated with radiation exposure. There are at least 10 different types of RET/PTC, all resulting from the fusion of the tyrosine kinase domain of RET to the 5′ portion of different genes. RET/PTC1 and RET/PTC3 are the most common types, accounting for >90% of all rearrangements. There is some evidence that different types of RET/PTC may be associated with distinct biologic properties of papillary carcinomas. RET/PTC1 tends to be more common in tumors with typical papillary growth and microcarcinomas and to have a more benign clinical course, whereas RET/PTC3 in some populations shows a strong correlation with the solid variant of papillary carcinoma and more aggressive tumor behavior. RET/PTC has recently been found in hyalinizing trabecular adenomas of the thyroid gland, providing molecular evidence in favor of this tumor to be a variant of papillary carcinoma. The occurrence of RET/PTC in Hashimoto thyroiditis and thyroid follicular adenomas and hyperplastic nodules reported in several studies has not been confirmed in other observations and remains controversial.     

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.     

Concurrent lymph node metastases of medullary and papillary thyroid carcinoma in a case with <Emphasis Type="Italic">RET</Emphasis> oncogene germline mutation

We report the case of a 72 yr-old woman who underwent total thyroidectomy and resection of neck lymph nodes because of a firm nodule in the right lobe, which was consistent with medullary thyroid carcinoma (MTC) on cytological examination. Histology showed multifocal bilateral MTC; a 2 mm papillary thyroid carcinoma (PTC) was also detected in the right lobe, next to a focus of MTC; five cervical lymph nodes contained MTC. In one right perithyroidal lymph node, concurrent metastases of MTC and PTC were demonstated. DNA analysis showed a point mutation in exon 14 at codon 804 of the RET proto-oncogene locus, as frequently found in cases of familial MTC (FMTC). To our knowledge, this case represents the first documented case of concurrent lymph node metastases of MTC and PTC in a patient with RET proto-oncogene germline mutation. We report this unique case, discuss related thyroid malignancies, and suggest possible underlying pathogenetic mechanisms.     

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     

RET Signaling in Endocrine Tumors: Delving Deeper into Molecular Mechanisms

The rearranged during transfection (RET) proto-oncogene encodes a receptor tyrosine kinase that is implicated in the development of endocrine tumors of the thyroid and adrenal glands. In humans, activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia 2 and in sporadic medullary and papillary thyroid carcinomas. The specific type and location of RET mutations are strongly correlated with the disease phenotype and have both diagnostic and prognostic value. Recent advances in the molecular characterization of the RET receptor and its mutants have begun to define the mechanisms underlying the transforming ability of the different RET mutant forms. This information has revealed key functional features of these mutant proteins that distinguish the different clinically recognized mutations and provide clues as to the functional origins of the phenotypes associated with specific RET mutations. The elucidation of molecular mechanisms involved in RET-mediated transformation is a key step in the development of much needed therapeutics that target RET’s oncogenic properties. Recent advances have begun to provide a deeper understanding of the receptor’s function, and dysfunction, in human tumors that may guide this process.     

Diversity of RET proto-oncogene mutations in familial and sporadic Hirschsprung disease

Hirschsprung disease (HSCR) is a common congenital malformation(1 in 5 000 live births) due to the absence of autonomic gangliain the terminal hindgut, and resulting in intestinal obstructionin neonates. Recently, a dominant gene for familial HSCR hasbeen mapped to chromosome sub-band 10q11.2 and the disease hasbeen ascribed to mutations in a tyrosine kinase receptor genemapping to this region, the RETproto-oncogene. Studying the20 exons of the RET gene by a combination of denaturating gradientgel electrophoresis and single strand conformation polymorphismin a large series of HSCR patients (45 sporadic cases and 35familial forms), we found mutations of the RET gene in 50% offamilial HSCR, regardless of the length of the aganglionic segment.The mean penetrance of the mutant allele in familial HSCR wassignificantly higher in males (72%) than in females (51%). Mostinterestingly, mutations at the RET locus accounted for at least1/3 of sporadic HSCR in our series. These mutations were scatteredalong the length of the gene. Finally, among the mutations identifiedin sporadic cases (16/45), seven proved to be de novo mutationssuggesting that new mutations at the RET locus significantlycontribute to sporadic HSCR. Taken together, the low penetranceof the mutant gene, the lack of genotype-phenotype correlation,the sex-dependent effect of RET mutations and the variable clinicalexpression of the disease support the existence of one or moremodifier genes in familial HSCR.     

Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours

The susceptibilIty locl for the three multiple endocrine neoplasIa(MEN) type 2 syndromes have been mapped to the region of chromosome10q11.2 containing the RET proto-oncogene, which codes for areceptor tyrosIne kinase. The majority of MEN 2A and familIalmedullary thyrold carcInoma results from mlssense mutationswithin one of five cysteIne codons In the extracellular domainof the RET proto-oncogene. We now report a missense mutation,resulting in the substitution of a threonlne for a methlonineat codon 918 in the tyrosIne kInase catalytIc domain, In thegermllne of 26 of 28 apparently distinct families with MEN 2B.DNA from five of 13 apparently sporadIc MTC and one of 12 apparentlysporadic phaeochromocytomas harboured a sImIlar mutation, butthe corresponding germllne DNA was wildtype In each case.     

Specific haplotypes of the RET proto-oncogene are over-represented in patients with sporadic papillary thyroid carcinoma

Background: Papillary thyroid carcinoma (PTC), which may be sporadic (95%) or familial (5%), has a prevalence adjusted for age in the general population of 1:100 000. Somatic rearrangements of the RET proto-oncogene are present in up to 66% of sporadic tumours, while they are rarely found in familial cases.

Purpose: In order to determine if some variants of this gene, or a combination of them, might predispose to PTC, we looked for an association of RET haplotype(s) in PTC cases and in controls from four countries matched for sex, age, and population.

Methods: Four single nucleotide polymorphisms (SNPs) across the RET coding sequence were typed and haplotype frequencies were estimated. Genotype and haplotype distributions were compared among these cases and controls.

Results: Ten haplotypes were observed, the seven most frequent of which have been previously described in sporadic Hirschsprung patients and controls. The single locus analyses suggested association of exon 2 and exon 13 SNPs with sporadic PTC. The haplotype analysis showed over-representation of one haplotype in French and Italian sporadic PTC, whereas a different haplotype was significantly under-represented in French familial PTC.

Conclusions: Our data suggest that some variants of RET and some specific haplotypes may act as low penetrance alleles in the predisposition to PTC.

     

Multiple Functional Effects of RET Kinase Domain Sequence Variants in Hirschsprung Disease

The REarranged during Transfection (RET) gene encodes a receptor tyrosine kinase required for maturation of the enteric nervous system. RET sequence variants occur in the congenital abnormality Hirschsprung disease (HSCR), characterized by absence of ganglia in the intestinal tract. Although HSCR‐RET variants are predicted to inactivate RET, the molecular mechanisms of these events are not well characterized. Using structure‐based models of RET, we predicted the molecular consequences of 23 HSCR‐associated missense variants and how they lead to receptor dysfunction. We validated our predictions in biochemical and cell‐based assays to explore mutational effects on RET protein functions. We found a minority of HSCR‐RET variants abrogated RET kinase function, while the remaining mutants were phosphorylated and transduced intracellular signals. HSCR‐RET sequence variants also impacted on maturation, stability, and degradation of RET proteins. We showed that each variant conferred a unique combination of effects that together impaired RET protein activity. However, all tested variants impaired RET‐mediated cellular functions, including cell transformation and migration. Our data indicate that the molecular mechanisms of impaired RET function in HSCR are highly variable. Although a subset of variants cause loss of RET kinase activity and downstream signaling, enzymatic inactivation is not the sole mechanism at play in HSCR.     

Presymptomatic genetic screening in families with multiple endocrine neoplasia type 2

Medullary thyroid carcinoma occurs sporadically or as a part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. The MEN 2 gene has been identified as the RET proto-oncogene on chromosome 10. In MEN 2A, RET mutations are detectable in one of five cysteine codons within exons 10 and 11 and in MEN 2B in codon 918 (exon 16). Direct DNA testing for RET proto-oncogene mutations is the method of first choice in presymptomatic screening of MEN 2 families. Gene carriers should be offered prophylactic thyroidectomy. The process of DNA analysis for RET proto-oncogene mutations is demonstrated in one family with hereditary medullary thyroid carcinoma. RET mutations were detectable in five of the nine family members at risk.Abbreviations MEN Multiple endocrine neoplasia - MTC Medullary thyroid carcinoma - FMTC Familial medullary thyroid carcinoma - PCR Polymerase chain reaction - C-cells Calcitonin-producing parafollicular cells     

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.     

Long segment and short segment familial Hirschsprung''s disease: variable clinical expression at the RET locus.

Hirschsprung's disease (aganglionic megacolon, HSCR) is a frequent condition of unknown origin (1/5000 live births) resulting in intestinal obstruction in neonates and severe constipation in infants and adults. In the majority of cases (80%), the aganglionic tract involves the rectum and the sigmoid colon only (short segment HSCR), while in 20% of cases it extends toward the proximal end of the colon (long segment HSCR). In a previous study, we mapped a gene for long segment familial HSCR to the proximal long arm of chromosome 10 (10q11.2). Further linkage analyses in familial HSCR have suggested tight linkage of the disease gene to the RET protoncogene mapped to chromosome 10q11.2. Recently, nonsense and missense mutations of RET have been identified in HSCR patients. However, the question of whether mutations of the RET gene account for both long segment and short segment familial HSCR remained unanswered. We have performed genetic linkage analyses in 11 long segment HSCR families and eight short segment HSCR families using microsatellite DNA markers of chromosome 10q. In both anatomical forms, tight pairwise linkage with no recombinant events was observed between the RET proto-oncogene locus and the disease locus (Zmax = 2.16 and Zmax = 5.38 for short segment and long segment HSCR respectively at 0 = 0%) Multipoint linkage analyses performed in the two groups showed that the maximum likelihood estimate was at the RET locus. Moreover, we show that point mutations of the RET proto-oncogene occur either in long segment or in short segment HSCR families and we provide evidence for incomplete penetrance of the disease causing mutation. These data suggest that the two anatomical forms of familial HSCR, which have been separated on the basis of clinical and genetic criteria, may be regarded as the variable clinical expression of mutations at the RET locus.     

Fusion of a novel gene,ELKS, to RET due to translocation t(10;12)(q11;p13) in a papillary thyroid carcinoma

In papillary thyroid carcinomas, the genes for receptor‐type tyrosine kinase, RET or TRKA, are sometimes rearranged, resulting in fusion of its tyrosine kinase domain to 5′ portions of several activating genes. In a papillary thyroid carcinoma, we identified a novel gene (ELKS), the 5′ portion of which is fused to the RET gene by gene rearrangement due to the translocation t(10;12)(q11;p13). Subsequent cloning of the ELKS cDNA revealed that ELKS encodes a novel 948 amino acid peptide and is expressed ubiquitously in human tissues. The presence of multiple coiled‐coil domains in the ELKS product suggests that the ELKS protein forms dimers. Since the tyrosine kinase of RET is activated by dimerization that occurs when its ligands bind to the receptor, fusion of RET with the 5′ dimerization domains of ELKS would activate its cytoplasmic tyrosine kinase constitutively in papillary thyroid carcinomas. Genes Chromosomes Cancer 25:97–103, 1999. © 1999 Wiley‐Liss, Inc.     

Molecular Screening for RET Proto-Oncogene Mutations in a German MEN2A Pedigree

Multiple endocrine neoplasia type 2A (MEN2A), a dominantly inherited cancer syndrome, is defined by the presence of medullary thyroid carcinoma (MTC), pheochromocytoma (pheo), and primary hyperparathyroidism (p-HPT). Along with multiple endocrine neoplasia type 2B (MEN2B) and familial medullary thyroid carcinoma (FMTC), it is associated with germline mutations of theRET proto-oncogene localized in 10q11.2. In FMTC and MEN2A, point mutations result in the substitution of one of five Cys residues in the extracellular domain ofRET. In a larger pedigree from Saarland, several individuals were observed with C-cell thyroid carcinoma. We screened 16 members of this extended family by single-strand conformation polymorphism analysis (SSCP), polymerase chain reaction (PCR), followed by restriction enzyme analysis, and by sequencing the mutated regions. In 7 family members, all of whom had been earlier operated on because of MTC, a DNA transition from T to C was observed, causing an amino acid substitution Cys⁶³⁴Arg. Nine members of the kindred did not carry the mutation and may be excluded from yearly biochemical testing. One of these persons seems to have been unnecessarily operated on owing to a borderline pentagastrin test.     

RET proto-oncogene mutations in French MEN 2A and FMTC families

Constitutional mutations of the RET proto-oncogene have beenidentified in multiple endocrine neoplasia type 2A (MEN 2A),type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC)families. We sequenced RET exons 10 and 11 in 86 unrelated patientswith an inherited predisposition to MTC (excluding MEN 2B).Germ-line mutations were Identified in 93% of the MEN 2A familiesand 67% of the FMTC families tested. All were missense mutationsaffecting one of three cysteines in the extracellular domainof the RET tyrosine kinase receptor. The prevalence of phaeochromocytomaand hyperparathyroidism was significantly higher in familieswith a mutation of cysteine 634. These data confirm the preferentiallocalisation of MEN 2A and FMTC associated mutations and thestrong correlation between clinical manifestations and the positionof RET mutation. Although direst sequencing of RET exons 10and 11 allows the identification of a constitutional mutationin a large proportion of MEN 2A and FMTC families, our datasustain the existence of other MTC predisposing mutations elsewherein RET coding or regulating region.