Rearranged During Transfection Proto-oncogene Research Articles

The clinical spectrum of RET proto-oncogene mutations in codon 790

Due to a strong genotype-phenotype correlation, the timing of prophylactic thyroidectomy in rearranged during transfection (RET) gene mutation carriers is usually dictated by genetic analysis. We report a nationwide retrospective study of the clinical data of 77 French patients from 19 families with a mutation in codon 790 of the RET proto-oncogene. The average age at diagnosis was 35.6 years ± 20.5. Thirty-nine patients were women. Fifty-five patients underwent operations for the treatment of medullary thyroid carcinoma (MTC) at the mean age of 38 years (4-82 years). The mean follow-up duration was 89 months. TNM staging was as follows: T0NxMx in 19, TxNxMx in 1, T1NxMx in 22, T1N1Mx in 8, T2N1Mx in 1 and T3N1Mx in four patients. In the T1/x-Nx group, 96% were considered cured after surgery. In the N1 group (n=13), six patients had multifocal disease and five patients were cured. Age and gender were not significant predictors of remission. Twenty-two patients did not undergo an operation (age 1.5-78 years); among them, 11 patients had a mean basal calcitonin (CT) level of 9.8 pg/ml (2-24) after 53 months of follow-up. One patient had been operated on for phaeochromocytoma (PHEO), and their CT level remained normal for 262 months. This study confirms that RET 790 mutation is associated with a non-aggressive form of multiple endocrine neoplasia type 2, as 28% of the patients were followed up without thyroidectomy, 25% had been thyroidectomised with no tumour being detected and even patients with MTC had slow-evolving disease. Moreover, only one patient had PHEO, and no-one had primary hyperparathyroidism.

Abstract 745: Role of G-quadruplex structures in the human RET promoter region in the regulation of this gene.

Abstract The medullary thyroid carcinoma (MTC) is a tumor type characterized by germline point mutations of the RET (REarranged during Transfection) proto-oncogene. Since these mutations are known to induce oncogenic activation of RET tyrosine kinase, various therapeutic strategies interfering with the oncogenic function of RET protein have been developed and tested for treating RET-associated cancers in several preclinical and clinical studies. Our previous study has demonstrated that the G-rich strand in the polypurine/polypyrimidine (pPu/pPy) region of the RET proximal promoter is capable of forming intramolecular G-quadruplex structures in vitro in the presence of K+, which can be further stabilized by a G-quadruplex interactive agents. Thus, the detailed mechanism by which G-quadruplex structures regulate RET transcription has been studied using mutation analysis and promoter reporter assays together with a detailed analysis of transcription factor binding to the RET promoter in vivo. To this end, we have explored the Flp-In cell system using homologous recombination, which allows integration of a single copy of a wild type or G-quadruplex-mutant RET promoter–luciferase construct into a specific genomic recombination locus (FRT site) in a host Flp-In the human embryonic kidney 293 (HEK293) cell line. Thus, differences in reporter expression levels from isolated isogenic cell clones can be ascribed more confidently to promoter-specific effects, rather than enhancer/silencer artifacts due to variations in integration sites. Using these isogenic cell lines, we are able to provide strong evidence for G-quadruplex-dependent transcriptional repression of the RET gene in vivo. Citation Format: Daekyu Sun, Yoon-Joo Shin. Role of G-quadruplex structures in the human RET promoter region in the regulation of this gene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 745. doi:10.1158/1538-7445.AM2013-745

Update on the Management of Unusual Neuroendocrine Tumors: Pheochromocytoma and Paraganglioma, Medullary Thyroid Cancer and Adrenocortical Carcinoma

Pheochromocytomas, paragangliomas, and medullary thyroid carcinomas (MTCs) originate in cells that share a common neuroectodermal origin. Like other neuroendocrine neoplasms, they are characterized by a propensity to secrete amines (epinephrine and norepinephrine) and peptide hormones (calcitonin). Improved understanding of underlying molecular pathways, such as mutations of the RET (rearranged during transfection) proto-oncogene, has led to new rational targeted therapies. Adrenocortical carcinomas (ACCs) originate in the steroid hormone-producing adrenal cortex. While tumors of the adrenal cortex are not, strictly speaking, part the "diffuse neuroendocrine system," they are often included in neuroendocrine tumor guidelines due to their orphan status. In this update on management of unusual neuroendocrine tumors, we review the biology and treatment of these rare neoplasms.

Inhibition of RET Activated Pathways: Novel Strategies for Therapeutic Intervention in Human Cancers

The REarranged during Transfection (RET) proto-oncogene and its activated signalling pathways have been shown to play an important role in cancer. RET genetic alterations including germline, somatic mutations and gene rearrangements have been demonstrated in several solid tumours, and numerous clinical trials using multikinase inhibitors containing RET as a target have shown significant activity against RET. Sorafenib and sunitinib have been approved for the treatment of renal, hepatocellular, gastrointestinal and pancreatic neuoendocrine carcinomas. Vandetanib has recently been approved for the treatment of unresectable locally advanced or metastatic medullary thyroid carcinomas. Novel genomic rearrangements and RET signalling interactions are now being studied in a variety of tumours and will provide the basis for new therapeutic strategies. Combination or sequential targeted therapies that are based on solid preclinical data regarding the inhibition of RET-mediated parallel or different -signalling pathways will likely be more effective. Keywords: RET proto-oncogene, cancer, RET-activated signalling pathways, RET interacting proteins, tyrosine kinase inhibitors, targeted therapies, clinical trials, therapeutic strategies, sorafenib, sunitinib

A Pilot Characterization of Human Lung NSCLC by Protein Pathway Activation Mapping

An understanding of the activated protein signaling architecture in non-small-cell lung cancer (NSCLC) is of critical importance to the development of new therapeutic approaches and identification of predictive and prognostic biomarkers for patient stratification. We used reverse-phase protein microarrays to map the activated protein signaling networks of 47 NSCLC tumors, 28 of which were node negative, which were subjected to tumor cellular enrichment using laser capture microdissection. The phosphorylation/cleavage levels of 111 key signaling proteins and total levels of 17 proteins were measured for broadscale signaling analysis. Pathway activation mapping of NSCLC revealed distinct subgroups composed of epidermal growth factor receptor (ERBB1), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), v-erb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4), v-akt murine thymoma viral oncogene homolog 1- mammalian target of rapamycin (AKT-mTOR), protein kinase, AMP-activated, alpha 2 catalytic subunit (AMPK), and autophagy-related signaling, along with transforming growth factor-beta-signaling protein 1 (SMAD), insulin-line growth factor receptor (IGFR), rearranged during transfection proto-oncogene (RET), and activated CDC42-associated kinase (ACK) activation. Investigation of epidermal growth factor receptor (EGFR)-driven signaling identified a unique cohort of tumors with low EGFR protein expression yet high relative levels of phosphorylated EGFR and high EGFR total protein with low relative levels of phosphorylation. Last, mapping analysis of patients with NSCLC with N0 disease revealed a pilot pathway activation signature composed of linked epidermal growth factor receptor family (HER)-AMPK-AKT-mTOR signaling network along with focal adhesion kinase- LIM domain kinase-1 (FAK-LIMK) and janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways that correlated with short-term survival and aggressive disease. Functional protein pathway activation mapping of NSCLC reveals distinct activation subgroups that are underpinned by important therapeutic targets and that patients with early-stage node negative disease and poor prognosis may be identified by activation of defined, biochemically linked protein signaling events. Such findings, if confirmed in larger study sets, could help select and stratify patients for personalized targeted therapies.

Altered gene expression profiles in the hippocampus and prefrontal cortex of type 2 diabetic rats

BackgroundThere has been an increasing body of epidemiologic and biochemical evidence implying the role of cerebral insulin resistance in Alzheimer-type dementia. For a better understanding of the insulin effect on the central nervous system, we performed microarray-based global gene expression profiling in the hippocampus, striatum and prefrontal cortex of streptozotocin-induced and spontaneously diabetic Goto-Kakizaki rats as model animals for type 1 and type 2 diabetes, respectively.ResultsFollowing pathway analysis and validation of gene lists by real-time polymerase chain reaction, 30 genes from the hippocampus, such as the inhibitory neuropeptide galanin, synuclein gamma and uncoupling protein 2, and 22 genes from the prefrontal cortex, e.g. galanin receptor 2, protein kinase C gamma and epsilon, ABCA1 (ATP-Binding Cassette A1), CD47 (Cluster of Differentiation 47) and the RET (Rearranged During Transfection) protooncogene, were found to exhibit altered expression levels in type 2 diabetic model animals in comparison to non-diabetic control animals. These gene lists proved to be partly overlapping and encompassed genes related to neurotransmission, lipid metabolism, neuronal development, insulin secretion, oxidative damage and DNA repair. On the other hand, no significant alterations were found in the transcriptomes of the corpus striatum in the same animals. Changes in the cerebral gene expression profiles seemed to be specific for the type 2 diabetic model, as no such alterations were found in streptozotocin-treated animals.ConclusionsAccording to our knowledge this is the first characterization of the whole-genome expression changes of specific brain regions in a diabetic model. Our findings shed light on the complex role of insulin signaling in fine-tuning brain functions, and provide further experimental evidence in support of the recently elaborated theory of type 3 diabetes.

Case report: a p.C618S RET proto-oncogene germline mutation in a large Chinese pedigree with familial medullary thyroid carcinoma

We report a Chinese pedigree with familial medullary thyroid carcinoma. Direct sequencing of the entire coding sequences of Rearranged during Transfection (RET) identified a recurrent c.T1852A (p.C618S) mutation in 13 of 23 members. The polymorphisms c.A135G (p.A45A), c.A1296G (p.A432A), c.T2307G (p.L769L) and IVS19 + 15T > C were also found in 13 carriers, and c.G2073A (p.G691S) was found in 1 carrier. Of the 13 carriers, seven (mean age: 42.6 years, range: 27-64) presented MTC as the isolated clinical phenotype, with elevated basal serum calcitonin (average: 1077.9 ng/L, range: 504-2,652) and a mean diameter of thyroid nodules of 2.97 cm (range: 1.6-4.3); they underwent a total thyroidectomy with modified bilateral/unilateral neck dissection and/or level VI lymph node dissection. The other 6 carriers did not accept surgery (4 rejected, 2 awaited). These were 2 older patients (63 and 32 years) with elevated calcitonin (1359 and 41.4 ng/L) and multi-centric hypoechoic nodules (1.5 and 0.6 cm) with calcifications in both/left thyroid lobes; and Doppler ultrasound showed normal bilateral thyroids in 4 younger carriers (median age: 8.3 years, range: 4-12) but with increased calcitonin (average: 9.7 ng/L, range: 7.87-12.2) in 3 of them. The phenotype here is consistent with the clinical symptoms reported worldwide. We recommend that screening of hotspot regions of RET should be preferentially carried out, while whole-exon sequencing should be performed when clinical signs fail to reveal hotspot mutations or different phenotype discrepancies. Moreover, we strongly suggest prophylactic thyroidectomy should be performed before age 5 in carriers with p.C618S to prevent the occurrence and metastasis of MTC.

RET Germline Mutations Identified by Exome Sequencing in a Chinese Multiple Endocrine Neoplasia Type 2A/Familial Medullary Thyroid Carcinoma Family

BackgroundWhole exome sequencing provides a labor-saving and direct means of genetic diagnosis of hereditary disorders in which the pathogenic gene harbors a large cohort of exons. We set out to demonstrate a suitable example of genetic diagnosis of MEN 2A/FMTC (multiple endocrine neoplasia type 2/familial medullary thyroid carcinoma) using this approach.Methodology/Principal FindingsWe sequenced the whole exome of six individuals from a large Chinese MEN2A/FMTC pedigree to identify the variants of the RET (REarranged during Transfection) protooncogene and followed this by validation. Then prophylactic or surgical thyroidectomy with modified or level VI lymph node dissection and adrenalectomy were performed for the carriers. The cases were closely followed up. Massively parallel sequencing revealed four missense mutations of RET. We unexpectedly discovered that the proband's daughter with MEN 2A-related MTC presented a novel p.C634Y/V292M/R67H/R982C compound mutation, due to the involvement of p.C634Y in the proband with MEN 2A and p.V292M/R67H/R982C in the proband's husband with FMTC. In the maternal origin, p.C634Y caused bilateral MTC in all 5 cases and bilateral pheochromocytoma in 2 of the 5; the earliest onset age was 28 years. In the paternal origin, one of the six p.V292M/R67H/R982C carriers presented bilateral MTC (70 years old), one only had bilateral C-cell hyperplasia (44 years), two had bilateral multi-nodules (46 and 48 years) and two showed no abnormality (22 and 19 years).Conclusions/SignificanceThe results confirmed the successful clinical utility of whole exome sequencing, and our data suggested that the p.C634Y/V292M/R67H/R982C mutation of RET exhibited a more aggressive clinical phenotype than p.C634Y or p.V292M/R67H/R982C, while p.V292M/R67H/R982C presented a relatively milder pathogenicity of MTC and likely predisposed to FMTC.

Abstract 4547: Treatment of human medullary thyroid carcinoma (MTC) with either proteasome (Pr) or histone deacetylase (HDAC) inhibitors leads to a fall in RET mRNA levels and, in turn, a decrease in RET protein expression providing alternate strategies to reduce RET expression in a tyrosine-kinase driven disease

Abstract Medullary thyroid carcinoma (MTC), a neuro-endocrine cancer of parafollicular C-cells accounts for 4-10% of all thyroid cancers and has low response rates to “cytotoxic chemotherapy”. The RET proto-oncogene encodes the RET (Rearranged during Transfection) transmembrane tyrosine kinase (TK) receptor, a pivotal player and a logical target for treatment of MTC. While trials with vandetanib (V) a RET TK inhibitor (TKI) in patients with MTC have been promising, we recognized a TKI is unlikely to have maximum benefit administered alone. We sought to combine V + a drug with a broad activity profile and no overlapping toxicities. Recognizing the failure of traditional “cytotoxic” agents we investigated the proteasome (Pr) inhibitor, bortezomib (B) not yet explored clinically in MTC. Initial experiments using TT MTC cells with a mutant C634W RET, found V + B non-antagonistic in cytotoxicity assays, and V showed decreased phospho-RET expression in TT cells with 0.5 or 1 µM in 1.5h. But while V did not change RET protein levels, B decreased RET protein 7 fold and secondarily phospho-RET levels. That this was secondary to Pr inhibition and not unique to B was confirmed by treating TT cells 6h or 24h with three other Pr inhibitors – epoxomycin, lactacystin or MG132. We observed 5-9 fold decreases in RET protein, which followed a time course and a dose response, with greater reduction at 24h than at 6h. To assess if the decrease in RET protein was due to decreased RET mRNA levels, we assessed RET mRNA expression and were surprised to find a 4-9 fold decrease with Pr inhibitors. Since Pr inhibitors reduced RET by reducing mRNA levels, and reasoning this would provide an alternate strategy to impact RET function, we studied the effect of three HDAC inhibitors on RET mRNA and protein expression. Treatment of TT cells with vorinostat, belinostat, or romidepsin (R) decreased RET mRNA 3-4 fold at 24h and protein levels 4 and 7-14 fold, at 24 and 48h respectively. Furthermore B + R had greater impact than either drug separately, depressing RET protein in TT cells an additional 2-3 fold to levels 4-12 fold less than that in untreated cells, suggesting mechanisms whereby Pr and HDAC inhibitors reduce mRNA levels might differ. We have pursued the latter by examining mRNA and protein levels of 3 candidate transcription factors identified in a literature search. To date we have shown TTF1 and E2F1 mRNA levels are decreased 4-58 fold by both Pr and HDAC inhibitors whereas EGR1 levels only fall with Pr inhibitors. siRNA studies that modulate transcription factor levels will clarify their possible roles in the Pr and HDAC inhibitor drug effects and help elucidate whether these inhibitors, either alone or in combination, can prove useful in MTC therapy by reducing the crucial RET TK. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4547. doi:10.1158/1538-7445.AM2011-4547

Mega-Intestine After Subtotal Colectomy for Constipation

t A male with multiple endocrine neoplasia (MEN) type 2B presented with chronic abdominal distention and onstipation. He had previously undergone total thyroidectomy or medullary thyroid carcinoma, and a subtotal colectomy with leosigmoidostomy for megacolon. Examination revealed a maranoid body habitus, neuromas on the tip of the tongue, pectus xcavatum, and severe abdominal distention. Laboratory studies ere unremarkable, including negative urine catecholamines creening for pheochromocytoma. Abdominal x-ray showed muliple dilated loops of small bowel (Figure A). At laparotomy, ilated loops of small bowel with maximum diameter of 8 cm were oted (Figure B). The patient underwent sigmoid resection with nd ileostomy. On microscopic examination, the sigmoid and erminal ileum revealed hyperplasia of the nerve plexuses and an ncreased density of ganglia, many of which showed dysplastic hanges (Figure C, inset, yellow arrow). Hyperplastic subserosal erve fibers were also identified (Figure C, inset, black arrow). Intestinal ganglioneuromatosis associated with MEN type 2B as confirmed by the identification of dysplastic ganglion cells in oth resected sigmoid and terminal ileum. Intestinal ganglioneuomatosis (gangliocytic dysplasia) is characterized by hyperplasia f myenteric or submucosal nerve plexuses with increased number nd size of dysplastic ganglia, and hyperplastic subserosal nerves.1 MEN 2B results from an autosomal dominant mutation with a high degree of penetrance. The mutation involves a gain of function of the RET (rearranged during transfection) proto-oncogene (that encodes a tyrosine kinase receptor) on chromosome 10. The mutation appears to induce growth and differentiation in several developing tissues including those derived from the neural crest.2 In a 28-patient series, 93% of patients with MEN 2B had gastrointestinal symptoms 1 to 24 years before the diagnosis of MEN 2B. Symptoms included flatulence (86%), abdominal distention or being underweight as a child (64%), abdominal pain (54%), constipation or diarrhea (43%), difficulty swallowing (39%), vomiting (14%), and 29% underwent surgery.2 In a series of 16 patients, 90% had colonic disturbances, typically chronic constipation from birth. Megacolon developed in 14 patients, and 8 required colonic surgery.3 Postoperatively, the patient received oral pyridostigmine 60 mg 4 times a day, and supplemental parenteral nutrition. One month later, he was asymptomatic, on regular (full) oral diet and pyridostigmine, and intestinal loops were not dilated on abdominal radiograph. Management of abdominal distension and constipation in MEN 2B is usually conservative with laxatives; however some patients require total colectomy and oral prokinetic agents. Our patient responded to acetyl cholinesterase inhibition (pyridostigmine). The potential efficacy of 5-hydroxytryptamine (5HT)4 agonists, other prokinetics, and stem cell therapy for intesinal ganglioneuromatosis associated with MEN 2B are unclear.

Targeting the RET Pathway in Thyroid Cancer

The RET (rearranged during transfection) protooncogene encodes a single pass transmembrane receptor that is expressed in cells derived from the neural crest and the urogenital tract. As part of a cell-surface complex, RET binds glial derived neurotrophic factor (GDNF) ligands in conjunction with GDNF-family alpha co-receptors (GFRalpha). Ligand-induced activation induces dimerization and tyrosine phosphorylation of the RET receptor with downstream activation of several signal transduction pathways. Activating germline RET mutations play a central role in the development of the multiple endocrine neoplasia (MEN) syndromes MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) and also in the development of the congenital abnormality Hirschsprung's disease. Approximately 50% of patients with sporadic MTC have somatic RET mutations, and a significant portion of papillary thyroid carcinomas result from chromosomal inversions or translocations, which activate RET (RET/PTC oncogenes). The RET protooncogene has a significant place in cancer prevention and treatment. Timely thyroidectomy in kindred members who have inherited a mutated RET allele, characteristic of MEN2A, MEN2B, or FMTC, can prevent MTC, the most common cause of death in these syndromes. Also, recently developed molecular therapeutics that target the RET pathway have shown activity in clinical trials of patients with advanced MTC, a disease for which there has been no effective therapy.

Mutational Spectrum of Multiple Endocrine Neoplasia Type 2 and Sporadic Medullary Thyroid Carcinoma in Taiwan

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant cancer predisposition syndrome, and > 95% of MEN 2 patients carry rearranged during transfection (RET) protooncogene mutants. We aimed to elucidate the genotype and phenotype relationship of RET proto-oncogene mutations in Taiwanese subjects with medullary thyroid cancer (MTC). We genotyped the MEN-2-associated germ-line mutations by PCR-based sequencing of the RET gene. DNA was extracted from a total of 69 members from eight unrelated families with individuals affected by MTC, and from seven sporadic cases of MTC. RET mutations were found in four MEN 2A families, all at codon 634 (one with C>R, two with C>F, and one with C>W). One MEN 2A patient carried a de novo mutation at codon 634 (C>R). In two families of MEN 2B, all carried the mutation at codon 918 (M>T). These two cases of MEN 2B were all de novo mutations. One family of familial MTC or unclassified MEN 2 carried the codon 620 (C>F) mutation. Among the seven sporadic cases of MTC, none was found to carry any mutation in hotspot exons. Only two non-synonymous variants (T278N/exon 4 and D489N/exon 7) were found in two cases. However, these two variants were not uncommon in our elderly population. We found that all eight MTC patients with a family history or with the other phenotypes of MEN 2 had RET mutations, whereas no significant RET mutation was found in seven patients with isolated MTC without family history and other endocrine diseases. Molecular scanning of the RET gene in MEN 2 and MTC in Taiwanese patients probably should be limited to exons 10, 11 and 16, initially to be cost-effective.

Routine screening for germline RET mutations is recommended for all patients with medullary thyroid cancer

All patients with medullary thyroid cancer (MTC) are routinely screened for germline mutations in the rearranged during transfection (RET) proto-oncogene. In this Practice Point commentary, we discuss a cross-sectional study by Machens and Dralle, who performed germline RET proto-oncogene mutation analysis in patients with MTC at a tertiary referral centre in Germany to reveal patterns of familial disease presentation over the generations. The study identified a typical distribution of germline RET mutations (subclassified into three accepted risk categories) in the study population, consistent with the reported literature. The investigators also attempted to obtain data on the time of appearance of RET germline mutations in previous generations of the families. The natural history of MTC and penetrance of specific RET mutations could not be clearly identified in each family because of the cross-sectional nature of the study. Nevertheless, the study re-establishes the importance of routine screening for germline RET mutations in all new cases of MTC.

Does the RET variant G691S influence the features of sporadic medullary thyroid carcinoma?

The RET (rearranged during transfection) proto-oncogene G691S variant is over-represented in the germline of patients with sporadic medullary thyroid carcinoma (sMTC) vs. normal controls but so far is not associated with any medical or pathological features of the tumour. The aim of our study was to assess the influence of this variant on the age of onset, clinical, biological and pathological features of sMTC. One hundred patients with histologically proven MTC, for whom the germline genetic analysis of RET was negative and medical records were available, were included in the study. Patients with the heterozygous GS variant or the homozygous SS variant (n = 36) were on average 8.0 years younger than patients with the wild-type GG variant (n = 64, mean age 43.9 vs. 51.9 years, P < 0.01). The former group did not differ from the wild-type group in terms of MTC size, prevalence of C-cell hyperplasia (CCH) or papillary thyroid carcinoma (PTC). However, the prevalence of an increased preoperative basal calcitonin (bCT) level (> 1000 pg/ml) was 2.75-fold higher in the patients with the GS or SS variant than in those with the wild-type variant (P < 0.001). The proportion of patients with lymph node metastases was also higher in the former group (P < 0.05). Multivariate analysis confirmed that the presence of the RET variant is independently associated with higher preoperative bCT values (P = 0.011). Our data demonstrate that the RET G691S variant could modulate the age of onset of sMTC as demonstrated previously for familial tumours. Moreover, this variant is an independent predictor of a higher basal calcitonin synthesis rate in patients with sMTC.

NEW ERA: PROPHYLACTIC SURGERY FOR PATIENTS WITH MULTIPLE ENDOCRINE NEOPLASIA‐2A

The surgical management of patients with multiple endocrine neoplasia-2A (MEN-2A) continues to evolve with specific genotype-phenotype correlations allowing for a more tailored approach. In this study, we report the surgical management of one of the largest MEN-2A families with a rearranged during transfection (RET) codon 804 mutation. This is a cohort study comprising all at-risk kindred within a single known MEN-2A family. Prophylactic total thyroidectomy with lymph node dissection was recommended to all mutation carriers aged 5 years and older. There were a total of 48 at-risk individuals in the MEN-2A kindred, with 22 patients undergoing thyroidectomy after appropriate preoperative evaluation. A total of 9 patients had medullary thyroid cancer including 5 with a normal preoperative calcitonin level. A total of 11 patients had C-cell hyperplasia and 7 showed histological evidence of parathyroid disease. Only the index case had a phaeochromocytoma. Genetic testing for germline mutations in the RET proto-oncogene has allowed precise identification of affected RET carriers and provided the opportunity for prophylactic or 'preclinical' surgery to treat and in fact to prevent medullary thyroid cancer. This concept of prophylactic surgery based on a genetic test is likely to be applied more widely as the tools of molecular biology advance.

Polymerase chain reaction-single strand conformational polymorphism analysis of rearranged during transfection proto-oncogene in Chinese familial hirschsprung’s disease

To investigate the relationship between mutations of rearranged during transfection (RET) proto-oncogene and Chinese patients with Hirschsprung's disease (HD), and to elucidate the genetic mechanism of familial HD patient at the molecular level. Genomic DNA was extracted from venous blood of probands and their relatives in two genealogies. Polymerase chain reaction (PCR) products, which were amplified using specific primers (RET, exons 11, 13, 15 and 17), were electrophoresed to analyze the single-strand conformational polymorphism (SSCP) patterns. The positive amplified products were sequenced. Forty-eight sporadic HD patients and 30 normal children were screened for mutations of RET proto-oncogene simultaneously. Three cases with HD in one family were found to have a G heterozygous insertion at nucleotide 18,974 in exon 13 of RET cDNA (18,974insG), which resulted in a frameshift mutation. In another family, a heterozygosity for T to G transition at nucleotide 18,888 in the same exon which resulted in a synonymous mutation of Leu at codon 745 was detected in the proband and his father. Eight RET mutations were confirmed in 48 sporadic HD patients. Mutations of RET proto-oncogene may play an important role in the pathogenesis of Chinese patients with HD. Detection of mutated RET proto-oncogene carriers may be used for genetic counseling of potential risk for HD in the affected families.

Emergence of medullary thyroid carcinoma in a family with the Cys630Arg RET germline mutation

Background Individual germline mutations in the RET (REarranged during Transfection) proto-oncogene may set the time window for malignant progression from C-cell hyperplasia to familial medullary thyroid carcinoma. Owing to the close genotype–phenotype correlation, genetic information may lend to individual timing of prophylactic thyroidectomy according to RET genotype. Limited information exists on the Cys630 RET genotype. Most of the few published carriers of this genotype who developed medullary thyroid carcinomas (MTCs) were in their mid-30s. Methods This case series of a German RET family with the Cys630Arg genotype was assembled to study malignant progression of MTC in this rare RET genotype. Results There was considerable variability of malignant progression from C-cell hyperplasia to MTC in carriers of the Cys630Arg genotype. In these persons, MTCs had developed by the age of 32 years (index patient, pT2bN0M0), and 15 years and 1 year (non-index patients; pT1apN1bM0 and pT1bpN0M0, respectively). The Cys630Arg genotype always segregated with the familial medullary thyroid carcinoma phenotype. Conclusions The Cys630 RET genotype may have a more vigorous transforming activity than currently thought and can cause MTC in RET gene carriers within the first year of life. Starting in early infancy, identified RET gene carriers should be scrutinized until stimulated serum calcitonin levels become positive or, when these remain normal, should undergo prophylactic thyroidectomy before they reach 5 years of age.

Screening of six risk exons of the RET proto-oncogene in families with medullary thyroid carcinoma in the Czech Republic

Medullary thyroid carcinoma (MTC) occurs as a sporadic form (75%) or as an autosomal dominant inherited familial disorder (25%) called familial MTC (FMTC) or as multiple endocrine neoplasia type 2 (MEN2) syndromes. Germ-line mutations in the rearranged during transfection (RET) proto-oncogene in exons 10, 11, 13, 14, 15 and 16 are known to be a cause of most of the familial forms. In this paper we report molecular genetic testing of 106 families with MTC (358 tested persons) from the Czech Republic in which we directly sequenced these six exons of the RET proto-oncogene. We detected germ-line mutations in 100% of MEN2B families (4/4 families), 90% of MEN2A families (9/10), 40% of FMTC families (4/10) and 7% of apparently sporadic MTC (6/82). Eleven different germ-line mutations were revealed. MEN2B was associated with mutation Met918 Thr in exon 16. In one MEN2B family beside this mutation the Tyr791 Phe was also found, which has not yet been reported. MEN2A was restricted to different mutations in exon 11 (codon 634). In FMTC and 'sporadic' MTC families the mutations in exons 10, 11, 13 and 14 were detected. The genotype/phenotype correlations are given. Genetic testing revealed germ-line mutations in 23 index patients, 24 family members and excluded them in 53 relatives.

Novel RET mutations in Hirschsprung's disease patients from the diverse South African population.

Hirschsprung's disease (HSCR) is a common cause of intestinal obstruction in neonates with an incidence of one in 5000 live births. The disease occurs due to the absence of parasympathetic neuronal ganglia in the hindgut, resulting in irregular or sustained contraction of the affected segment. DNA samples of 40 unrelated subjects with HSCR were subjected to mutation screening of the RET (REarranged during Transfection) proto-oncogene, the major susceptibility gene for HSCR. Five novel (V202M, E480K, IVS10-2A/G, D771N, IVS19-9C/T) and one previously described mutation (P973L) were identified. Only two of the mutation-positive patients (from different ethnic groups) displayed total colonic aganglionosis, and both were heterozygous for mutation D771N. The potential disease-causing mutations occurred in 20% of individuals, with more males (22.5% representing seven of 31 males) affected than females (12.5% representing one of eight females). This study represents the first comprehensive genetic analysis of this disease in the diverse South African population.

RET/PTC activation in hyalinizing trabecular tumors of the thyroid.

Hyalinizing trabecular tumor (HTT) of the thyroid is a neoplasm of follicular derivation with a histogenesis that is still the subject of debate. Morphologic affinities between HTT and papillary carcinoma, including nuclear pseudoinclusions and grooves, suggest that these tumors may be of similar origin. The authors investigated the relationship between these two types of tumors by assessing HTT for the presence of rearrangements of the proto-oncogene rearranged during transfection (RET) that, in thyroid tumors, are specific for papillary carcinoma. A series of 14 HTTs, including two cases associated with classic papillary carcinoma, was studied by means of immunohistochemistry and reverse transcription-polymerase chain reaction. Seven follicular adenomas with focal hyalinized trabecular areas served as control cases. Three of the 14 HTT cases under consideration displayed rearrangements of RET generating the RET/papillary thyroid carcinoma type 1 (PTC1) oncogene. In another case, RET expression was detected focally by immunohistochemistry alone. Finally, in one mixed HTT-papillary carcinoma sample, RET/PTC1 expression was detected, but only in the papillary component. None of the control follicular adenomas contained rearrangements of RET/PTC. These findings demonstrate that a comparable percentage (28.6%) of HTTs and papillary carcinomas exhibit the same RET proto-oncogene alterations. Thus, HTT may represent the "hyalinizing trabecular" variant of papillary carcinoma rather than a separate entity.