Update on the MEN 2A c804 RET mutation: is prophylactic thyroidectomy indicated?

BACKGROUND: Mutations of the RET proto-oncogene co-segregate with multiple endocrine neoplasia type 2A. A rare sequence abnormality at codon 804 (c804) has been reported in 6 kindreds and linked to mild C-cell disease, which raises the question of the appropriateness of thyroidectomy in childhood. The purpose of this study was to (1) report the clinical correlates of 5 additional c804 kindreds, and (2) clarify therapeutic options in children. METHODS: Thirty-eight members from five c804 kindreds underwent genetic analysis. Biochemical, operative, and pathology reports were reviewed. RESULTS: Twenty-three gene carriers were identified, of whom 14 had thyroidectomy. Medullary thyroid carcinoma was found in 7 patients (aged 5-56 years), C-cell hyperplasia in 6 patients (aged 13-40 years), and normal histology in a single patient (aged 27 years). One patient with medullary thyroid carcinoma died of metastases (aged 12 years). Nine of the 23 gene carriers delayed operation, 4 of whom had calcitonin testing. Three of the 4 patients had abnormal calcitonin levels and a single patient was negative (aged 40 years). Of the remaining 9 patients, 2 await thyroidectomy, and 3 have refused evaluation. CONCLUSIONS: Penetrance of the c804 mutation is highly variable. Medullary thyroid carcinoma associated with this genotype has aggressive potential. Prophylactic thyroidectomy in childhood is a viable approach.

A codon 891 exon 15 RET proto-oncogene mutation in familial medullary thyroid carcinoma: a detection strategy.

A ser891ala RET proto-oncogene mutation has been previously discovered in a single kindred with familial medullary thyroid carcinoma (FMTC). An additional two probands with this mutation and with medullary thyroid carcinoma (MTC) without any other manifestations of MEN 2 have been identified. In one of thse families, two other individuals also had the mutant sequence and FMTC. Analysis of both cases showed cosegregation of the mutation and MTC. To facilitate detection of this mutation, a primer was engineered which creates a Hha I recognition site in the presence of the mutant sequence. As a result, this codon 891 exon 15 mutation can be identified with a restriction enzyme digestion.

Occurrence of MEN 2a in familial Hirschsprung's disease: a new indication for genetic testing of the RET proto-oncogene.

PURPOSE: The association of the rare hereditary cancer syndrome, multiple endocrine neoplasia type 2a (MEN 2a) with Hirschsprung's disease, both linked to germline mutations in the RET proto-oncogene, has been reported recently. With the widespread availability of genetic screening for MEN 2a, it is necessary to define the indications for genetic testing of MEN 2a and population subgroups at high risk for inheriting the disease. The purpose of this study was to assess the prevalence of Hirschsprung's disease in MEN 2a and investigate the value of genetic analysis for MEN 2a in children with familial Hirschsprung's disease. METHODS: The ethnically diverse study group consisted of unselected consecutive patients (n=426) at risk for hereditary medullary thyroid cancer (MTC) referred to a single laboratory for genetic testing. Analysis used genomic DNA and a polymerase chain reaction-based heteroduplex mutation detection strategy for exons 10, 11, 13, and 14 of the RET proto-oncogene followed by direct DNA sequencing. Significance of RET genotype-phenotype correlation was determined by Fisher's two-tailed Exact test and a 2 x 2 contingency table. RESULTS: Thirty-six distinctly new MEN 2a kindreds were identified. Hirschsprung's disease cosegregated among siblings with MEN 2a in 15 patients from 6 of the 36 (17%) families. The extent of aganglionosis in the 15 patients ranged from midrectum to duodenum. Of the 15 patients with Hirschsprung's disease, 10 (six boys, four girls) underwent thyroidectomy for MTC (n=5) or C-cell hyperplasia (n = 5) at ages 2 to 47 years (mean, 15.6 years), and the remaining five patients died in childhood of complications related to the aganglionosis. In retrospect, Hirschsprung's disease was the presenting feature of MEN 2a in five of the six families rather than MTC or pheochromocytoma. In all six MEN 2a families expressing Hirschsprung's disease, the RET mutation predisposing to the combined phenotype occurred in exon 10 at codons 609 (n=2), 618 (n=3), or 620 (n = 1). By contrast, the MEN 2a with Hirschsprung's phenotype was not found in any of the 22 families with a RETexon 11, 13, or 14 mutation (P=.0007). CONCLUSIONS: The authors conclude that Hirschsprung's disease is a phenotypic marker for MEN 2a and possibly more common than originally appreciated. The expression of Hirschsprung's disease with MEN 2a may be uniquely linked to RETexon 10 mutations. The authors recommend that (1) patients affected with MEN 2a may be counseled regarding the potential risk of Hirschsprung's disease in offspring and (2) a family history of MTC be explored in children with familial Hirschsprung's disease and genetic screening for MEN 2a be considered.

Hirschsprung disease in MEN 2A: increased spectrum of RET exon 10 genotypes and strong genotype-phenotype correlation.

The RET proto-oncogene encodes a transmembrane receptor with tyrosine kinase activity. Germline mutations in RET are responsible for a number of inherited diseases. These include the dominantly inherited cancer syndromes multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC), as well as some cases of familial Hirschsprung disease (HSCR1). RET mutations in HSCR1 have been shown to cause a loss of RET function, while the cancer syndromes result in RET oncogenic activation. Occasionally MEN 2A or FMTC occurs in association with HSCR1, albeit with low penetrance. An initial report linked HSCR1 in MEN 2A solely to the C618R and C620R RET mutations. In this study we have analyzed 44 families with MEN 2A. HSCR1 co-segregated with MEN 2A in seven (16%) of the 44 families. The predisposing RET mutation in all seven families had been previously reported in MEN 2A or FMTC and occurred in exon 10 at codons 609, 618 or 620, resulting in C609Y, C618S, C620R or C620W substitution. MEN 2A families with RET exon 10 Cys mutations had a substantially greater risk of developing HSCR1 than those with the more common RET exon 11 Cys634 or exon 14 c804 mutations (P = 0.0005). These findings suggest that expression of HSCR1 in MEN 2A may be peculiar to RET exon 10 Cys mutations . However, HSCR1 in MEN 2A is not exclusive to C618R or C620R RET mutations and can occur with other exon 10 Cys amino acid substitutions. The strong correlation between disease phenotype and position of the MEN 2A RET mutation suggests that oncogenic activation of RET alone is insufficient to account for co-expression of the diseases.

Prophylactic surgery for multiple endocrine neoplasia type IIa after genetic diagnosis: is parathyroid transplantation indicated?

Identification of germline mutations in the RET proto-oncogene predisposing to multiple endocrine neoplasia type IIa (MEN-IIa) has allowed a DNA-based approach to diagnosis and treatment by prophylactic thyroidectomy in children testing genetically positive. Although total thyroidectomy is the accepted operation for C cell disease, the necessity of routine total parathyroidectomy and autotransplantation as previously described in these asymptomatic children is questionable, particularly given the low occurrence of hyperparathyroidism in MEN-IIa (10-20%). Thirty-six children (ages 1 month to 12 years) from four MEN-IIa kindreds at risk for disease underwent genetic testing. Mutational analysis was done using a highly sensitive PCR-based denaturing gradient gel electrophoresis technique. Parathyroid or serum calcium concentrations were determined preoperatively. Of the 36 children at risk, 18 were found to have a MEN-IIa mutation; 11 have undergone prophylactic thyroidectomy at ages ranging from 2 to 12 years (mean 7.5 years). In each case, there was no biochemical evidence of hypercalcemia preoperatively, and all parathyroid glands were identified and were found to be grossly normal at exploration. Glands were carefully dissected and left in situ. Postoperatively, 10 of the 11 children maintained normocalcemia, allowing discharge within 24 to 36 hours. Resected thyroid glands contained C cell hyperplasia in nine, medullary carcinoma in one, and normal histology in one. We conclude that an alternative to routine parathyroidectomy may be desirable for prophylactic treatment of MEN-IIa. In situ parathyroid preservation can be safely achieved without compromising the completeness of the thyroid resection. This conservative approach obviates the potential morbidity associated with total parathyroidectomy and autotransplantation.

Oncogenic RET receptors display different autophosphorylation sites and substrate binding specificities.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in neural crest as well as kidney development. Genetic studies have demonstrated that germ line mutations in the ret oncogene are the direct cause of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease. However, despite the large body of genetic and biological evidence suggesting the importance of RET in development and neoplastic processes, the signal transduction mechanisms of RET remain unknown. To begin to understand the molecular mechanisms of the disease states caused by mutations in RET, the patterns of autophosphorylation of the wild-type RET and the MEN mutants were studied using site-directed mutagenesis and phosphopeptide mapping. Among the 6 autophosphorylation sites found in the wild-type RET receptor, the MEN2B mutant lacked phosphorylation at Tyr-1096, leading to decreased Grb2 binding, while simultaneously creating a new phosphorylation site. These changes in autophosphorylation suggest that the MEN2B mutation may result in the more aggressive MEN2B phenotype by altering the receptor's signaling capabilities.

Detection of RET mutations in multiple endocrine neoplasia type 2a and familial medullary thyroid carcinoma by denaturing gradient gel electrophoresis.

Germline missense mutations within the coding region of the RET proto-oncogene have recently been described in patients with the dominantly inherited cancer syndromes, multiple endocrine neoplasia type 2a (MEN 2a) and familial medullary thyroid carcinoma (FMTC). To date, the sequence variations occur in RET exons 10 and 11 and alter highly conserved cysteine residues in the proposed extracellular domain at codons 609, 611, 618, 620, and 634. To expedite rapid screening of populations at risk of MEN 2a or FMTC, we developed a PCR-based denaturing gradient gel electrophoresis (DGGE) strategy that detects polymorphisms occurring at all five Cys codons in both RET exons using identical gel conditions. In this report, the screening results from DGGE analysis of 15 distinct MEN 2a and FMTC mutations are shown. Each mutation generated a clearly distinguishable and unique homo- and heteroduplex band pattern. Given the highly efficient, reproducible, and sensitive nature of this approach, DGGE is particularly appropriate for rapid, large-scale screening of patients. Since prior knowledge of the RET mutation is unnecessary for analysis, DGGE is potentially valuable for distinguishing germline from seemingly sporadic medullary thyroid cancer as well as identifying novel sequence changes.

Progress in genetic screening of multiple endocrine neoplasia type 2A: is calcitonin testing obsolete?

BACKGROUND: Recent identification of RET mutations in multiple endocrine neoplasia type 2A (MEN 2A) allows a DNA-based approach to diagnosis in lieu of calcitonin sampling. To prospectively evaluate the efficacy of mutational analysis, genetic screening was performed in 124 patients (53 male, 71 female; age, 1 month to 80 years) at risk for MEN 2A referred over 3 months. METHODS: Analysis used genomic DNA and a polymerase chain reaction-based denaturing gradient gel electrophoresis strategy for mutation detection at RET codons 609, 611, 618, 620, and 634. Ninety-three of 124 patients were from established MEN 2A kindreds (group A), and screening replaced calcitonin testing. Twenty-one of 124 patients (group B) represented index cases of medullary thyroid carcinoma (MTC), and DNA analysis was performed to distinguish sporadic from hereditary disease. Ten patients (group C) had modest calcitonin elevations or had undergone thyroidectomy without confirming pathologic results, and testing was undertaken to clarify status. RESULTS: Group A: RET mutations occurred in 29 (median age, 10 years) of 93 patients, 14 of whom underwent thyroidectomy. No false-positive results were observed. Group B: five (24%) of 21 patients with seemingly sporadic MTC had RET mutations at codons 618 (one), 620 (one), or 634 (three). Group C: Nine of 10 patients with alleged MEN 2A had genetically negative results. CONCLUSIONS: Denaturing gradient gel electrophoresis reliably detects MEN 2A. Modest calcitonin elevations may lead to a false-positive diagnosis of MTC. DNA testing is the optimal approach to evaluating MEN 2A. Index cases of sporadic MTC should also undergo DNA analysis.

Mutational analysis of multiple endocrine neoplasia type 2A associated with Hirschsprung's disease.

BACKGROUND: The clinical association of multiple endocrine neoplasia type 2A (MEN 2A) and Hirschsprung's disease (HD), although rare, has been previously observed. Recently, germline mutations in the RET proto-oncogene, a transmembrane receptor with tyrosine kinase activity, have been detected in patients with familial HD. RET is also the predisposition gene for the inherited cancer syndrome MEN 2A. METHODS: We describe a DNA sequence variation within the coding region of RET in two large unrelated kindreds with MEN 2A (with 83 and 42 persons affected) in which HD cosegregated with MEN 2A in seven patients. Mutational analysis was performed with a highly sensitive polymerase chain reaction-based denaturing gradient gel electrophoresis technique followed by direct sequencing of mutants. RESULTS: Genetic analysis by denaturing gradient gel electrophoresis detected mutant bands in RET exon 10 in patients with MEN 2A from both kindreds. Direct DNA sequencing of mutants revealed a thymine-to-adenine base change in codon 618, resulting in a cysteine-to-serine substitution. The identical mutation was present in all seven children with HD. Of these children five underwent thyroidectomy for C-cell abnormalities; one 3-year-old child is awaiting thyroid surgery, and the remaining patient died at age of 12 weeks. CONCLUSIONS: The RET codon 618 Ser mutation could predispose patients with MEN 2A to HD. RET may assume a critical role in embryologic enteric nerve migration and tumorigenesis of cells from neural crest lineage.

Genetic aspects of multiple endocrine neoplasia types 1 and 2.

Multiple endocrine neoplasia (MEN) type 1 is an autosomal, dominantly inherited predisposition to develop neoplastic lesions of the parathyroid glands, the neuroendocrine pancreas-duodenum, and the anterior pituitary. The genetic defect was mapped to the centromeric part of the long arm of chromosome 11 based on studies of somatic deletions in MEN-1-associated tumors and linkage analysis in families in whom the disease is segregated. Combined family and tumor analysis has shown that tumorigenesis in MEN-1 involves loss of the wild-type chromosome, indicating that the putative MEN-1 gene is a tumor suppressor gene. Similar deletions are also seen in a proportion of sporadic parathyroid and pancreatic tumors, suggesting that tumorigenesis involves related mechanisms in both sporadic and familial cases. Based on results from linkage analysis in more than 40 MEN-1 families, predictive testing for MEN-1 using DNA polymorphisms can now be performed with high accuracy. Hence, biochemical screening programs can focus on individuals at risk to identify early signs of tumor development. MEN-2, an autosomal dominant cancer syndrome of variable expressivity, has previously been localized to chromosome 10q11.2 by positional cloning tactics. The RET protooncogene mapping to the MEN2 susceptibility locus has recently emerged as a candidate gene for MEN-2A. RET, a transmembrane receptor protein, has a large glycosylated extracellular domain containing clustered cysteine residues and calcium-binding motifs, a single hydrophobic transmembrane domain, and a cytoplasmic domain with tyrosine kinase catalytic activity. Several germline missense mutations in a codon specifying one of these highly conserved cysteine residues have been detected in patients affected with MEN-2A.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of papillary thyroid carcinoma in multiple endocrine neoplasia type 2A.

BACKGROUND: The ret protooncogene (RET), shown to be rearranged in human papillary thyroid cancers (PTC), has been mapped by in situ hybridization to 10q11.2 near the predisposition locus for the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). To date PTC has not been an observed characteristic of MEN 2; however, linkage studies in affected families have shown no meiotic recombinants between the MEN 2A gene and RET suggesting tight linkage between loci. Furthermore, RET appears to be expressed in medullary thyroid carcinoma (MTC) and pheochromocytoma and for these reasons has emerged as a candidate gene for MEN 2. METHODS: Two patients from a single kindred with MEN 2A (18 affected) are presented in which expression of PTC appeared to cosegregate with the MEN2 gene. In both patients the diagnosis of occult C-cell disease was suspected by an elevation in the basal and pentagastrin-stimulated peak calcitonin levels. Histologic examination of the thyroid gland after operation for MTC revealed tumor nodules consistent with PTC. There was no history of radiation exposure. Characteristics of MEN 2A syndrome in the kindred in addition to MTC and PTC include hyperparathyroidism and Hirschsprung's disease in three and two patients, respectively. RESULTS: Two-point linkage analysis with a new highly polymorphic DNA marker, LGfd01, derived from a cosmid clone mapping to 10q11.2 assigns the MEN 2 predisposition locus in this kindred to chromosome 10q11.2 (0 = 0.00; maximum LOD, 4.78). Recombination between MEN 2A and a polymorphic microsatellite from the RET locus could not be shown among informative meioses. CONCLUSIONS: The observed association of MEN 2A and PTC is intriguing and suggests that the variation in expression of the syndrome may be due to the presence of a structural alteration affecting several contiguous genes spanning the putative MEN 2 region.

Molecular genetics of APUDomas.

It is tempting to speculate that genetic studies of the human APUDomas, particularly those of a hereditary nature, may ultimately provide valuable clues to the molecular basis of malignant transformation in cells of all types, uncover the mechanisms responsible for tumor progression, and perhaps decipher the signals important in the differentiation of normal neural crest-derived tissue. Generally, several strategies have been used in the genetic analysis of these tumors with success. These include (1) cytogenetic examination of recurring chromosomal abnormalities in hopes of pinpointing critical neighboring growth regulatory sequences important in tumor evolution, (2) identification of dominant acting oncogenes in tumor cells, (3) search for recessive inactivated suppressor genes that may regulate cell growth by analyzing tumors for loss of heterozygosity (LOH), and (4) genetic linkage studies of kindreds affected with familial APUDomas to identify and subsequently characterize the predisposition gene using a positional or functional cloning approach. The results of these strategies as they have been employed in the investigation of cutaneous malignant melanoma (CMM), the dysplastic nevus syndrome (DNS), and the multiple endocrine neoplasia (MEN) syndromes are summarized.

Long-term follow-up of a large North American kindred with multiple endocrine neoplasia type 2A.

BACKGROUND: Sixty-one patients with multiple endocrine neoplasia (MEN) type 2A (27 men; 34 women; mean age, 39 years) from a single kindred (76 affected, 49 at risk) were followed 1 month to 33 years (median, 14 years) after diagnosis for disease expression. METHODS: Peripheral basal and pentagastrin-stimulated calcitonin, parathyroid hormone, and calcium levels were measured in 60 patients, 58 of whom had undergone previous thyroidectomy; the calcitonin concentration in four patients was concomitantly determined in the hepatic and internal jugular veins. Biochemical or radiographic screening for pheochromocytoma was performed in 58 patients. RESULTS: Recurrence of medullary thyroid carcinoma (MTC) developed in nineteen (33%) of 58 patients after they underwent thyroidectomies. In 14 of 19 patients regional metastases were inapparent at initial operation, and lymphadenectomy was not undertaken. Three patients underwent neck reexploration with removal of micrometastases after selective venous studies were performed. One patient, 33 years of age, died of MTC, and two patients who refused thyroidectomy are alive at 76 and 83 years of age. Fifteen patients are alive with disease 8 to 33 years after they underwent thyroidectomies. All patients identified by prospective screening remain pentagastrin negative. Pheochromocytoma developed in six patients (10%), and four patients have Hirschsprung's disease. Hyperparathyroidism was present in seven patients and did not occur in those with minimal MTC. CONCLUSIONS: These observations suggest that (1) the course of MTC in MEN 2A is highly variable, (2) early treatment of C-cell disease can be curative, (3) routine lymphadenectomy for occult micrometastases may be necessary for cure of MTC, and (4) the hyperparathyroidism of MEN 2A may not be a primary genetic event.

Linkage mapping of human chromosome 10 microsatellite polymorphisms.

Ten microsatellite DNA polymorphisms located on human chromosome 10 were regionally mapped using subchromosomal somatic cell hybrids and linkage analysis. The resulting order of the markers from pter-qter was [D10S89, D10S111], D10S107, D10S109, [D10S91, D10S110, D10S108, D10S88, D10S168], and D10S169. Order of the markers within brackets was uncertain, although the order given was most likely. The microsatellites were distributed along the chromosome from the proximal p arm to near qter, with an unlinked gap between D10S168 and D10S169.

Eastern Cooperative Oncology Group study 1879: mitotane and adriamycin in patients with advanced adrenocortical carcinoma.

From November 1979 to July 1986, 52 patients (27 women and 25 men; median age 52 years) with advanced adrenocortical carcinoma entered a prospective, nonrandomized study evaluating moderate-dose mitotane and doxorubicin hydrochloride (Adriamycin). Thirty-two tumors (62%) were well differentiated and evidence of hormone production was present in 24 patients (46%). Patients with well-differentiated or functional tumors received mitotane, 6 gm daily; patients for whom mitotane failed or those with poorly differentiated, non-hormone-producing tumors received Adriamycin, 60 mg/m2 every 3 weeks. Initially, 36 patients were treated with mitotane and 16 patients with Adriamycin. Eight patients (22%) responded to mitotane and three (19%) responded to Adriamycin. No response was noted in the 15 patients for whom mitotane failed and who then received Adriamycin. Severe toxicity occurred in 36% of patients who received mitotane and in 26% who received Adriamycin. Overall median survival after onset of treatment was 14 months. We conclude that mitotane or Adriamycin used initially can induce tumor regression in about 22% and 19% of selected patients, respectively. However, Adriamycin is ineffective as second-line chemotherapy for patients with well-differentiated or functioning tumors for whom mitotane is ineffective.

Adrenocortical carcinoma.

Ten patients, seven women and three men, ages 47 to 76 years (mean 58.6 years), treated for adrenal cortical carcinoma between 1971 and 1989, were reviewed. Three (30%) of the tumors were nonfunctioning. The remaining seven (70%) were functioning, six of them occurring in women. Common presenting features were hormonal excess, distant metastases, weight loss, and abdominal pain. The primary tumor was resected in all patients, only two of whom had disease confined to the adrenal gland. Tumor diameter ranged from 9 to 21 cm (mean 15.7 cm). Inferior vena caval or right atrial extension of tumor thrombus was present in two patients. Excluding two deaths from postoperative complications, seven patients died of their disease after a mean survival of 25 months (range 2 to 84 months). Of seven patients who received o,p'-DDD treatment for metastatic or recurrent tumor, three (43%) had an objective response. In two patients, tumor regression was complete and was associated with prolonged survival. The first patient underwent resection of recurrent tumor on two occasions in addition to receiving o,p'-DDD and survived 84 months. The second patient had complete regression of pulmonary and liver metastases confirmed at laparotomy and thoracotomy and remains free of disease at 78 months. None of the five patients treated with various combinations of cytotoxic chemotherapy had an observable response, and no measurable effect was seen in a single patient following abdominal radiotherapy. It is concluded that resection for local recurrence may prolong survival and that significant and lasting tumor regression is possible with o,p'-DDD administration. Beneficial results from cytotoxic agents, however, could not be demonstrated.