Germline mutations in the multiple endocrine neoplasia type 1 gene: evidence for frequent splicing defects.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial cancer syndrome characterized by parathyroid hyperplasia, pituitary adenomas, and neuroendocrine tumors of the pancreas and duodenum. In 1997, the MEN1 tumor suppressor gene was identified, and numerous germline mutations have been reported to be distributed throughout the gene. We used single strand conformational variant (SSCV) analysis to search for germline mutations in the members of 33 kindreds with a confirmed diagnosis of MEN 1. SSCV analysis revealed 25 conformational variants representing germline mutations that are predicted to result in loss of normal menin function. Twenty different disease-associated mutations were identified: five resulting in potential abnormal RNA splicing, two missense mutations, seven nonsense mutations, and six frameshift mutations. The aberrant splice products were identified and confirmed by RT-PCR and direct sequence analysis for two of the five splice mutations. Sixteen of the 20 (80%) mutations identified have not been previously reported. Mutations were not identified in eight kindreds with signs and symptoms consistent with MEN 1. The SSCV analysis revealed mutations in 76% (25 of 33) of the kindreds investigated, thus showing SSCV analysis to be a reliable mutation detection strategy. One-fifth of the mutations identified in this study involve intron sequences, therefore, highlighting the importance of including intron sequences in the search for germline mutations in the MEN1 gene. The need to investigate the entire gene when characterizing new MEN 1 families presents challenges in the translation of genetic studies to efficient clinical diagnostic tests.

Surgical management of patients with persistent or recurrent medullary thyroid cancer.

Residual or recurrent medullary thyroid carcinoma (MTC), manifested by elevated calcitonin levels, occurs commonly following primary treatment of MTC. Re-operation in appropriately selected patients is the only treatment modality which consistently and reliably reduces stimulated calcitonin levels, and results in excellent local disease control. We report improved results of surgical management of recurrent MTC in two consecutive series of patients. In our most recent series (1992-96), 38% of patients (17 out of 45) had normal postoperative stimulated calcitonin levels, compared to 28% (nine of 32) in our first series (1990-92). In the most recent series, only 13% (six of 45) of patients had no decrease in calcitonin levels following re-operation, compared to 31% (10 of 32) in our first series (P = 0.07, Fisher's exact test). This improvement has mainly occurred through better preoperative selection of patients, and the institution of routine laparoscopic liver examination preoperatively, which identified metastases in 10 patients, nine of whom had normal CT or MRI imaging.

Improved results of cervical reoperation for medullary thyroid carcinoma.

OBJECTIVE: The purpose of the study is to determine whether reoperation for medullary thyroid carcinoma (MTC), performed with low morbidity in carefully selected patients, consistently results in improvement as determined by lowering of stimulated calcitonin levels. BACKGROUND: Persistent or recurrent elevation of stimulated plasma calcitonin levels occurs in > 50% of patients after primary operation for MTC. Success of reoperation with clearance of metastatic cervical nodal disease has been hampered by failure to identify patients with distant metastases and by inadequate removal of involved nodal groups. METHODS: Since 1992, the authors have evaluated 115 patients with recurrent or residual MTC. Fifty-three patients have not undergone operation because of extent of disease, previous extensive treatment, medical condition, or patient choice. Sixty-two patients underwent surgery. Ten patients had laparoscopic or open examination of the liver, the results of which showed liver metastases. Seven patients had palliative debulking of cervical tumor. In 45 patients without evidence of distant metastases, cervical operation was carried out with curative intent. Removal of central, upper mediastinal, and lateral nodes (levels II, III, IV, VI, and VII) was done. RESULTS: Seven of eight patients who had palliative resections are alive without symptoms. In patients who underwent curative resections, postoperative stimulated calcitonin levels were in the normal range in 17 patients (38%) and were not significantly lowered in 6 patients (13%). There were no deaths, and no transfusions were used. CONCLUSIONS: These results are a significant improvement over the authors' previous series and reflect better preoperative identification of patients with disease confined to the neck and improved operative strategy based on knowledge of the pattern of nodal spread of MTC.

Progression of postoperative residual medullary thyroid carcinoma as monitored by plasma calcitonin levels.

BACKGROUND: Patients operated on for medullary thyroid carcinoma (MTC) frequently have persistent elevated plasma calcitonin concentrations after operation, indicating remaining tumor. The plasma calcitonin concentration in a patient with MTC roughly reflects the endogenous tumor burden. The only effective treatment for MTC is surgical. The decision about whether a patient with persistent MTC should have a repeat operation would be influenced by knowledge of the natural course of the disease. METHODS: Forty patients with persistently elevated peak plasma calcitonin concentrations after thyroidectomy for MTC were monitored for a mean of 6 years. Serial determinations of plasma calcitonin levels were obtained before and after intravenous injection of calcium and pentagastrin. RESULTS: At the first postoperative test 63% of the patients had undetectable basal calcitonin values, although their stimulated plasma calcitonin concentrations were elevated. The mean annual increase in stimulated plasma calcitonin concentrations was 117%, but plasma calcitonin concentrations were stable in three patients and decreased in one patient. Five patients are known to have experienced distant metastases. CONCLUSIONS: MTC is a progressive disease in most patients with persistent hypercalcitoninemia after thyroidectomy. Stimulated peak plasma calcitonin levels are more meaningful than basal levels in the serial postoperative evaluation of patients with persistent hypercalcitoninemia after thyroidectomy for MTC.

Predictive DNA testing and prophylactic thyroidectomy in patients at risk for multiple endocrine neoplasia type 2A.

BACKGROUND: Missense germ-line mutations in the RET protooncogene are associated with multiple endocrine neoplasia type 2A (MEN 2A). Detection of these mutant alleles in kindred members predicts disease inheritance and provides the basis for preventative thyroidectomy. METHODS: A polymerase chain reaction (PCR)-based genetic test for the 19 known RET mutations was designed to study 132 members of 7 kindreds with MEN 2A. Haplotypes also were constructed using genetic markers flanking the MEN 2A locus. Plasma calcitonin (CT) concentrations were determined before and after provocative testing. RESULTS: Direct DNA testing and haplotype analysis showed that 21 of 58 kindred members at risk for disease had inherited a mutation in the RET protooncogene associated with MEN 2A. Plasma CT concentrations were elevated in 9 of the 21 family members, but were normal in 12. After genetic counseling, 13 of the 21 kindred members (6 with normal and seven with elevated plasma CT levels), consented to immediate thyroidectomy. In each patient, the resected thyroid gland showed C-cell hyperplasia with or without medullary thyroid carcinoma. There were no metastases to regional lymph nodes, and postoperative stimulated plasma CT levels were normal. CONCLUSION: The PCR-based direct DNA test for RET mutations is accurate, rapid, and reproducible. For all 132 individuals evaluated, the results of direct DNA analysis were consistent with haplotype studies. The direct test for mutations in the RET protooncogene is the preferred method for screening MEN 2A kindreds. In family members who have inherited a RET mutation, total thyroidectomy is indicated, regardless of the plasma CT values.

Reoperation for recurrent or persistent medullary thyroid cancer.

BACKGROUND: Initial operations for medullary thyroid cancer (MTC) frequently do not eradicate all disease, as evidenced by persistently elevated levels of stimulated plasma calcitonin. METHODS: Thirty-two patients with MTC and elevated stimulated plasma calcitonin levels after thyroidectomy were studied between 1990 and 1993. Thirty-five repeat neck explorations and microdissections were performed. Four patients also underwent a median sternotomy and mediastinal dissection. RESULTS: In nine patients (group 1), stimulated plasma calcitonin levels were undetectable after reoperation, whereas in 13 cases (group 2) the calcitonin levels decreased by 40% or more. In 10 cases (group 3) the CT levels did not decrease. Primary tumors that exhibited invasive features (invasion of adjacent structures or extranodal or extracapsular spread) were found more often in patients from group 3 than in patients from groups 1 or 2 (p < 0.05, Fisher's exact test). CONCLUSIONS: Reoperation resulted in normalization of calcitonin levels in 28% of patients and a decrease in calcitonin levels by 40% or more in another 42% of patients. The data also suggest that patients whose primary MTC has invaded tissues beyond the thyroid gland or a lymph node capsule are less likely to benefit from repeat operation.

Presymptomatic identification of carriers of the multiple endocrine neoplasia type 2A gene using flanking DNA markers.

BACKGROUND: Because the predisposition locus for multiple endocrine neoplasia type 2A (MEN2A) has been mapped to chromosome 10 by genetic linkage analysis, it has become possible to identify gene carriers by following the transmission of linked genetic markers from affected parents to offspring at risk for MEN2A. We have applied a highly accurate genetic test to presymptomatic diagnosis of gene carriers in several large kindreds with MEN2A. METHODS: DNA was extracted from 300 individuals in six kindreds with MEN2A and used for genotyping studies with DNA markers flanking the MEN2A locus. Genotype data were used to predict the inheritance of the MEN2A gene in kindred members at risk according to previously calculated map distances and the program LINKAGE: RESULTS: Ninety-five percent of individuals were informative with markers flanking the MEN2A locus. Of 130 patients at risk, 26 (20%) were predicted to be MEN2A gene carriers, 100% (77%) were noncarriers, and 4 (3%) were recombinant and their gene carrier status could not be determined. Gene carrier prediction probabilities were calculated at greater than 98% in 94% of these patients. CONCLUSIONS: We conclude that genetic testing with flanking DNA markers is a highly accurate method for the presymptomatic identification of MEN2A gene carriers and allows for diagnosis at an earlier stage than does traditional calcitonin testing.

Secretion of breast gross cystic disease fluid proteins by T47D breast cancer cells in culture--modulation by steroid hormones.

The effect of steroid hormones on modulating the secretion rates of three human breast gross cystic disease fluid proteins (GCDFP-15, GCDFP-24, and GCDFP-44) by T47D breast carcinoma cells in tissue culture was evaluated. Androgens (dihydrotestosterone or fluoxymesterone) were capable of stimulating the secretion rates for all three GCDFP's while showing a minimal trend toward slowing the growth rate of T47D cells. This is the first study which shows that androgens can specifically stimulate all three of the major breast GCDFP's concomitantly. Progesterone, and three synthetic progestins, all showed inhibition of the growth rate of T47D cells while causing enhancement of the secretion of GCDFP-15 and GCDFP-44, and only minimal effect on the secretion rate of GCDFP-24. Estradiol was essentially neutral to the growth rate of the T47D cells in our test system. Estradiol did cause a mild enhancement of GCDFP-44 secretion rate, with no appreciable effect on GCDFP-15 or GCDFP-24 secretion rates. These findings suggest that an androgenic stimulus may be involved in the secretion of GCDFP's associated with breast gross cystic disease.

Familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2B map to the same region of chromosome 10 as multiple endocrine neoplasia type 2A.

Medullary thyroid carcinoma (MTC) occurs as a component of three well-described autosomal dominant familial cancer syndromes. Multiple endocrine neoplasia type 2A (MEN 2A) is characterized by MTC, pheochromocytomas, and parathyroid hyperplasia. Patients with the rarer multiple endocrine neoplasia type 2B (MEN 2B) syndrome develop MTC and pheochromocytomas, as well as mucosal neuromas, ganglioneuromatosis of the gastrointestinal tract, and a characteristic "marfanoid" habitus. Finally, MTC is transmitted in an autosomal dominant pattern in some families without associated pheochromocytomas or parathyroid hyperplasia (familial medullary thyroid carcinoma, MTC1(2). Sixty-one members of two well-characterized kindreds segregating MTC1 and 34 [corrected] members of six families segregating MEN2B were genotyped using a panel of RFLP probes from the pericentromeric region of chromosome 10 near a locus for MEN 2A. Statistically significant linkage was observed between the chromosome 10 centromere-specific marker D10Z1 and MTC1 (maximum pairwise lod score 5.88 with 0% recombination) and D10Z1 and MEN2B (maximum pairwise lod score 3.58 with 0% recombination). A maximum multipoint lod score of 4.08 was obtained for MEN2B at the position of D10Z1. In addition, 92 members of a previously unreported large MEN2A kindred were genotyped, and linkage to the pericentromeric region of chromosome 10 is reported (maximum pairwise lod score of 11.33 with 0% recombination between MEN2A and RBP3). These results demonstrate that both a locus for familial MTC and a locus for MEN 2B map to the pericentromeric region of chromosome 10, in the same region as a locus for MEN 2A. The finding that each of these three clinically distinct familial cancer syndromes maps to the same chromosomal region suggests that all are allelic mutations at the same locus or represent a cluster of genes involved in the regulation of neuroendocrine tissue development.

Immunologic and steroid binding properties of the GCDFP-24 protein isolated from human breast gross cystic disease fluid.

A major protein of human breast cyst fluid, termed GCDFP-24, has the property of specifically binding progestins. The purified glycoprotein, of 24,000 apparent molecular weight, bound pregnenolone and progesterone with highest affinity. The association constant for binding of progesterone was 1 X 10(6)L/mol by Scatchard analysis, and there was one binding site per molecule. Changes to the progesterone structure at C-17, C-20, or C-21 interfered with binding. The pH optimum for binding was 4-4.5. The purified protein was highly stable and was not irreversibly denatured by 50% methanol or 3M guanidine. However, dithiothreitol reversibly interfered with progesterone binding. Rabbit antiserum produced against the glycoprotein recognized an immunologically identical component in normal human sera, and partially cross-reacting components in normal monkey and baboon sera. The component in human sera was present in Cohn fractions IV and VI.

Human parathyroid antigen: characterization and localization with monoclonal antibodies.

A cell surface antigen on human parathyroid cells was identified by monoclonal antibodies. The antigen, called parathyroid antigen (PTA), is found on both of two major polypeptides (190 kDa and 160 kDa) apparently associated exclusively with parathyroid cells. To determine whether PTA was a suitable target for in vivo imaging, 125I-labeled anti-PTA was injected into nude mice bearing human parathyroid xenografts. IgG1 anti-PTA antibody showed excellent radiolocalization of the grafts, whereas IgM anti-PTA, containing the same variable domains as the IgG1 antibody, showed little specific binding. These results suggest that antibody isotype is an important parameter for the in vivo immunoscintigraphy of specific cellular antigens.

Familial medullary thyroid carcinoma without associated endocrinopathies: a distinct clinical entity.

In an evaluation of 213 patients from 15 kindreds with familial medullary thyroid carcinoma (MTC), we detected 41 subjects from two kindreds (L and O) who had MTC but no extra-thyroidal manifestations (hyperparathyroidism, phaeochromocytomas or mucosal neuromas) of multiple endocrine neoplasia (MEN) type IIa or IIb. In screening 178 members of the L and O kindreds, we found no evidence that any of them had died from MTC. To assess whether the malignancy was relatively indolent in these families, 20 selected subjects from the two kindreds were compared with 33 MEN IIa subjects. Both groups had clinically occult disease which was diagnosed biochemically by documenting elevated plasma calcitonin (CT) levels following stimulation with intravenous calcium and pentagastrin. There were no differences in the peak stimulated plasma CT levels at the time of diagnosis (1055 +/- 236 pg/ml versus 1096 +/- 191 pg/ml) or the incidence of regional lymph node metastases (0/20 versus 1/33) in the two groups. The mean age at diagnosis, however, was significantly higher in patients of the L and O kindreds than in patients with MEN IIa (43.1 +/- 3.4 years versus 21.1 +/- 2.2 years; P less than 0.001) indicating that in the two kindreds the MTC either developed at a later age or grew more slowly. This study demonstrates that MTC may occur in a familial pattern distinct from its presentation as MEN IIa or MEN IIb. In this setting it appears to be the least aggressive form of MTC yet described.

Stimulation of insulin secretion by a rapid intravenous calcium infusion in patients with beta-cell neoplasms of the pancreas.

The effects of calcium on fasting plasma insulin and glucose levels were compared in 16 normal subjects and 11 patients with beta-cell neoplasms of the pancreas. Calcium was administered iv either as a rapid calcium infusion (RCI; 2 mg/kg in 1 min) or as a long calcium infusion (LCI; 12 mg/kg in 3 h). In normal subjects, the RCI produced a rise in mean plasma insulin from 11 +/- 1 (+/- SEM) microU/ml basally to a peak of 18 +/- 2 microU/ml (P less than 0.001). No consistent pattern of change in insulin levels occurred during the LCI, and plasma glucose levels did not change significantly with either test. In the patients with beta-cell neoplasms, the RCI resulted in a rapid increase in mean plasma insulin from 36 +/- 6 microU/ml to a peak level of 312 +/- 67 microU/ml (P less than 0.002). With the LCI, a more gradual rise in insulin from 35 +/- 11 to 92 +/- 36 microU/ml occurred (P less than 0.002). The mean increase in insulin in the patients with beta-cell neoplasms was significantly greater for the RCI than for the LCI (P less than 0.01). Pronounced increments in plasma insulin occurred in all 11 patients after the RCI, but in only 3 of 8 patients during the LCI. Plasma glucose levels declined significantly from 69 +/- 7 to 56 +/- 8 mg/dl during the RCI (P less than 0.05) and from 69 +/- 8 to 49 +/- 7 mg/dl during the LCI (P less than 0.005). Symptomatic hypoglycemia developed in 3 patients during the LCI but did not occur after the RCI. These data indicate that calcium is a more effective insulin secretagogue in patients with beta-cell neoplasms when administered as an RCI than as an LCI, and suggest that the RCI may be a useful test for the diagnosis of insulin-secreting tumors.

Fluoxymesterone stimulation of tumor marker secretion in patients with breast carcinoma.

The gross cystic disease fluid protein of 15,000 MW (GCDFP-15) has been demonstrated to be a circulating glycoprotein tumor marker for breast carcinoma in approximately 40% of patients with advanced disease. A recent retrospective analysis of plasma GCDFP-15 levels in patients with advanced breast cancer suggested that androgen therapy could cause significant increases in plasma levels in the absence of disease progression. In order to evaluate the frequency, time course, and intensity of the androgen effect on GCDFP-15 production, a prospective study was initiated. Twenty-nine patients with stage IV breast carcinoma were treated with fluoxymesterone (20 or 30 mg/d). Plasma levels of GCDFP-15 and carcinoembryonic antigen (CEA) were measured by radioimmunoassay before and at various times during therapy. By day 6 of therapy, plasma GCDFP-15 had increased significantly (p = 0.03) from a mean basal level of 58 +/- 12 ng/ml to 160 +/- 60 ng/ml. By contrast, the mean CEA levels in the same patients increased only from 36 +/- 14 ng/ml. The distribution of percent increases in plasma GCDFP-15 was not uniform, but patients with high (greater than 82 ng/ml) basal levels had marked (greater than or equal to 75%) increases in 6/6 (100%) cases, whereas patients with low (less than 30 ng/ml) basal levels had similar increases in only 2/15 (13%) cases. Urinary excretion of GCDFP-15 usually paralleled the increases in plasma levels of the glycoprotein during the first six days of therapy. A linear correlation between percent change in plasma and percent change in urinary GCDFP-15 was demonstrated. A permanent cell line of human breast carcinoma, T47-D, was stimulated to secrete GCDFP-15 in vitro by androgen, but not by estrogen. From these data, we conclude that androgens can specifically stimulate secretion of GCDFP-15 by breast carcinoma tissue in most patients with elevated plasma levels of GCDFP-15, and in some patients with normal levels. The stimulation occurs within days and is not associated with clinical signs of tumor growth.

Hyperparathyroidism associated with the enlargement of two or three parathyroid glands.

Eighty-five (23%) of 375 patients undergoing surgery for primary hyperparathyroidism were found to have enlargement (greater than 50 mg) of two or three parathyroid glands. Of 76 patients followed from 12 to 140 months after surgery, eight (10.5%) developed hypercalcemia at 1, 4, 45, 64, 74, 79, 84, and 133 months. In a comparison of pertinent preoperative biochemical and pathologic data between 55 patients with two- or three-gland hyperparathyroidism and 55 age- and sex-matched patients with single-gland hyperparathyroidism, only the preoperative serum phosphate differed significantly, being lower in the patients with single-gland disease (2.4 +/- 0.1 vs. 2.6 +/- 0.1; p less than 0.04). In the eight patients with two- or three-gland hyperparathyroidism who developed postoperative hypercalcemia, the preoperative concentrations of serum calcium were lower (10.8 +/- 0.2 vs. 11.5 +/- 0.2; p less than 0.019), the preoperative concentrations of serum phosphate were higher (3.1 +/- 0.2 vs. 2.5 +/- 0.1; p less than 0.020), and the weights of the excised parathyroid tissues were less (356 +/- 72 mg vs. 1354 +/- 215 mg; p less than 0.02) than those of patients with two- or three-gland disease who did not develop postoperative hypercalcemia, indicating a milder form of hyperparathyroidism. In the 68 patients without recurrent hypercalcemia, there was no tendency for the serum calcium concentration to increase with time. Patients with primary hyperparathyroidism associated with two or three enlarged parathyroid glands have an appreciable incidence of persistent or recurrent hypercalcemia, which may increase even further with longer observation.

Comparison of four provocative tests for the diagnosis of gastrinoma.

In an attempt to determine the best provocative test for the diagnosis of gastrinoma, ten normal subjects, 13 patients with known gastrinoma, and one patient with presumed gastrinoma were administered four regimens: (1) rapid calcium infusion (2 mg Ca++/kg/min), (2) secretin (2 clinical units (CU)/kg/bolus), (3) long calcium infusion (12 mg Ca++/kg/3 h) and (4) a combination test consisting of a rapid calcium infusion followed immediately by secretin. Blood was drawn for serum gastrin levels before and following infusion of the test agents. The administration of rapid calcium followed by secretin provoked the greatest increases in serum gastrin above basal levels in both normals (29%) and patients (362%). Peak gastrin levels in patients were similar following the long calcium infusion (341%) but were less following the rapid calcium infusion alone (124%) and secretin alone (207%). There were no false-positive or false-negative tests with the calcium plus secretin when the criterion for diagnosis was either a 50% increase or a 200 pg/ml increase above the basal gastrin level. The distinct advantages (short test period, low patient morbidity, and relatively great potency) of the calcium plus secretin test make it an attractive alternative to other previously described provocative tests for the diagnosis of gastrinoma.