William G. Dilley

Predictive DNA Testing and Prophylactic Thyroidectomy in Patients at Risk for Multiple Endocrine Neoplasia Type 2A

BackgroundMissense 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. MethodsA 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. ResultsDirect 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. ConclusionThe 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.

Long-term evaluation of patients with primary parathyroid hyperplasia managed by total parathyroidectomy and heterotopic autotransplantation.

Since 1973, we have performed total parathyroidectomy and forearm parathyroid autotransplantation in 36 patients with generalized (four gland) primary parathyroid hyperplasia. Twenty (56%) patients had nonfamilial parathyroid hyperplasia (NFPH) and 16 (44%) patients had familial parathyroid hyperplasia (FPH). Twenty-one patients (Group A) were undergoing operation for the first time and 15 (Group B) were having either second, third or fourth re-explorations for persistent hyperparathyroidism. All patients in Group A and nine patients in Group B had parathyroid resection and immediate autotransplantation as a single procedure. Six Group B patients had hyperfunctioning parathyroid tissue resected, cryopreserved, and subsequently grafted when it was evident that they had been rendered aparathyroid. A sustained differential elevation (13.7 fold ± 2.7) of parathyroid hormone was detected in the antecubital vein of the grafted compared to the nongrafted arm in 35 (97%) patients. Two (5.6%) of the 36 patients (both with FPH; one Group A and one Group B) required permanent oral calcium and vitamin D replacement therapy and one (3%) patient (NFPH: Group A) had persistent hypercalcemia postoperatively, presumably due to a supernumerary gland. The remaining 33 (92%) patients became normocalcemic after surgery and 23 (70%) of them remained so. Ten (30%) of the 33 patients developed recurrent graft dependent hyperparathyroidism. Eight patients were from the group with FPH (8/14, 57%) and two were from the group with NFPH (2/19, 11%) (FPH vs. NFPH, p < 0.005). Because of symptoms of hypercalcemia or a serum calcium concentration exceeding 11 mg/dl, partial graft resection was performed in five patients and four became normocalcemic. Patients with generalized primary parathyroid hyperplasia may be difficult to cure, especially if the disease is familial. The technique of total parathyroidectomy and heterotopic autotransplantation of fresh or cryopreserved parathyroid tissue offers distinct advantages over alternative techniques.

Medullary thyroid carcinoma: Relationship of method of diagnosis to pathologic staging

Medullary thyroid carcinoma (MTC) develops in virtually all patients affected with multiple endocrine neoplasia type II (MEN II), a disease inherited as an autosomal dominant trait. The thyroid tumor cells secrete calcitonin (CT) and the detection of elevated plasma levels (>300 pg/ml) of this hormone in MEN II kindred members strongly suggests the presence of MTC even though it may not be evident clinically. Intravenously administered calcium ion (Ca++) and pentagastrin (Pg) are potent CT secretagogues which are of particular value in establishing the early diagnosis of MTC. In evaluating seven kindreds with MEN II, we detected 90 patients with MTC. Depending on the method of diagnosis, they could be divided into three categories: Group 1; patients with no clinical evidence of MTC whose undetectable basal plasma calcitonin levels became elevated following intravenous Ca++ or Pg, Group II; patients with no clinical evidence of MTC who had elevated basal plasma CT levels, and Group III; patients with clinically evident MTC. At the time of diagnosis of MTC, the patients in Group I were younger (20.5 ± 1.9 years) than the patients in Group II (32.5 ± 4.7 years, p < 0.005) and Group III (34.3 ± 2.0, p < 0.00005). The incidence of residual MTC, as indicated by an elevated plasma CT level following provocative testing post operatively, was less frequent in patients diagnosed biochemically ([6/34]; Group I, 4/26 and Group II, 2/8) than in those diagnosed clinically (Group III, 15/26, p<0.002). Furthermore, regional nodes were involved less often in patients diagnosed biochemically ([5/28]; Group I, 2/22 and Group II, 3/6) than in those diagnosed clinically (Group III, 15/24, p < 0.02). Distant metastases were only evident in Group III patients. Patients with MEN II who had the diagnosis of MTC established biochemically rather than clinically, had a more favorable pathological stage of disease at the tune of thyroidectomy. This was especially true if the biochemical diagnosis had been by provocative testing.

The importance of early diagnosis in patients with hereditary medullary thyroid carcinoma.

Ninety-two patients from 12 kindreds with hereditary medullary thyroid carcinoma (MTC) were evaluated. We sought to determine if the stimulated plasma calcitonin (CT) level at the time of diagnosis was of prognostic significance. The patients were divided into four groups according to their preoperative stimulated plasma CT levels (1) 250–1,000 pg/ml (n = 25); (2) 1,000–5,000 pg/ml (n = 36); (3) 5,000–10,000 pg/ml (n = 8); (4) > 10,000 pg/ml (n = 23). Compared between the four groups were several parameters, including incidence of regional lymph node metastases, incidence of residual MTC post-thyroidectomy (as indicated by increased (>300 pg/ml) plasma CT levels after operation), incidence of distant metastases, and incidence of death. Also compared were the incidences of microscopic or gross MTC in thyroidectomy specimens. The incidence of regional lymph node involvement ranged from a minimum of one (4%) of 25 patients in Group 1 to 13 (57%) of 23 patients in Group 4. Similarly, plasma CT levels were elevated in only one (4%) of 25 patients in Group 1 compared to 14 (61%) of 23 patients in Group 4. There was no evidence of distant metastases or death in the patients in Groups 1, 2, or 3. In the 23 patients in Group 4, however, four (17.4%) had distant metastases and two (8.7%) died of disease during the period of observation. Of the 25 patients in Group 1, MTC was evident only by microscopic examination in 14 (56%). Eleven (44%) of the patients in Group 1 had macroscopically evident medullary thyroid carcinoma. This is in contrast with patients in Group 4 where all 23 had grossly evident MTC. These data indicate that the stimulated plasma CT level at the time of diagnosis is an excellent prognostic indicator of the extent of a disease in patients with hereditary MTC. Aggressive screening of kindred members at risk is of critical importance for establishing the diagnosis and instituting therapy at a time when the neoplasm is confined to the thyroid gland.

Clinical Genetic Testing and Early Surgical Intervention in Patients With Multiple Endocrine Neoplasia Type 1 (MEN 1)

Objective:We sought to develop a comprehensive program for clinical genetic testing in a large group of extended families with multiple endocrine neoplasia type 1 (MEN 1), with the ultimate aim of early tumor detection and surgical intervention. Summary Background Data:Germline mutations in the MEN1 tumor suppressor gene are responsible for the MEN 1 syndrome. Direct genetic testing for a disease-associated MEN1 mutation is now possible in selected families. The neuroendocrine tumors of the pancreas/duodenum and the intrathoracic neuroendocrine tumors that occur in MEN 1 carry a malignant potential. Importantly, these tumors arise in otherwise young healthy patients and are complicated by the potential for multifocality and involvement of multiple target tissues. The optimal screening methods and indications for early surgical intervention in genetically positive patients have yet to be defined. Methods:Nine MEN 1 kindreds were included in the study. The mutations for each kindred were initially identified in the research laboratory. Subsequently, mutation detection was independently validated in the clinical Molecular Diagnostic Laboratory. Each patient in the study underwent formal genetic counseling before testing. Results:Genetic testing was performed in 56 at-risk patients. Patients were stratified according to risk: Group I (n = 25), 50% risk, younger than 30 years old; Group II (n = 20), 50% risk, 30 years old or older; Group III (n = 11) 25% risk. Seven patients (age, 12 to 42 years; mean, 20.6 ± 3.8 years) had a positive genetic test. Patients with a novel positive genetic test were in either Group I (n = 6) or Group II (n = 1) and have been followed for 35.8 ± 2.0 months. Of the 7 genetically positive patients, hypercalcemia was either present at the time of diagnosis or developed during the period of follow-up in 6 patients. Four patients have undergone parathyroidectomy as early as age 16 years. One genetically positive patient has not yet developed hyperparathyroidism. Intensive biochemical screening in this select group of patients identified an elevated pancreatic polypeptide level and pancreatic tail mass lesion in a 15-year-old male who is asymptomatic and currently normocalcemic. Conclusions:Genetic testing identifies patients harboring an MEN1 mutation before the development of clinical signs or symptoms of endocrine disease. When genetically positive patients are carefully studied prospectively, biochemical evidence of neoplasia can be detected an average of 10 years before clinically evident disease, allowing for early surgical intervention. Genetically positive individuals should undergo focused cancer surveillance for early detection of the potentially malignant neuroendocrine tumors that account for most of the disease-related morbidity and mortality.

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. Hum Mutat 13:175–185, 1999.

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.

Medical adrenalectomy with aminoglutethimide: clinical studies in postmenopausal patients with metastatic breast carcinoma.

The use of adrenalectomy and hypophysectomy in the management of postmenopausal patients with metastatic breast carcinoma is reserved for highly selected patients. As an alternate approach, a pharmacologic method of inhibiting adrenal cortical secretion was developed which consisted of the daily administration of 1000 mg of aminoglutethimide to block steroidogenesis and either dexamethasone (2.0–3.0 mg/day) or hydrocortisone (40–60 mg/day) as replacement glucocorticoid. This regimen markedly suppressed plasma levels of DHA-S, androstenedione, estrone, and estradiol, and urinary levels of aldosterone. Of 50 patients treated, 19 (38%) demonstrated either a complete (8/19) or a partial (11/19) objective disease remission which lasted for 18.05 ± 3.1 months (mean ± SEM). In 10 (20%) patients, there was stabilization of disease (7.8 ± 1.2 months), accompanied by symptomatic relief of bone pain in six (12%). There was disease progression in 20 (40%) patients. The acute side effects of aminoglutethimide therapy were significant and consisted of transient lethargy (41.5%) and a cutaneous rash (35.8%). Chronic toxicity was negligible. The medical adrenalectomy regimen of aminoglutethimide plus glucocorticoid offers a suitable alternative to surgical adrenalectomy or hypophysectomy in the management of postmenopausal patients with metastatic breast carcinoma.

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

SummaryA 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 × 106 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.

Global gene expression in neuroendocrine tumors from patients with the MEN1 syndrome

BackgroundMultiple Endocrine Neoplasia type 1 (MEN1, OMIM 131100) is an autosomal dominant disorder characterized by endocrine tumors of the parathyroids, pancreatic islets and pituitary. The disease is caused by the functional loss of the tumor suppressor protein menin, coded by the MEN1 gene. The protein sequence has no significant homology to known consensus motifs. In vitro studies have shown menin binding to JunD, Pem, Smad3, NF-kappaB, nm23H1, and RPA2 proteins. However, none of these binding studies have led to a convincing theory of how loss-of-menin leads to neoplasia.ResultsGlobal gene expression studies on eight neuroendocrine tumors from MEN1 patients and 4 normal islet controls was performed utilizing Affymetrix U95Av2 chips. Overall hierarchical clustering placed all tumors in one group separate from the group of normal islets. Within the group of tumors, those of the same type were mostly clustered together. The clustering analysis also revealed 19 apoptosis-related genes that were under-expressed in the group of tumors. There were 193 genes that were increased/decreased by at least 2-fold in the tumors relative to the normal islets and that had a t-test significance value of p < = 0.005. Forty-five of these genes were increased and 148 were decreased in the tumors relative to the controls. One hundred and four of the genes could be classified as being involved in cell growth, cell death, or signal transduction. The results from 11 genes were selected for validation by quantitative RT-PCR. The average correlation coefficient was 0.655 (range 0.235–0.964).ConclusionThis is the first analysis of global gene expression in MEN1-associated neuroendocrine tumors. Many genes were identified which were differentially expressed in neuroendocrine tumors arising in patients with the MEN1 syndrome, as compared with normal human islet cells. The expression of a group of apoptosis-related genes was significantly suppressed, suggesting that these genes may play crucial roles in tumorigenesis in this syndrome. We identified a number of genes which are attractive candidates for further investigation into the mechanisms by which menin loss causes tumors in pancreatic islets. Of particular interest are: FGF9 which may stimulate the growth of prostate cancer, brain cancer and endometrium; and IER3 (IEX-1), PHLDA2 (TSS3), IAPP (amylin), and SST, all of which may play roles in apoptosis.