Histologically Proven Bronchial Neuroendocrine Tumors in MEN1: A GTE 51-Case Cohort Study.

OBJECTIVE: To evaluate the natural history of MEN1-related bronchial endocrine tumors (br-NETs) and to determine their histological characteristics, survival and causes of death. br-NETs frequency ranges from 3 to 13% and may reach 32% depending on the number of patients evaluated and on the criteria required for diagnosis. METHODS: The 1023-patient series of symptomatic MEN1 patients followed up in a median of 48.7 [35.5-59.6] years by the Groupe d'étude des Tumeurs Endocrines was analyzed using time-to-event techniques. RESULTS: br-NETs were found in 51 patients (4.8%, [95% CI 3.6-6.2%]) and were discovered by imaging in 86% of cases (CT scan, Octreoscan, Chest X-ray, MRI). Median age at diagnosis was 45 years [28-66]. Histological examination showed 27 (53%) typical carcinoids (TC), 16 (31%) atypical carcinoids (AC), 2 (4%) large cell neuroendocrine carcinomas (LCNEC), 3(6%) small cell neuroendocrine carcinomas (SCLC), 3(6%) TC associated with AC. Overall survival was not different from the rest of the cohort (HR 0.29, [95% CI 0.02-5.14]). AC tended to have a worse prognosis than TC (p = 0.08). Seven deaths were directly related to br-NETs (three AC, three SCLC and one LCNEC). Patients who underwent surgery survived longer (p = 10-4) and were metastasis free, while 8 of 14 non-operated patients were metastatic. There were no operative deaths. CONCLUSIONS: Around 5% of MEN1 patients develop br-NETs. br-NETs do not decrease overall survival in MEN1 patients, but poorly differentiated and aggressive br-NETs can cause death. br-NETs must be screened carefully. A biopsy is essential to operate on patients in time.

Sunitinib in pancreatic neuroendocrine tumors: updated progression-free survival and final overall survival from a phase III randomized study.

BACKGROUND: In a phase III trial in patients with advanced, well-differentiated, progressive pancreatic neuroendocrine tumors, sunitinib 37.5 mg/day improved investigator-assessed progression-free survival (PFS) versus placebo (11.4 versus 5.5 months; HR, 0.42; P < 0.001). Here, we present PFS using retrospective blinded independent central review (BICR) and final median overall survival (OS), including an assessment highlighting the impact of patient crossover from placebo to sunitinib. PATIENTS AND METHODS: In this randomized, double-blind, placebo-controlled study, cross-sectional imaging from patients was evaluated retrospectively by blinded third-party radiologists using a two-reader, two-time-point lock, followed by a sequential locked-read, batch-mode paradigm. OS was summarized using the Kaplan-Meier method and Cox proportional hazards model. Crossover-adjusted OS effect was derived using rank-preserving structural failure time (RPSFT) analyses. RESULTS: Of 171 randomized patients (sunitinib, n = 86; placebo, n = 85), 160 (94%) had complete scan sets/time points. By BICR, median (95% confidence interval [CI]) PFS was 12.6 (11.1-20.6) months for sunitinib and 5.8 (3.8-7.2) months for placebo (HR, 0.32; 95% CI 0.18-0.55; P = 0.000015). Five years after study closure, median (95% CI) OS was 38.6 (25.6-56.4) months for sunitinib and 29.1 (16.4-36.8) months for placebo (HR, 0.73; 95% CI 0.50-1.06; P = 0.094), with 69% of placebo patients having crossed over to sunitinib. RPSFT analysis confirmed an OS benefit for sunitinib. CONCLUSIONS: BICR confirmed the doubling of PFS with sunitinib compared with placebo. Although the observed median OS improved by nearly 10 months, the effect estimate did not reach statistical significance, potentially due to crossover from placebo to sunitinib. TRIAL REGISTRATION NUMBER: NCT00428597.

Unraveling the intrafamilial correlations and heritability of tumor types in MEN1: a Groupe d'étude des Tumeurs Endocrines study.

BACKGROUND: MEN1, which is secondary to the mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Most studies demonstrated the absence of direct genotype-phenotype correlations. The existence of a higher risk of death in the Groupe d'étude des Tumeurs Endocrines-cohort associated with a mutation in the JunD interacting domain suggests heterogeneity across families in disease expressivity. This study aims to assess the existence of modifying genetic factors by estimating the intrafamilial correlations and heritability of the six main tumor types in MEN1. METHODS: The study included 797 patients from 265 kindred and studied seven phenotypic criteria: parathyroid and pancreatic neuroendocrine tumors (NETs) and pituitary, adrenal, bronchial, and thymic (thNET) tumors and the presence of metastasis. Intrafamilial correlations and heritability estimates were calculated from family tree data using specific validated statistical analysis software. RESULTS: Intrafamilial correlations were significant and decreased along parental degrees distance for pituitary, adrenal and thNETs. The heritability of these three tumor types was consistently strong and significant with 64% (s.e.m.=0.13; P<0.001) for pituitary tumor, 65% (s.e.m.=0.21; P<0.001) for adrenal tumors, and 97% (s.e.m.=0.41; P=0.006) for thNETs. CONCLUSION: The present study shows the existence of modifying genetic factors for thymus, adrenal, and pituitary MEN1 tumor types. The identification of at-risk subgroups of individuals within cohorts is the first step toward personalization of care. Next generation sequencing on this subset of tumors will help identify the molecular basis of MEN1 variable genetic expressivity.

MEN1 disease occurring before 21 years old: a 160-patient cohort study from the Groupe d'étude des Tumeurs Endocrines.

CONTEXT: Multiple endocrine neoplasia Type-1 (MEN1) in young patients is only described by case reports. OBJECTIVE: To improve the knowledge of MEN1 natural history before 21 years old. METHODS: Obtain a description of the first symptoms occurring before 21 years old (clinical symptoms, biological or imaging abnormalities), surgical outcomes related to MEN1 Neuro Endocrine Tumors (NETs) occurring in a group of 160 patients extracted from the "Groupe d'étude des Tumeurs Endocrines" MEN1 cohort. RESULTS: The first symptoms were related to hyperparathyroidism in 122 cases (75%), pituitary adenoma in 55 cases (34%), nonsecreting pancreatic tumor (NSPT) in 14 cases (9%), insulinoma in 20 cases (12%), gastrinoma in three cases (2%), malignant adrenal tumors in 2 cases (1%), and malignant thymic-NET in one case (1%). Hyperparathyrodism was the first lesion in 90 cases (56%). The first symptoms occurred before 10 years old in 22 cases (14%) and before 5 years old in five cases (3%). Surgery was performed before age 21 in 66 patients (41%) with a total of 74 operations: pituitary adenoma (n = 9, 16%), hyperparathyroidism (n = 38, 31%), gastrinoma (n = 1, 33%), NSPT (n = 5, 36%), and all cases of insulinoma, adrenal tumors, and thymic-NET. One patient died before age 21 due to a thymic-NET. Overall, lesions were malignant in four cases. CONCLUSIONS: Various MEN1 lesions occurred frequently before 21 years old, but mainly after 10 years of age. Rare, aggressive tumors may develop at any age. Hyperparathyroidism was the most frequently encountered lesion but was not always the first biological or clinical abnormality to appear during the course of MEN1.

Adrenal involvement in MEN1. Analysis of 715 cases from the Groupe d'etude des Tumeurs Endocrines database.

OBJECTIVE: Limited data regarding adrenal involvement in multiple endocrine neoplasia type 1 (MEN1) is available. We describe the characteristics of MEN1-associated adrenal lesions in a large cohort to provide a rationale for their management. METHODS: Analysis of records from 715 MEN1 patients from a multicentre database between 1956 and 2008. Adrenal lesions were compared with those from a multicentre cohort of 144 patients with adrenal sporadic incidentalomas. RESULTS: Adrenal enlargement was reported in 20.4% (146/715) of patients. Adrenal tumours (>10 mm in size) accounted for 58.1% of these cases (10.1% of the whole patient cohort). Tumours were bilateral and >40 mm in size in 12.5 and 19.4% of cases respectively. Hormonal hypersecretion was restricted to patients with tumours and occurred in 15.3% of them. Compared with incidentalomas, MEN1-related tumours exhibited more cases of primary hyperaldosteronism, fewer pheochromocytomas and more adrenocortical carcinomas (ACCs; 13.8 vs 1.3%). Ten ACCs occurred in eight patients. Interestingly, ACCs occurred after several years of follow-up of small adrenal tumours in two of the eight affected patients. Nine of the ten ACCs were classified as stage I or II according to the European Network for the Study of Adrenal Tumors. No evident genotype/phenotype correlation was found for the occurrence of adrenal lesions, endocrine hypersecretion or ACC. CONCLUSIONS: Adrenal pathology in MEN1 differs from that observed in sporadic incidentalomas. In the absence of relevant symptoms, endocrine biology can be restricted to patients with adrenal tumours and should focus on steroid secretion including the aldosterone-renin system. MEN1 is a high-risk condition for the occurrence of ACCs. It should be considered regardless of the size of the tumour.

Gender-related differences in MEN1 lesion occurrence and diagnosis: a cohort study of 734 cases from the Groupe d'etude des Tumeurs Endocrines.

CONTEXT: Multiple endocrine neoplasia type 1 (MEN1) disease is an autosomal dominant syndrome that is believed to equally affect men and women. This assumption has never been confirmed. OBJECTIVE: The aims of this study were to evaluate the impact of gender on the prevalence of MEN1 lesions, on their lifetime probability of occurrence, and on the diagnosis of MEN1. DESIGN: Data regarding a study of 734 cases of MEN1 from the multicenter 'Groupe d'étude des Tumeurs Endocrines' were analyzed. RESULTS: There were 57.8% females. The prevalence and probability of pancreatic tumors were higher in males than in females (P=0.06, P=0.0004). This difference was due to gastrinomas. The prevalence and probability of developing pituitary tumors were significantly greater in females (P<0.001, P<0.0001). Thymic tumors were exclusively found in men. There were no significant gender differences in the prevalence and the probability of developing hyperparathyroidism, or adrenal and bronchial tumors, or in the proportion of positive genetic tests. A family history of MEN1 was more frequently found in men than in women at the time of diagnosis (P=0.02). In the case of pituitary tumor, the proportion of patients diagnosed with MEN1 at the time of the first lesion was lower in women (44.2%) than in men (67.3%). CONCLUSION: The phenotype expression of the MEN1 disease gene was different in males and females. In female patients, the possibility of MEN1 is not sufficiently taken into account. Any patient presenting a lesion that belongs to the MEN1 spectrum, such as a pituitary tumor, should be closely questioned about their family history and should be tested for hypercalcemia.

Prognostic factors of disease-free survival after thyroidectomy in 170 young patients with a RET germline mutation: a multicenter study of the Groupe Francais d'Etude des Tumeurs Endocrines.

BACKGROUND: In hereditary medullary thyroid carcinoma (HMTC), prophylactic surgery is the only curative option, which should be properly defined both in time and extent. OBJECTIVES: To identify and characterize prognostic factors associated with disease-free survival (DFS) in children from HMTC families. DESIGN: We conducted a retrospective analysis of a multi-center cohort of 170 patients below age 21 at surgery. Demographic, clinical, genetic, biological data [basal and pentagastrine-stimulated calcitonin (CT and CT/Pg, respectively)], and tumor node metastasis (TNM) status were collected. DFS was assessed based on basal CT levels. Kaplan-Meier curves, Cox regression, and logistic regression models were used to determine factors associated with DFS and TNM staging. RESULTS: No patients with a preoperative basal CT <31 ng/ml had persistent or recurrent disease. Medullary thyroid carcinoma defined by a diameter ≥10 mm [hazard ratio (HR): 6.0; 95% confidence interval (95% CI): 1.8-19.8] and N1 status (HR: 20.8; 95% CI: 3.9-109.8) were independently associated with DFS. Class D genotype [odds ratio (OR): 48.5, 95% CI: 10.6-225.1], preoperative basal CT >30 ng/liter (OR: 43.4, 95% CI: 5.2-359.8), and age >10 (OR: 5.5, 95% CI: 1.4-21.8) were associated with medullary thyroid carcinoma ≥10 mm. No patient with a preoperative basal CT <31 ng/ml had a N1 status. Class D genotype (OR: 48.6, 95% CI: 8.6-274.1), and age >10 (OR: 4.6, 95% CI: 1.1-19.0) were associated with N1 status. CONCLUSION: In HMTC patients, DFS is best predicted by TNM staging and preoperative basal CT level below 30 pg/ml. Basal CT, class D genotype, and age constitute key determinants to decide preoperatively timely surgery.

Could primary hyperparathyroidism-related hypercalcemia induce hypercalcitoninemia?

AIMS: To determine if primary hyperparathyroidism (pHPT) per se may be responsible of hypercalcitoninemia. pHPT induces chronic hypercalcemia that should be expected to be a potential stimulatory pathway of calcitonin (CT) secretion and to cause hypercalcitoninemia. METHOD: We studied relationships between CT and pHPT-related chronic hypercalcemia in 122 patients aged 25-83 years who underwent parathyroid surgery. CT, calcium and PTH plasma levels were measured in all patients preoperatively. CT was measured by a current immunometric assay specific of mature CT monomer. RESULTS: Of our 122 patients with pHPT-related hypercalcemia, 120 (98.4%) had normal CT values of less than 10 pg/ml and two (1.6%) exhibited a mildly increased CT above 10 pg/ml (11 and 12 pg/ml, respectively). We evidenced no relationship between CT and calcium level or PTH level. CONCLUSIONS: Chronic pHPT-related hypercalcemia per se does not cause hypercalcitoninemia. The finding of pHPT concomitant with high CT levels should raise suspicion of multiple endocrine neoplasia type 2A.

[Early therapeutic management of patients genetically predisposed to medullary thyroid cancer]. / Prise en charge thérapeutique précoce des sujets prédisposés génétiquement au cancer médullaire de la thyroïde. Le Groupe d'Etude des Tumeurs à Calcitonine.

STUDY: The aim of our study was to study therapeutic results after thyroidectomy in patients positive for predictive genetic analysis and with preoperative calcitonin (CT) response to pentagastlin (Pg) < 150 pg/ml. MATERIAL AND METHODS: 36 patients (13 F, 23 M) were selected: 13 F-MTC from 8 families, 22 MEN 2A from 15 families and 1 MEN 2B. They were positive for direct RET mutation analysis. CT was assayed by immunoradiometric method before and after Pg. Pg test results before and after thyroidectomy, age at operation and histologic results were analysed. RESULTS: Mean preoperative peak CT was 82.5 +/- 34.0 pg/ml (22-133): among these 36 patients preoperative basal and peak CT were normal in 16 and 2 patients respectively. F-MTC and MEN 2A patients were different according to their preoperative peak CT levels (58.1 +/- 24.0 vs 97.6 +/- 31.3) pg/ml, p < 0.01) and age at thyroidectomy (20.4 +/- 10.5 vs 11.6 +/- 7.6 years, p < 0.01 by Mann-Whitney test). Total thyroidectomy was performed in all patients at a mean age of 14.8 +/- 9.8 years (2.5-41.7) and was associated with lymph node dissection in 30 cases. The 2 F-MTC patients with normal preoperative peak CT levels had bilateral C-cell hyperplasia (CCH) associated with uni or bilateral micro-MTC. Other patients had uni or bilateral micro MTC except 4 who had isolated CCH without carcinoma. The age of two MEN-2A and 1 MEN 2B patients with micro-MTC ranged from 2.5 to 4.7 yr. Micro MTC was present in 100% of MEN-2A cases after the age of 10 yr. There were no lymph nodes metastases. During postoperative survey, the last PG tests (n = 33) were performed 27.5 months (1-92) after thyroidectomy: peak CT values were always < 10 pg/ml. IN CONCLUSION: Thyroidectomy should be performed at a very young age in RET mutation carriers, regardless of the plasma CT values. This choice is justified in NEM-2A and NEM-2B patients but must be discussed in F-MTC families with less aggressive forms of the disease.

Germ-line mutation analysis in patients with multiple endocrine neoplasia type 1 and related disorders.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome predisposing to tumors of the parathyroid, endocrine pancreas, anterior pituitary, adrenal glands, and diffuse neuroendocrine tissues. The MEN1 gene has been assigned, by linkage analysis and loss of heterozygosity, to chromosome 11q13 and recently has been identified by positional cloning. In this study, a total of 84 families and/or isolated patients with either MEN1 or MEN1-related inherited endocrine tumors were screened for MEN1 germ-line mutations, by heteroduplex and sequence analysis of the MEN1 gene-coding region and untranslated exon 1. Germ-line MEN1 alterations were identified in 47/54 (87%) MEN1 families, in 9/11 (82%) isolated MEN1 patients, and in only 6/19 (31.5%) atypical MEN1-related inherited cases. We characterized 52 distinct mutations in a total of 62 MEN1 germ-line alterations. Thirty-five of the 52 mutations were frameshifts and nonsense mutations predicted to encode for a truncated MEN1 protein. We identified eight missense mutations and five in-frame deletions over the entire coding sequence. Six mutations were observed more than once in familial MEN1. Haplotype analysis in families with identical mutations indicate that these occurrences reflected mainly independent mutational events. No MEN1 germ-line mutations were found in 7/54 (13%) MEN1 families, in 2/11 (18%) isolated MEN1 cases, in 13/19 (68. 5%) MEN1-related cases, and in a kindred with familial isolated hyperparathyroidism. Two hundred twenty gene carriers (167 affected and 53 unaffected) were identified. No evidence of genotype-phenotype correlation was found. Age-related penetrance was estimated to be >95% at age >30 years. Our results add to the diversity of MEN1 germ-line mutations and provide new tools in genetic screening of MEN1 and clinically related cases.

Pentagastrin testing in patients with renal insufficiency: normal responsivity of mature calcitonin.

Calcitonin (CT) is the most sensitive tumor marker for medullary thyroid carcinoma available, but it lacks specificity. Chronic renal failure (CRF) is known to be associated with elevations of serum immunoreactive calcitonin. Using an immunoradiometric assay to detect only mature CT, we evaluated the basal CT level and its response to pentagastrin in 30 patients with CRF and compared these data with those obtained in 71 controls. Basal mature CT was significantly higher (p < 0.05) in patients with CRF (3.55 pg/mL) than in controls (2.00). Among these patients, 20% had basal CT levels more than 10 pg/mL with a maximum of 51 pg/mL. Peak CT values (highest value obtained 3 or 5 minutes after pentagastrin) were comparable in the two groups. Among patients with CRF, 10% had peak CT values greater than 30 pg/mL with a maximum of 53 pg/mL. In this group of patients, no correlation was found between CT (at any time during the test) and parathyroid hormone, calcium, phosphate, or creatinine clearance. Men had significantly higher CT values compared with women at each time point tested, including peak values. Patients with CRF, who have not yet undergone dialysis, have moderately elevated basal CT levels, but have normal pentagastrin-stimulated peak CT levels.

Human thyroperoxidase in its alternatively spliced form (TPO2) is enzymatically inactive and exhibits changes in intracellular processing and trafficking.

Thyroid peroxidase (TPO1) is a membrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. We generated stable cell lines expressing TPO1 and the alternatively spliced isoform TPO2. Pulse-chase studies showed that TPO2 half-life was dramatically decreased as compared with TPO1. The sensitivity of TPO2 to endo-beta-N-acetylglucosaminidase H indicated that the protein is processed through the endoplasmic reticulum and bears high mannose-type structures. Cell surface biotinylation experiments showed that the two isoforms also differ in their intracellular trafficking. TPO2 was totally retained in the cell, whereas 15% of TPO1 reached the cell surface. The inability of TPO2 to come out of the intracellular compartments was related to structural changes in the molecule. Evidence of these changes was obtained through the lack of recognition of TPO2 by half of the 13 TPO monoclonal antibodies tested in immunoprecipitation experiments. Our data suggest that because of an improper folding, TPO2 is trapped in the endoplasmic reticulum and rapidly degraded. The failure of incorporation of [14C]aminolevulinic acid in the cultured cells showed that TPO2 did not bind to heme, whereas TPO1 did. This result was confirmed through a guaiacol assay showing that TPO2 is enzymatically inactive.

Interest of routine measurement of serum calcitonin: study in a large series of thyroidectomized patients. The French Medullary Study Group.

The aim of our study was to assess the ability of routine calcitonin (CT) measurement to improve the preoperative diagnosis of medullary thyroid carcinoma (MTC) in nodular thyroid diseases. We systematically determined basal CT in 1167 patients before thyroid surgery and performed a pentagastrin (Pg) CT stimulation test in 121 of these patients whose basal CT level was normal. Sixteen MTC (1.37%) were found on histopathological examination of surgical specimens: 14 in the 34 patients (41.1%) with abnormal basal CT levels and 2 in the 1133 patients with normal basal CT levels (0.17%). An abnormal increase in Pg-stimulated CT was observed in 7 of the 121 patients tested and was related to microscopic MTC in 2 cases. Among 1167 thyroidectomized patients with nodular thyroid diseases, the prevalence of MTC was 1.37% and reached 41.1% when the basal CT level was abnormal (3% of the patients). CT evaluation detected MTC, whereas other procedures, such as fine needle aspiration cytology, failed, thus allowing early radical surgery. CT measurement should thus become a routine part of the diagnostic evaluation of nodular thyroid diseases.

Multiple endocrine neoplasia type 2: management of patients and subjects at risk. French Study Group on Calcitonin-Secreting Tumors (GETC).

Multiple endocrine neoplasia type 2 (MEN-2) is an inherited multiglandular disease with age-related penetrance and variable expression. The prognosis of MEN-2 is linked to the carcinological evolution of medullary thyroid cancer (MTC), which depends mainly on the stage of discovery, and to the incidents related to pheochromocytomas. The emphasizes the need for early diagnosis and management of MEN-2. Since 1993, mutations evidenced on the protooncogene RET have allowed subjects at risk to be identified, thus leading to a three-step management of these patients. (1) For all the potentially affected members of a MEN-2 family, screening by molecular genetics of the ret gene enables one to identify the subjects at risk who bear the mutation. When no mutation is observed, a linkage analysis study may be proposed. (2) In the subjects at risk, early screening for the various types of endocrine lesions may then start in childhood and be performed using specific biological markers of MTC, pheochromocytoma and primary hyperparathyroidism, and particularly, basal and pentagastrin-stimulated calcitonin measurement, which is known to be the most sensitive marker for the monitoring of MTC. (3) This step of biological investigations enables the earliest possible treatment of any endocrine lesion detected before it is expressed clinically, thus improving the prognosis of MEN-2. When genetic screening cannot be performed, only annual clinical and biological monitoring remain available in all members of a family affected with MEN-2.

Results of the calcitonin stimulation test in normal volunteers compared with genetically unaffected members of MEN 2A and familial medullary thyroid carcinoma families.

In the multiple endocrine neoplasia (MEN) type 2A and in the familial medullary thyroid carcinoma (FMTC), the recent development of genetic testing has provided new methods of identifying disease gene carriers. The use of sensitive immunoradiometric calcitonin (CT) assays led us to evaluate reference ranges of plasma CT responses after pentagastrin in healthy subjects in order to discuss the place of pentagastrin testing in the management of hereditary MTC. Basal and pentagastrin-stimulated CT concentrations were measured in 71 healthy volunteers--aged 20-67 years--and 76 genetically unaffected members of families with hereditary MTC--aged 4-61 years. In healthy subjects, CT peak values were below 30 ng/l in 68 cases and below 50 ng/l in 3 cases. In the genetically unaffected patients, CT peak values were below 15 ng/l in young patients and below 30 ng/l in patients older than 19 year excepted 5 men with stimulated CT levels ranging from 36.5 to 52 ng/l. In 2 of these 5, thyroidectomy revealed C-cell hyperplasia. Borderline test results are not sufficient to establish diagnosis of MTC and in these familial syndromes, management has to be based on the results of genetic testing. However, the determination of the upper normal limit for stimulated CT concentrations in young patients (< 15 ng/l) may help to identify the optimal moment for surgery.

Dityrosine bridge formation and thyroid hormone synthesis are tightly linked and are both dependent on N-glycans.

Formation of dityrosine bridges is a ubiquitous process mainly attributed to oxidative stress leading to protein degradation and cellular damages. Here we show that dityrosine formation is involved in a physiological process, thyroid hormone synthesis, and is strictly dependent on structural characteristics, namely N-glycans, presented by the protein acting as the prothyroid hormone. We used two isoforms of the N-terminal thyroid hormone forming domain (NTD) of human thyroglobulin: one without N-glycan (19 kDa isoform) and the other with high mannose type structures (25 kDa isoform). Both isoforms were able to form iodotyrosines after in vitro iodination. However, iodotyrosine coupling to form thyroxine did not occur with the unglycosylated 19 kDa NTD. In contrast, the 25 kDa isoform formed thyroxine. Strikingly, thyroxine synthesis was accompanied by dimerization of the 25 kDa isoform and formation of a dityrosine bridge; none of this was observed with the 19 kDa isoform. Taken as a whole, our results indicate that dimerization through dityrosine bridging accompanies and could have a role in thyroid hormone synthesis.

[Early diagnosis of sporadic medullary cancers of the thyroid: value of systematic assay of calcitonin]. / Diagnostic précoce des cancers médullaires sporadiques de la thyroïde: intérêt du dosage systématique de la calcitonine.

OBJECTIVE: Sporadic medullary cancer of the thyroid is often diagnosed late beyond the surgically curable stage. The aim of this work was to assess the capacity of routine calcitonin assay as an early diagnosis test for medullary cancer in patients with a thyroid lesion. METHODS: Calcitonin was assayed (normal < 10 pg/ml) as a routine test from 1993-1995 in a series of 2975 patients seen for thyroid exploration. When baseline level was above 10 pg/ml, a pentagastrine test was performed (normal < 30 pg/ml). All patients with a calcitonin peak > or = 100 pg/ml after pentagastrin underwent surgery for suspected medullary cancer. Surgery for suspected malignancy, hyperthyroidism or locoregional functional disorders was also performed in 1494 of the included patients, independent of calcitonin level. Patients with personal or familial history of multiple endocrine disease were excluded. Fine needle aspiration was done in all patients with an unique or predominant thyroid nodule. RESULTS: Medullary cancer of the thyroid was demonstrated in 14 patients (0.47%). Among 8 patients with clinically patent tumor, the diagnosis was established in 3 on the basis of cytology results and elevated calcitonin level; in the 5 other cases, initial cytology was incorrect (anaplastic, papillary, thyroiditis) but correct diagnosis was established on the basis of high calcitonin levels. Diagnosis was suspected preoperatively in the 6 others solely because of high calcitonin; these patients had microlesions measuring 1.2-9 mm. None of the 7 patients with a medullary cancer measuring < 10 mm had node extension at surgery and all 7 attained biological cure. Among the 7 other patients with a lesion > 10 mm, calcitonin level returned to normal level in 3 and remained high in 2; the 2 others died with distant metastasis. CONCLUSION: Routine assay of calcitonin in all patients with a thyroid nodule can improve preoperative diagnosis of medullary cancer of the thyroid and allows early diagnosis of latent infraclinical tumors.

Interest of epitopic dissection in immunoanalysis of proteins and peptides: review of theoretical and practical aspects.

The literature abounds with reports showing discrepancies in immunoassays of proteins and peptides. Whereas the isomorphism and polymorphism of proteins remains largely hidden in immunoassays making use of polyclonal antibodies, the use of monoclonal antibodies uncovered the difficulty of accurately assaying microheterogeneous analytes. Indeed, most proteic hormones are not entities with unique structures but rather mixtures of molecular forms with slight differences in structure which may reflect large variations in biological and immunological activities; the monoclonal antibodies appeared clearly less suited than the polyclonal for testing a mixture of isoforms. Protein microheterogeneity also has an impact on assay standardisation, since reference preparations may contain several isoforms of the analyte. Using recombinant glycoprotein does not solve the problem. Regarding the problem of discrepancy in immunoanalysis of proteins and peptides, we could establish, in a previous work, that discrepancy among lutropin assay kits may be related to various causes: i) differences in standard preparation and calibration curves; ii) microheterogeneity of lutropin molecules leading to missing some isoforms due to the restricted epitopic specificity of the monoclonal antibodies used in the kits. The epitopic dissection we engaged in appeared thus instrumental in explaining these discrepancies. It allowed us to enumerate epitopes on the surface of lutropin molecules, to elucidate the immunological structure and, finally, to characterize monoclonal antibodies used in commercially available lutropin assay kits with regard to their epitopic specificity. This work allowed us to interpret the discrepancy in serum lutropin concentration which was related to the use of monoclonal antibody with given specificity. Epitopic dissection may thus be instrumental in explaining discrepancy among immunoassays of proteins and peptides and in improving the accuracy of kits.

European collaborative study of LH assay: 3. relationship of immunological reactivity, biological activity and charge of human luteinizing hormone.

This report describes the results of the third part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The aim of the present work was to establish the link between immunoreactivity and biological activity of human LH, thus allowing to determine the antigenic domains of the molecule involved in the induction of the biological effect. The relationship between immunoreactivity and electric charge of hLH was also studied. This work allowed to further apprehend hLH isomorphism and its role in discrepancies observed among hLH assays and clinical status. It also made the feasibility of measuring biologically active isoforms by an immunological method to be assessed. The effect of 36 mAb with known epitopic specificity, was evaluated on both hLH binding to rat membrane receptor and hLH induced production of testosterone by porcine Leydig cells. All the epitopes located on the beta subunit were found to be strongly involved in the biological activity whereas 4/9 and 10/18 epitopes present on the alpha subunit or specific for the holomolecule respectively appeared weakly involved. Assaying biological hLH using immunological method would require that mAb specific for all the epitopes involved in the receptor activation be tested, and thus appears presently unsuitable for routine clinical evaluation. In the previous work some LH immunoassays were found to underestimate LH concentrations (J. Endocrinol. Invest 1994, 17: 397-406 and 407-416). The mAb used in liquid phase in these kits were found in the present work to be directed against the domains of LH weakly involved in the activation of the receptor and would suggest that bioactive LH would be misevaluated by these kits. The immunoreactivity of hLH isoforms separated by isoelectric focusing (IEF) in liquid phase was also determined. IEF allowed to separate three groups of hLH isoforms but none of them exhibited a specific discriminating pattern of immunoreactivity when they were tested against a panel of mAb. It suggests that, in our experimental conditions, the electric charge and the immunoreactivity of hLH were not closely linked.

Polymerase chain reaction-based site-directed mutagenesis using magnetic beads.

Thyroglobulin double mutants with various substitutions were obtained with a new polymerase chain reaction-based mutagenesis technique. Maximum length of megaprimer and efficiency of mutagenesis were improved by purification of single-stranded DNA, using the avidin-biotin interaction. This method might allow the construction of large libraries of DNA, mutated at different sites.