Changes in TNM stage, reoperation and 131-I ablation rate during the use of newer methods for the preoperative diagnosis of differentiated thyroid carcinoma.

OBJECTIVE: To define and discuss the changes of important risk factors and TNM staging over the last 40 years in patients operated on for differentiated thyroid carcinoma (DTC), resulting from the introduction of newer sensitive diagnostic procedures in the preoperative evaluation of thyroid nodules. PATIENTS: We reviewed the medical records of 1251 patients with postoperative diagnosis of DTC who had undergone initial diagnosis, before surgery, at our unit, between 1971 and 2010. According to the period of diagnosis, the patients were divided into four groups (I, II, III, IV) corresponding to the four decades. RESULTS: The mean age at diagnosis was unchanged over time for both papillary (PTC) and follicular thyroid cancer (FTC). A decrease in the proportion of FTC (group I vs group IV P < 0·01) and a concomitant increase in PTC/FTC ratio was observed particularly in group IV. The significant decrease in the proportion of tumour size of PTC (group I vs group IV, P < 0·01), the increase in the proportion of microcarcinomas, from 22·4% in group I to 53·0% in group IV, P < 0·001, and the decrease in the number of cases with features of aggressiveness have changed the TNM stage towards stages I and II. The overall frequency of patients at high risk was significantly decreased, from 8·0% in group I to 1·8% in group IV. The number of patients who underwent reoperation for the completion of tumour resection and/or radioiodine therapy significantly decreased over time. CONCLUSIONS: The evaluation of thyroid nodules using the newer diagnostic methods was useful in identifying DTC early. Consequently, the reoperation and thyroid remnant ablation rates were reduced.

Effect of an oral glucose load on PTH, 250HD3, calcium, and phosphorus homeostasis in postmenopausal women.

INTRODUCTION. Previous studies particularly in children and neonates have shown that serum calcium declines and parathyroid hormone (PTH) rises during an oral glucose load. However, there is not a general agreement in this regard. This study was carried out to evaluate the effects of an oral glucose load on calcium and phosphorus homeostasis in postmenopausal women along with serum insulin, PTH, and 250HD3 changes. PATIENTS AND METHODS. After an overnight fasting, an oral glucose tolerance test was performed in 50 postmenopausal women; and glucose, insulin, PTH, and D3 were measured at baseline and every 30 min during the 2 hours of the test. RESULTS. Serum glucose and insulin increased as expected and reached their peak values at 60 and 90 min, respectively. PTH and phosphorus decreased significantly and the maximum decline was observed at 30 and 120 min after glucose load (p < 0.0001), respectively. Serum calcium, magnesium, and D3 levels showed no significant changes at any time measured. Serum PTH values had a significant negative correlation with glucose and insulin values (p = 0.026 and p = 0.031, respectively). Serum D3 also correlated negatively with glucose (p = 0.002). CONCLUSION. Our study shows that an oral glucose load induced hyperglycemia/hyperinsulinemia promotes a significant decline in serum PTH and phosphorus levels without changes in calcium or 250HD3 in postmenopausal women.

Diurnal variation of endothelial function and arterial stiffness in hypertension.

The onset of cardiovascular events presents a circadian variation that may be mediated by similar temporal patterns of vascular function. Blood pressure also follows circadian variation. We investigated the possible diurnal variation of endothelial function and arterial stiffness in patients with hypertension. Thirty-five individuals with recently diagnosed hypertension (mean age 48.3 years, range 30-60 years, 14 men) were examined. Flow-mediated vasodilatation (FMD), nitrate-mediated vasodilatation (NMD) and carotid-femoral (cf) pulse wave velocity (PWV) were measured at three different occasions: at 0700 hours immediately after awaking, at 1200 hours and at 2100 hours. FMD was markedly lower in the morning (0700 hours, 2.22+/-1.58%; 1200 hours, 4.37+/-2.25%; 2100 hours, 4.28+/-2.12%; P<0.001), whereas NMD was similar at the same time points. This difference remained significant after adjustment for baseline brachial artery diameter and reactive hyperaemia. PWVcf progressively increased from morning to evening (0700 hours, 9.8+/-1.9 m s(-1); 1200 hours, 10.2+/-2.2 m s(-1); 0900 hours, 10.5+/-1.9 m s(-1); P=0.013 for linear trend). Similar temporal patterns were observed in systolic and diastolic blood pressures peaking in the evening. PWVcf changes lost significance after adjustment for changes in mean blood pressure. Endothelial function is decreased in the early morning in hypertensive patients, whereas arterial stiffness is increased in the evening. Changes in BP-dependent passive artery distension may be involved in this phenomenon.

Heterophilic antibodies causing falsely high serum calcitonin values.

Heterophilic antibodies (HA) may interfere in some immunoassays, causing falsely high hormone values, of which practitioners should be aware when measuring calcitonin (CT) used as tumor marker for medullary thyroid carcinoma (MTC). We studied four patients with thyroid nodules, three of whom underwent surgical neck exploration, after an erroneous diagnosis of MTC because of falsely high serum CT eventually proved to be due to HA. One patient had a lingual thyroid, two autoimmune thyroiditis and the fourth a colloid goiter. The minimal incremental CT response to calcium infusion raised our suspicion of possible false high CT values due to HA. There was no linearity of the CT values obtained by testing serial dilutions of the sera in the CT assay, which employs two monoclonal mouse anti-CT antibodies. Addition of normal mouse gamma globulin eliminated the interference by HA in the sera of two patients. Serum assayed in a polyclonal radioimmunoassay using goat anti-CT antibodies gave normal CT values. Finally, incubation of the sera in Heterophilic Blocking Tubes (HBT) eliminated the false CT immunoreactivity. A spontaneous change of the CT serum concentrations was noticed in three patients over several months, apparently due to changing titles of HA. We suggest that, in patients a) whose CT response to calcium or pentagastrin infusion is minimal despite high basal CT values, b) with autoimmune thyroiditis and c) in whom an unexpected change in serum CT concentrations occurs, the possibility of spuriously high CT values because of circulating HA should be considered.

Ras oncogenes and p53 tumor suppressor gene analysis in cardiac myxomas.

Although ras oncogenes and p53 tumor suppressor gene mutations are implicated in the development of several human tumors, little is known about their role in the pathogenesis of primary cardiac tumors. Paraffin-embedded tissue from 19 cardiac myxomas were investigated for the presence of ras oncogenes and p53 tumor suppressor gene abnormalities. Immunohistochemical analysis was used to identify the accumulation of p21-ras and p53 proteins. A polymerase chain reaction was used to amplify exons 1 and 2 of the ras genes and exons 5 to 8 of the p53 gene. The PCR products were analyzed by single strand conformation polymorphism analysis and by direct DNA sequencing. Three of 19 myxomas showed strong positive staining for the ras p21 protein. In contrast, nuclear p53 was not detectable in any of the myxomas. Among the ras p21 immunopositive myxomas, 2 were heterozygous for a missense point mutation of the K-ras, Gly 12Asp. Further screening of the remaining myxomas showed no mutation or even silent polymorphism in any exon of the ras and p53. The results suggest that although genetic alterations of ras oncogenes and p53 are uncommon events in cardiac myxomas, ras mutations may be involved in the pathogenesis of a subgroup of this type of tumor.

The role of cytokines and cortisol in the non-thyroidal illness syndrome following acute myocardial infarction.

OBJECTIVE: A number of different hormone changes have been described during the acute myocardial infarction (AMI), including those of the non-thyroidal illness syndrome (NTIS). DESIGN AND METHODS: We assessed the alterations of serum thyroid hormones, cytokines and cortisol levels in 30 patients with a first episode of AMI 4, 24, 48h and 10 days (240h) after the onset of the chest pain and we investigated the possible relationship of these alterations with the severity of AMI. RESULTS: Fifteen patients had left ventricular ejection fraction (LVEF) 50% (group II). A transient decrease of total tri-iodothyronine (T(3)), more prominent in group I (P<0.05, t-test) with a concomitant rise of reverse T(3 )(rT(3)) occurred at 24h. Total thyroxine (T(4)), free T(4) (FT(4)) and free T(4) index did not change significantly, but tended to be higher in group I patients, whereas TSH significantly increased in group II at 48h. Interleukin-6 (IL-6) increased significantly at 24h only in group I and declined thereafter (24 vs 240h, P<0.001) and this temporal change of IL-6 was associated with similar changes of creatine phosphokinase and creatine kinase isoenzyme MB (CK-MB). Tumor necrosis factor-alpha and IL-1beta remained low in both groups. Cortisol was higher at 4h and in 12 patients was above the normal values. Negative correlation was found between LVEF and IL-6 (P<0. 001), whereas T(3), T(4) or cortisol levels were not correlated with the LVEF. CONCLUSIONS: Our data indicate that NTIS, in association with increase of IL-6, occurs in the early post-infarction period. In the NTIS following AMI the high level of IL-6 is the best predictor, among several parameters, of the severity of AMI as assessed by the LVEF and the rise of CK-MB.

Germ line mutation analysis in families with multiple endocrine neoplasia type 2A or familial medullary thyroid carcinoma.

The RET proto-oncogene has been identified as the multiple endocrine neoplasia type 2 disease gene. An association between specific RET mutation and disease phenotype has been reported. We present the phenotype-genotype of 12 Greek families with multiple endocrine neoplasia type 2A (MEN 2A) or familial medullary thyroid carcinoma (FMTC). Seventy members were studied and DNA analysis for RET mutations was performed in fifty-eight of them. Exons 10, 11, 13, 14 and 16 of the RET proto-oncogene were analyzed by single strand conformation polymorphism analysis, direct DNA sequencing and/or restriction enzyme analysis. No mutations of the RET proto-oncogene were identified in 1 of 9 families with MEN 2A and in the 3 families with FMTC. In 7 MEN 2A families, the mutation was demonstrated in codon 634 and in 1 family it was demonstrated in codon 620. There was a low frequency, about 8%, of hyperparathyroidism associated with MEN 2A. The specific causative mutations for pararthyroid disease were C634R or C634Y. Among the MEN 2A individuals there was one case with de novo C634R mutation and one case, C634Y, with cutaneous lichen amyloidosis which predated by 24 years the diagnosis of MEN 2A. In 2 children who were MEN 2A gene carriers, microscopic medullary thyroid carcinomas were found. These data show a low frequency of hyperparathyroidism in our cases and provide further evidence that individuals with C634R as well as with C634Y mutations of the RET proto-oncogene could be at risk for parathyroid disease. Cutaneous lichen amyloidosis could be an early feature of MEN 2A. Additionally, direct DNA testing provided an opportunity to resect medullary thyroid carcinoma at an early stage.

Identical complementary deoxyribonucleic acids encode a human renal and bone parathyroid hormone (PTH)/PTH-related peptide receptor.

Identical complementary DNAs (cDNAs) that encode a 593-amino acid human PTH (PTH)/PTH-related peptide (PTHrP) receptor were isolated by hybridization techniques from two cDNA libraries which had been constructed from human kidney and human osteoblast-like osteosarcoma cells (SaOS-2). Northern blot analysis of total RNA from human bone- and kidney-derived tissue revealed one single major messenger RNA species of about 2.5 kilobases in both tissues. The human PTH/PTHrP receptor has 91% and 81% identity, respectively, with the previously cloned rat and opossum receptors, indicating a high degree of conservation among mammals. Despite this striking degree of amino-acid conservation, the human PTH/PTHrP receptor has several unique biological properties when transiently expressed in COS-7 cells. The apparent dissociation constants for [Nle8,18,Tyr34] bovine PTH(1-34) amide [bPTH(1-34)] are similar for the human and the rat receptor (approximately 8 vs. approximately 15 nM) whereas [Tyr36]PTHrP(1-36) amide has a slightly lower affinity for the human (15-40 nM) than for the rat receptor (approximately 15 nM). Both ligands stimulate efficiently and with similar efficacy the accumulation of intracellular cAMP. The affinities for the antagonists [Nle8,18,Tyr34] bPTH(3.34) amide [bPTH(3-34)] and in particular for [Nle8,18,Tyr34] bPTH(7-34) amide [bPTH(7-34)] are considerably higher for the human receptor, e.g. approximately 8 nM vs. 30 nM for bPTH(3-34) and approximately 100 nM vs. 5000 nM for bPTH(7-34), respectively. Similar biological findings were previously attributed to differences in species- and/or organ-specific PTH/PTHrP receptors. The expression of the recombinant, highly homologous rat and human receptors in a uniform environment indicate that the moderate differences in the primary receptor structure have profound consequences for the receptor binding affinity of amino-terminally truncated PTH analogs. Furthermore, the molecular cloning of identical cDNAs encoding a human PTH/PTHrP receptor from the two major target organs for PTH, bone and kidney, provides strong evidence for one single PTH/PTHrP receptor in both organs, although additional and/or alternatively spliced receptors cannot be excluded.

Transcriptional regulation of G-protein alpha i subunit genes in LLC-PK1 renal cells and characterization of the porcine G alpha 1-3 gene promoter.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) function as signal transducers for a variety of hormone-coupled enzyme and ion transport systems in eukaryotic cells. The expression of pertussis toxin-sensitive G-proteins (Gi) which couple their cognate receptors and effectors are regulated by cell cycle-dependent events in porcine LLC-PK1 renal epithelial cells. G alpha i-2 and G alpha i-3 isoforms are detected in these cells, and like G alpha i-2 (Holtzman, E. J., Soper, B. W., Stow, L. L., Ausiello, D. A., and Ercolani, L. (1991) J. Biol. Chem. 266, 1763-1771), we now demonstrate that G alpha i-3 mRNA and protein is coordinately expressed in these cells during differentiation. To gain further insights into these events, the porcine G alpha i-3 gene minimal promoter was characterized and found 67 base pairs upstream from the major transcription start site. The 56-base pair minimal promoter lacked TATAAA and GC boxes but did contain a sequence GGAAGTG conserved in both the human and porcine gene that could potentially bind an adenovirus E4TF1 transcription factor. In cells stably transfected with G alpha i-2 or G alpha i-3 gene 5'-flanking sequences fused to firefly luciferase cDNA reporter, temporal 10-15-fold transcriptional activation of both genes occurred before cellular polarization. Utilizing mobility shift assays which compared nuclear extracts from cells before and after cell polarization, a motif in the 5' region of the gene promoter GTACTTCCGCT was identified that bound an induced nuclear protein complex during transcriptional activation. In polarized cells complemented with the human glucocorticoid receptor, dexamethasone decreased G alpha i-2 but increased G alpha i-3 basal transcription and mRNA content 3-fold. These studies demonstrate that both G alpha i genes are dynamically regulated in LLC-PK1 cells by both growth, differentiation, and hormone signals.

Parathyroid hormone (PTH) stimulates adrenocorticotropin release in AtT-20 cells stably expressing a common receptor for PTH and PTH-related peptide.

Complementary DNA encoding a rat bone PTH/PTHrP receptor was stably expressed in the murine corticotroph cell line, AtT-20. Several clones, expressing variable numbers of PTH/PTHrP receptors, were developed. In contrast to the relatively low binding affinity (apparent Kd = 15 nM) observed in COS-7 cells transiently expressing the PTH/PTHrP receptor, all AtT-20 stable transfectants bound [Nle8,18,Tyr34]bPTH(1-34)NH2 (NlePTH) with an affinity that was indistinguishable from that observed in ROS 17/2.8 cells expressing native PTH/PTHrP receptors. Additionally, NlePTH dramatically increased cAMP accumulation and ACTH release in AtT-20 cells expressing the PTH/PTHrP receptor with an ED50 of 0.6 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The high binding affinity and the high efficacy of NlePTH in stimulating cAMP accumulation and ACTH release indicate that the PTH/PTHrP receptor is efficiently coupled to the intracellular signalling system responsible for stimulation of ACTH release in AtT-20 cells. No additivity of cAMP accumulation or of ACTH release was observed when these cells were treated with maximally active concentrations of both NlePTH and CRF. This suggests that the receptors for both of these hormones share the same intracellular effectors, and that intracellular signaling in AtT-20 cells is not compartmentalized. Additionally, the ability of NlePTH to stimulate ACTH release in AtT-20 cells, a function that is normally performed by CRF, demonstrates promiscuity between activated receptors and distal biological functions.

Ras mutations in human pituitary tumors.

The cellular basis for pituitary neoplasia is poorly understood. Mutations that activate the ras protooncogenes have been identified in a number of different types of human cancers and potentially represent one of the genetic alterations that occur in pituitary tumors. In this study we examined 19 pituitary tumors for the occurrence of ras mutations. The tumor types included 11 nonfunctioning adenomas, 6 somatotroph adenomas, and 2 prolactinomas. Each of the three ras genes (K-ras, N-ras, and H-ras) was amplified from pituitary tumor DNA using the polymerase chain reaction. Oligonucleotide-specific hybridization was used to screen for mutations that inhibit GTPase activity and cause activation of the ras oncogene. No ras mutations were observed in 18 of the pituitary adenomas. However, a mutation was identified in codon 12 of the H-ras gene (Gly to Val) in a recurrent prolactinoma that was highly invasive and ultimately proved to be fatal. We conclude that ras mutations are uncommon in pituitary adenomas, but may provide a marker for highly invasive tumors.

Ras oncogene mutations in benign and malignant thyroid neoplasms.

Current models for tumorigenesis propose that a series of genetic alterations occur during the progression from the normal cell to the malignant phenotype. Mutations in each of the three ras genes (K-ras, H-ras, and N-ras) have been identified in many human neoplasms, including thyroid cancer. In this study we examined genomic DNA from benign and malignant thyroid neoplasms for mutations that are known to activate the ras oncogenes (codons 12, 13, and 61). DNA from frozen surgically excised tissue (n = 8) and from formalin-fixed paraffin-embedded tissue (n = 30) was amplified by the polymerase chain reaction and screened for mutations using oligonucleotide-specific hybridization. No mutations were identified in follicular adenomas (n = 9). In follicular carcinomas, 2 of 14 tumors contained mutations (N-ras 61, Gln to Arg), and both of these patients had bone metastases. One of 15 papillary carcinomas had a ras mutation (H-ras 12, Gly to Ser). In contrast to other studies, we found that ras mutations are relatively uncommon in both benign and malignant thyroid neoplasms. Studies of larger numbers of tumors and comparisons of different patient populations will be required to assess a possible association of mutations in N-ras 61 with clinically aggressive follicular cancer.

Digoxin-like substance(s) interfere(s) with serum estimations of the drug in cirrhotic patients.

We measured the levels of digoxin-like immunoreactivity in the serum of 40 volunteers (20 patients with liver cirrhosis and 20 healthy adults) before and after the administration of a 5-day standard regimen of digoxin. Serum digoxin levels (SDL) were evaluated with two different radioimmunoassay (RIA) kits--Amerlex Digoxin 125I RIA and Digoxin 125I RIA. Digoxin was detectable by each RIA kit in 10 and 15% of controls and 50 and 60% of cirrhotic patients before the administration of the drug, respectively. At the end of the treatment with digoxin, SDL were significantly higher in cirrhotics when compared with those of controls. This study provides evidence that digoxin-like substance(s) is (are) implicated in the detection of high SDL in patients with histologically confirmed liver cirrhosis.