Mario Andreoli

p53 re-expression inhibits proliferation and restores differentiation of human thyroid anaplastic carcinoma cells

Alterations of the tumor suppressor gene p53 are uncommon in differentiated thyroid neoplasia but are detected at high frequency in anaplastic thyroid carcinoma suggesting that impaired p53 function may contribute to the undifferentiated and highly aggressive phenotype of these tumors. Effects of wild type p53 (wt-p53) re-expression were investigated in a human anaplastic thyroid carcinoma cell line (ARO) expressing a mutated p53. ARO cells were stably transfected with the temperature-sensitive p53 Val135 gene (ts-p53) which exhibits wild type-like activity at 32°C. Exogenous wt-p53 function in ARO-tsp53 clones was assessed by evaluating its transcriptional activity on a CAT reporter vector containing p53 binding sites. At 32°C, a significant reduction in the proliferation rate (≈percnt;50%) was observed, with accumulation of cells in the G0/G1 phase of the cell cycle. This effect was accompanied by induction of the expression of the growth inhibitor p21/Waf1 gene. At 32°C, ARO-tsp53 clones also showed a marked impairment of their tumorigenic potential. Furthermore, transfected clones re-acquired the ability to respond to thyrotropin (TSH) stimulation showing an increased expression of thyroid-specific genes (thyroglobulin, thyroperoxidase and TSH receptor). In conclusion, re-expression of wt-p53 activity in ARO cells, inhibits cell proliferation and restores responsiveness to physiological stimuli.

Thyroid carcinoma in children and adolescents

Abstract A clinical and pathological study was undertaken to define the prevalence, clinical presentation and outcome of thyroid carcinoma in children and adolescents. Clinical records from 48 patients under 20 years of age at diagnosis, out of 372 patients with thyroid cancer examined between 1980 and 1994, were retrospectively reviewed. Female/male ratio was 3.8/1. None had a previous positive history of head and neck irradiation. Patients underwent near-total (44 patients) or partial (4 patients) thyroidectomy followed by 131I ablation of residual thyroid tissue. The mean follow up period was 58.4 months, ranging between 2 and 190 months. Clinically a thyroid mass was present in 41 patients, 28 of whom also showed neck lymph node involvement. Node metastases were present in 50% of the patients and lung metastases in 4.2%. Histological type was papillary in 41, follicular in 6, and medullary in 1 case. Surgical complications were observed in 19 patients (40%). In 3 patients papillary thyroid cancer was associated with chronic lymphocytic thyroiditis. All patients were treated with l-thyroxine suppressive therapy. Recurrence of cancer after surgical and radio-iodine treatment was observed only in one patient 8 months after surgery. Conclusion Our experience demonstrates that thyroid carcinoma in childhood cannot be considered a rare occurrence, since it represents about 13% of all thyroid cancers, and is frequently associated with lymph node but rarely with distant metastases. Nevertheless, the prognosis of thyroid carcinoma in childhood is fairly good.

PCR amplification and analysis of ras oncogenes from thyroid cytologic smears

Fine needle aspiration (FNA) cytology represents a reliable diagnostic procedure for preoperative identification of thyroid carcinoma. For improving its diagnostic accuracy, a technique that allows the analysis of cancer-related gene abnormalities on thyroid FNA smears has been developed. Cells were collected by scraping from thyroid smears, DNA-extracted and ras proto-oncogene sequences amplified by the polymerase chain reaction (PCR). Preliminary destaining of cytologic smears was essential for efficient PCR amplification. Twelve thyroid FNA cytologic smears, characterized by the indeterminate pattern of follicular neoplasia, were analysed for the presence of ras mutations known to confer an oncogenic potential. The same nucleotide substitution at codon 12 of the H-ras proto-oncogene was detected in two different thyroid nodules among six cases that, at final histology, were identified as follicular carcinomas. No ras mutations were observed in the remaining six cases that were diagnosed as follicular adenoma at histologic examination. Molecular analysis of FNA smears may provide additional information on the nature of the lesion underlying thyroid neoplasia, thus improving diagnostic accuracy of conventional FNA cytology.

Thyroid hormone formation in thyroglobulin synthesized in the Amphioxus (Branchiostoma lanceolatum pallas)

Abstract 1. 1. Thyroglobulin (Tg) biosynthesis in the endostyles of Amphioxus ( Branchiostoma lanceolatum Pallas) has been demonstrated. 2. 2. The protein molecule shows characteristics similar to those of Tg from ammocoetes of fresh-water lampreys, sedimenting with a coefficient of 17-19S and incorporating in vivo 125 I. 3. 3. Iodothyronine synthesis occurs in thyroglobulin in animals incorporating 125 I both after in vivo and after in vitro iodination with lactoperoxidase in presence of labelled iodide. 4. 4. Labelled T3 and T4 are present in the Tg molecule of Amphioxus as in control mammalian Tg and practically in the same molecular rates. 5. 5. Thus thyroid hormones are present in thyroglobulin included in the cells of endostyles. 6. 6. It is shown that in evolution of Chordates the synthesis of thyroglobulin, as the site of formation of thyroid hormones, preceeds the morphological differentiation of thyroid cells and the folliclular organization out (fishes) or into the thyroid glands of higher vertebrates.

Thyroglobulin biosynthesis in a larval (ammocoete) and adult freshwater lamprey (Lampetra planeri B1.)

1. The biosynthesis of 18-19S thyroglobulin has been studied in a larval and adult freshwater lamprey (Lampetra planeri Bl.). 2. In vivo and in vitro experiments have been performed by injecting into the coelomic cavity or by incubating branchial region labeled constituents of Tg of higher vertebrates (125I, [3H]leucine and various [3H]carbohydrates). 3. Larvae (ammocoetes) and adults incorporate all labels into thyroglobulin (18-19S Tg), containing a small proportion of labeled T3 and T4, as identified by paper chromatography, and very minute amounts of stable iodine. 4. In adults, the biosynthesis of 18-19S Tg proceeds much more rapidly and the labels are incorporated in higher percentage than in larvae. 5. The demonstration of the biosynthesis of the specific thyroid protein, 18-19S Tg, in larvae indicates that the biochemical mechanism of hormonogenesis is present in larval endostyle before the morphological differentiation of thyroid cells and follicles occurring during metamorphosis. 6. Some 18-19S Tg is apparently stored in the endostyle.

Neonatal screening in Italy for congenital hypothyroidism and metabolic disorders: hyperphenylalaninemia, maple syrup urine disease and homocystinuria

A multiple screening program to establish the frequency of congenital hypothyroidism (CH), phenylketonuria (PKU), maple syrup urine disease (MSUD), homocystinuria and hypertyrosinemia in endemic and sporadic goitrous regions of Italy is being carried out. Valine, methionine, leucine, isoleucine, tyrosine and phenylalanine, eluted from a single spot and separated by column chromatography, are measured, using whole blood adsorbed on filter paper. CH is detected by RIA assay of TSH eluted from dried blood spot. A cut-off of 100 gmU/ml for TSH is used providing a recall rate of 0.38%. Out of 116,000 newborn infants screened for àminoacidopathies (since 1974), 16 PKU patients, 3 affected by MSUD, 2 homocystinuric babies have been detected. Out of 25,400 newborn infants screened for CH, 5 patients were affected by permanent CH and 29 by transient hyperthyrotropinemia. Thus PKU shows a frequency of 1∶7,200 newborn infants, and permanent congenital hypothyroidism 1∶5,080. The coordination of screening programs for congenital metabolic diseases in a single central unit allows: — the unification of the input of samples and output of data in a single data bank; — a minimization of the physical and psychological stress to the patients and their families; — and a more satisfactory cost/benefit ratio.

CYTOFLUOROMETRIC ANALYSIS OF LYMPHOCYTE SUBSETS IN THYROID ASPIRATES FROM PATIENTS WITH AUTONOMOUSLY FUNCTIONING NODULE

A microscale method, based on two‐colour dye immunolabelling and flow sorting cytofluorometry, was used to characterize lymphocyte subsets in thyroid tissue specimens obtained by fine‐needle aspiration (FNA) in 21 patients with autonomously functioning thyroid nodule (AFTN) and five patients with cold thyroid nodule (CTN). Inversion of the ratio between CD4+ and CD8+ lymphocytes, due to a relative increase of CD8+ cells, was found among intrathyroidal lymphocytes in AFTN patients. The abnormal lymphocyte subset distribution was not observed in the peripheral blood of the same group of patients. In patients with CTN the lymphocyte subset distribution was normal, both in the thyroid and in the peripheral blood. In AFTN patients, a significant correlation was observed between the decrease of intrathyroidal CD4+/CD8+ ratio and the increase of plasma thyroid hormone levels. Whether the immunological abnormalities found in AFTN could play a pathogenetic role in the clinical presentation and outcome of the disease remains to be established. The FNA‐applied micromethod used in this study could be extended as a routine investigation to characterize the immunogenic substrate of thyroid disorders.

Human undifferentiated thyroid carcinoma synthesizes and secretes 19S thyroglobulin.

Thyroglobulin (Tg) biosynthesis and secretion have been studied in one case of undifferentiated small spindle cell human thyroid carcinoma. From tumor tissue were extracted 32 mg of soluble proteins per gram of wet tissue, compared with 54 mg from control gland; after ammonium sulfate fractionation, density gradient centrifugation, and immunoprecipitation, 1.3 mg of Tg per gram of wet tissue, compared with 31.2 mg from control, were purified. By incubating in vitro up to 3 hours tumor slices with tritiated leucine, galactose, and 125I, the rate of incorporation of leucine and galactose increased with time in tumor tissue. However 125I incorporation into soluble proteins from tumor, at 3 hours of incubation, was only 1.7% of control value. By density gradient centrifugation of labeled soluble proteins a well‐separated component sedimenting in the 19S peak was identified. No radioiodine was detected in the 19S fraction which showed the immunoreactive properties of 19s and was poor in 127I and sialic acid. In the incubation medium of tumor were secreted 0.4 mg of 19S Tg per gram of wet tissue compared with 48 mg of tissue in the control gland. Thus the undifferentiated thyroid carcinoma synthesizes a protein with the phys‐icochemical and immunologic properties of 19S Tg. The undifferentiation of thyroid cells does not affect the biochemical steps involved in the synthesis of Tg.