Fibrosis in undifferentiated (anaplastic) thyroid carcinomas: evidence for a dual action of tumour cells in collagen type I synthesis.

The mechanisms involved in stromal reactions and fibrosis in solid malignant tumours are incompletely understood. In the present study, collagen type I production was investigated in tissues and cell lines derived from human undifferentiated (anaplastic) thyroid carcinomas, a highly aggressive, often fibrotic malignancy with mesenchymal phenotype. In situ hybridization showed the expression of pro-alpha1(I) collagen mRNA throughout the stromal part of the tumours. However, immunofluorescence staining using an anti-pro-collagen type I antibody revealed the synthesis of pro-collagen type I protein mainly in stromal cells juxtaposed to nests of tumour cells. In one out of five tissue samples from human undifferentiated thyroid carcinomas, pro-alpha1(I) collagen mRNA expression was also found in a small number of tumour cells. Several well-characterized cell lines established from undifferentiated thyroid carcinomas, two from tumours included in the present study, expressed both pro-alpha1(I) collagen and prolyl 4-hydroxylase mRNA, and three of these cell lines also synthesized native triple-helical collagen type I. Taken together, these data suggest that stromal fibroblasts are the main producers of collagen type I in anaplastic thyroid tumours. The carcinoma cells seem to play a regulatory role, stimulating the synthesis of collagen type I protein in the surrounding stroma by increasing pro-alpha1(I) collagen mRNA translation. However, collagen type I production by the carcinoma cells might also contribute to the marked desmoplasia commonly seen in these tumours.

Integrins in thyroid tissue: upregulation of alpha2beta1 in anaplastic thyroid carcinoma.

OBJECTIVE: To evaluate the integrin pattern in the normal thyroid gland and in different pathological disorders including malignant tumors, because the aggressiveness of several malignant tumors correlates with alterations in the expression of one or more integrins. DESIGN: We examined the expression of integrins and E-cadherin immunohistochemically in a large and well-defined sample of normal and pathological human thyroid tissue. METHODS: Cryosections of 58 thyroid tissue specimens from normal tissue, thyrotoxicosis, nodular goiter, oxyphilic adenoma, follicular adenoma, follicular carcinoma, papillary carcinoma and anaplastic carcinoma, and three lymph node metastases were investigated immunohistochemically using monoclonal antibodies specifically recognizing the integrin beta1-, beta4-, alpha1-, alpha2-, alpha3-, alpha5- and alpha6-subunits, or E-cadherin. RESULTS: All thyroid epithelial cells expressed integrin beta1- and alpha3-subunits. Immunostaining of the beta4-subunit and the alpha6-subunits was found only in tumors. The staining pattern in the three lymph node metastases from papillary carcinomas did not differ from that in their primaries. Anaplastic carcinomas demonstrated neoexpression of the integrin alpha2-subunit. E-cadherin was detected in all tissues except anaplastic carcinomas. CONCLUSIONS: Neoexpression of alpha6beta4 was seen in most malignant tumors, whereas alpha2 was exclusively found in anaplastic carcinomas. In the latter, a loss of E-cadherin expression was also seen. These changes in cell adhesion molecule expression strongly suggest an association with the acquisition of proliferative and invasive properties.

Efficient detection of mutations in Wilson disease by manifold sequencing.

We have applied a solid support for parallel handling and direct loading of sequencing reactions--manifold sequencing--to analyze the coding sequence for the deficient copper transporting P-type ATPase in 24 families with Wilson disease. At least 100 different amplification reactions could be handled in parallel, with a minimal turnaround time of 12 h from isolated genomic DNA to identification of the mutations. Sixteen different mutations were found, accounting for 92% of the mutant genes. Ten of these mutations have not been previously described. Eleven were observed only in single families. Mutation His1069Gln, previously identified as the most prevalent mutation in Northern Europe, was found in one-third of the Northern European chromosomes in our material. Four patients were homozygous for this mutation, and three were homozygous for Thr977Met. The method allowed us to establish the diagnosis of Wilson disease in 24 h in a patient with acute hepatic failure.

EGF-receptors in human normal and pathological thyroid tissue.

Expression of the epidermal growth factor receptor (EGFR) was studied in cryosections from human thyroid tissues. Normal tissue (4 cases), nodular goitre (12), toxic goitre (9), adenoma (9), follicular carcinoma (1), papillary carcinoma (7) and poorly differentiated carcinoma (1) were used for immunohistochemistry. Northern blot analysis was performed in two nodular goitres, three adenomas, two papillary carcinomas, one follicular carcinoma and the adjacent normal tissue in five cases as well as in two cell lines from anaplastic carcinomas. Epidermal growth factor receptor immunoreactivity was detected in all tissues examined. The amount of EGFR mRNA did not differ between normal and abnormal tissues. However, the EGFR staining was weaker in normal thyroid tissue compared to the adjacent neoplastic areas suggesting an upregulation at the posttranslational level in the latter. A strong staining was also seen in hyperfunctioning thyroid glands. The EGFR location was mainly basal or basolateral in all thyroid tissues with normal histology and in toxic diffuse goitre. Pericellular and sometimes cytoplasmatic staining was seen in neoplastic tissues. In nodular goitre the staining was both basal, lateral and apical and varied in intensity. Our data suggest that a non-polarized location of EGFR probably indicates a loss of the normal epithelial cell polarity and could be interpreted as an early sign of dedifferentiation. Furthermore, a role for the EGFR is proposed, not only in the development of thyroid neoplasias but also in goitre formation.

Long-term treatment of Wilson's disease with triethylene tetramine dihydrochloride (trientine).

Long-term treatment with triethylene tetramine dihydrochloride, (trientine, TETA) was evaluated in 19 patients with Wilson's disease (WD). Two were given the drug as first choice and 17 after treatment with penicillamine. The change was made because of side-effects, lack of improvement or worsening of neurological symptoms. All penicillamine-induced side-effects reverted. Thirteen patients still receive trientine, and the mean total observation time on this treatment is 8.5 years/patient. Seven of the 13 are free from symptoms related to WD, five have mild to moderate neurological symptoms, mainly dysarthria. One patient with neurological symptoms who received trientine from the start of treatment deteriorated rapidly and is now severely dystonic. The symptoms initially worsened and later improved in one patient. All other patients improved during trientine treatment. Three patients died: two from a multifocal cancer including the liver and one non-complier from a ruptured spleen. Two patients underwent liver transplantation for progressive liver failure: one non-complier and one with liver cirrhosis whose liver function deteriorated despite treatment; both are now free from symptoms. Unexpectedly, two patients developed a serious colitis, one with duodenitis as well, that improved after withdrawal of the drug. No other unfavourable effects of trientine were recorded.

Calcium homeostasis in pregnancy during long-term heparin treatment.

OBJECTIVE: To investigate the effect of subcutaneous heparin treatment on calcium homeostasis in pregnancy. DESIGN: A longitudinal case-control observational study. SETTING: Department of Obstetrics and Gynaecology, Karolinska Hospital, Stockholm, Sweden. SUBJECTS: 36 pregnant women with previously verified thromboembolic complications and 23 healthy pregnant control women similar in age, parity, weight, and smoking habit. INTERVENTIONS: Thromboprophylaxis during pregnancy and 6 weeks post partum was given with subcutaneous heparin twice daily to the 36 women with a history of thromboembolic complications, 16 received an average dose of 24,500 IU/day and 20 a mean dose of 17,300 IU/day. Venous blood and urine samples were obtained every 4 weeks. MAIN OUTCOME MEASURES: Serum concentrations of total calcium, ionized calcium, calcitonin and urinary calcium. RESULTS: Women on high-dose heparin treatment showed significantly higher concentrations of total and ionized calcium and of calcitonin in serum and significantly lower concentrations of calcium in urine than did 23 normal pregnant controls. The differences were most pronounced in the third trimester. The results obtained in the low-dose heparin group were between those in the high-dose and the control groups. At 8 weeks postpartum there were no significant differences between the heparin-treated women and the controls. No significant differences were found during pregnancy in haematocrit, liver or renal function, serum levels of albumin, phosphate, magnesium, alkaline phosphatase, parathyroid hormone or urinary cyclic AMP. CONCLUSIONS: Heparin treatment during pregnancy results in changes in calcium homeostasis and a dose-dependent response is suggested.