DNA changes in progressive cervical intraepithelial neoplasia.

Cervical intra-epithelial neoplasm (CIN) is treated as a progressive lesion, even though most CIN will not progress to invasive cancer if left untreated. This study focussed on DNA-cytometric analysis of cytologic smears of patients who had developed invasive cancer after initial smears showing CIN. The first part of the study aimed at describing the DNA-cytometric changes in these progressive ('malignant') CIN lesions. In the second part a cluster analysis was performed on 'malignant' CIN III lesions and CIN III lesions, with 'unknown' malignant potential. The results indicated that 'malignant' CIN lesions developed high DNA-index (DI) values during malignant transformation, as demonstrated by increasing mean DI values, a high percentage of DNA-aneuploidy and 2.5c Exceeding Rates. Furthermore, a trend-like pattern of texture feature values occurred in 'malignant' CIN lesions with increasing severity. These findings provide objective quantitative confirmation of the evolution of nuclear changes during malignant transformation. Cluster analysis showed that it was possible, using a set of four cytometric features, to subdivide the 'unknown' CIN III lesions into a cluster of lesions with feature values similar to the vast majority of the 'malignant' CIN III lesions, and a second cluster of lesions with feature values dissimilar to 'malignant' CIN III. It is argued that the profile of 'malignant' CIN has become clearer and that the results of this study may serve as a basis for a more objective cytopathologic subdivision of premalignant CIN. It may be justified to follow up patients whose lesions do not yet fit this 'malignant' profile. Not treating the non-progressive lesion group will avoid putting these patients at risk.     

Molecular and biochemical screening for the diagnosis and management of medullary thyroid carcinoma in multiple endocrine neoplasia type 2A.

Patients with Multiple Endocrine Neoplasia (MEN) type 2A are at risk for early medullary thyroid carcinoma (MTC). We performed different screening tests for MTC--a recently reported biochemical screening test using omeprazole-induced calcitonin (CT) stimulation and DNA analysis--in fifteen members of two non-consanguineous Brazilian families with MEN 2A. RET proto-oncogene analysis was carried out by direct DNA sequencing of PCR-amplified products for exons 10 and 11. Family 1 showed a germline mutation (C634Y) in three individuals; a sister and a brother with symptomatic MTC; the former also presented with pheochromocytoma and hyperparathyroidism, and her son was a nine-year-old boy of previously unknown status. Family 2 showed the C634R mutation only in the index case, who presented with cutaneous lichen amyloidosis in addition to MTC, pheochromocytoma and hyperparathyroidism. Neither her parents nor her four brothers showed this genetic abnormality, suggesting a de novo RET proto-oncogene mutation in this patient. The controls and patients presented normal basal gastrin levels and a significant increase after omeprazole. Basal CT levels were elevated in patients with MTC and undetectable in control and asymptomatic family members. No subject showed any increase in CT levels after omeprazole treatment. In conclusion, the two most frequent RET proto-oncogene mutations in MEN 2A are present in Brazilian families. In addition, the specificity of basal and omeprazole-stimulated calcitonin is rather limited, and the efficacy of the omeprazole test still needs to be systematically examined. Therefore, RET proto-oncogene analysis must be the first choice for a screening procedure to identify gene carriers in MEN 2A family members and to permit early prophylactic treatment of MTC.     

Molecular pathology and thyroid FNA

This review summarises molecular pathological techniques applicable to thyroid FNA. The molecular pathology of thyroid tumours is now fairly well understood. Molecular methods may be used as a rule‐in test for diagnosis of malignancy in thyroid nodules, eg BRAF V600E point mutation, use of a seven‐gene mutational panel (BRAF V600E, RAS genes, RET/PTC or PAX8/PPARG rearrangement), or as a comprehensive multigene next‐generation sequencing panel, eg ThyroSeq v2. Molecular methods can also be applied as rule‐out tests for malignancy in thyroid nodules, eg Afirma or ThyroSeq v2 or as markers of prognosis, eg TERT promoter mutation or other gene mutations including BRAF V600E, TP53 and AKT1, and as tests for newly defined tumour entities such as non‐invasive follicular thyroid neoplasm with papillary like nuclei, or as a molecular marker(s) for targeted therapies. This review describes practical examples of molecular techniques as applied to thyroid FNA in routine clinical practice and the value of molecular diagnostics in thyroid FNA. It describes the range of molecular abnormalities identified in thyroid nodules and thyroid cancers with some practical applications of molecular methods to diagnosis and prognosis of thyroid nodules and thyroid cancer.     

Molecular mechanisms of thyroid dysgenesis

Thyroid dysgenesis (TD) is the most prevalent form of congenital hypothyroidism. Ttf-1, Ttf-2, Pax8 and the Tshr are expressed at early stages of thyroid development and are implicated in thyroid ontogeny. Mutations in these genes have been found in some cases of TD. The prevalence of familial forms of TD is significantly higher than expected if the disease was only sporadic, allowing to postulate a genetic basis of the disease. Linkage analysis and mutational screening of the four above-mentioned genes in familial forms of TD showed their exclusion as contributors to the disease in some families, implicating genetic heterogeneity and involving other genetic mechanisms. Strategies to uncover new genes involved in TD are therefore needed. We underscore differences in the temporal expression patterns during the human thyroid development with those in animal models. Further, the extrathyroid expression of these genes during human development enables to define the gene-specific malformations that may be present in patients bearing mutations. The data gathered on molecular thyroid development enable precise genetic counselling of affected families. By increasing our knowledge of thyroid development, we hope to uncover new perspectives of genetic screening and eventually of early in utero treatment.     

Changes in tight junctions of thyroid epithelium with changes in thyroid activity

The morphology of the tight junction of rat thyroid epithelium was examined in freeze-fractured material fixed in glutaraldehyde and briefly glycerinated. In normal thyroids the overall appearance of this junctional specialization resembled that of other cell types in many respects. Short-term changes in thyroid activity and hypophysectomy for 3 wk did not obviously affect the appearance of tight junctions. Feeding of the goitrogen, thiouracil, which stimulates secretion of thyroid-stimulating hormone, resulted in the appearance of some very narrow and some very wide, tight junctions or sometimes junctions with both wide and narrow regions within the same cell.     

Report of the committee on structural chromosome changes in neoplasia

An enormous amount of data on neoplasia-associated chromosome aberrations has accumulated over the past two years. More than 4,000 tumors with a chromosome anomaly identified by banding have been published since HGM9, and the total number of cases contained in the registry on which the Catalog of Chromosome Aberrations in Cancer (Mitelman, 1988) is based is now well above 12,000. The information presently available is, however, still in many respects incomplete. First, the data is heavily biased in favor of hematologic disorders. Solid tumors comprise only 20% of the total data base, which is totally disproportionate to the relative contribution of these disorders to human cancer morbidity and mortality. For example, malignant epithelial tumors (carcinomas), which cause almost 80% of all cancer deaths in man, comprise only 7% of the total. Second, our knowledge about early stage tumors is very limited. For example, the great majority of the solid tumors that have been studied cytogenetically have been metastatic lesions or effusions (advanced tumors usually have a large number of complex structural and numerical chromosome aberrations). Obviously, many more such neoplasms will have to be studied before the primary (pathogenetically essential) changes can be distinguished from the confusing variety of secondary abnormalities that may dominate the karyotype. It should be noted that secondary changes may also be nonrandom, and may be important for tumor progression. Therefore, no attempt has been made in this report to distinguish between primary and secondary changes. All nonrandomly occurring abnormalities that met the criteria for inclusion are listed in Table 1 irrespective of their presumed pathogenetic significance. Results of molecular genetic studies (e.g. the demonstration of loss of heterozygosity or gene amplification) were not considered, although they may be included in the HGM10.5 report. A total of 149 nonrandom chromosome changes were identified (Table 1) in 43 different types of neoplastic disorders, including hematologic diseases and malignant lymphomas, as well as tumors of epithelial, mesenchymal, neurogenic, germ cell, and melanocytic origin. The aberrations comprise a variety of structural chromosome rearrangements (translocations, inversions, insertions, deletions, duplications and isochromosomes), and all chromosomes, except the Y chromosome are involved. The great majority (121 of the 149 identified aberrations) represent well-defined, specific structural changes. More than half of them are consistently associated with a particular morphologic disease characteristic. Twenty-eight of the aberrations, although nonrandom, are not characterized as well. Most are deletions or translocations that only affect a certain chromosome region, often spanning several bands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural changes in thyroid epithelium during involution of the hyperplastic thyroid gland

The ultrastructure of the thyroid epithelial cell was examined at various time intervals after induction of involution of the hyperplastic thyroid gland. Thyroids were made hyperplastic by the feeding of thiouracil in a Remington low-iodine diet to male Fischer rats for 3 weeks. Involution was induced by replacing the thiouracil-containing diet with Purina Laboratory Chow, a high-iodine diet. During involution, organelles that play a role in the synthesis and secretion of thyroglobulin, such as the rough endoplasmic reticulum, Golgi apparatus, and apical vesicles, were well preserved and prominent features of the epithelial cell. The apical plasma membrane of many cells was highly irregular for approximately 2 weeks with signs suggesting rapid discharge of apical-vesical contents into the lumen of the follicle. Pseudopods and colloid droplets were present but were not very prominent features of the cell. No signs of extensive autophagy or obvious increased incidence of lysosomes were present, although there was an elevation in the incidence of small dense bodies starting about day 8, and prominent by 15 days. Some of these may be phagosomes formed from luminal debris. The observations indicate that involution of the hyperplastic thyroid in which there is maintenance of the protein synthetic apparatus and little sign of autophagy or death of the epithelial cells is remarkably different from phenomena occurring during involution of prostate or mammary glands.     

Molecular evolution of thyroid peroxidase.

Thyroid peroxidase is a member of a family of mammalian peroxidases that includes myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and salivary peroxidase. Protein sequences showing a high degree of sequence similarity with mammalian peroxidases have recently been observed in several invertebrate species. A multiple sequence alignment prepared with five mammalian and six invertebrate peroxidases shows complete conservation of amino acid residues considered to be important in the formation of peroxidase compound 1. These include the distal and proximal histidines, a catalytic arginine residue, and an asparagine residue hydrogen bonded to the proximal histidine. TPO-2, an alternatively spliced form of TPO, lacks the essential asparagine (Asn 579). It is now possible to speak more broadly of the family of animal peroxidases, rather than mammalian peroxidases. The animal peroxidases comprise a group of homologous proteins that differ markedly from the plant/fungal/bacterial peroxidases in primary, secondary and tertiary structure, but which share with them a common function. Animal peroxidases probably arose independently of the plant/fungal/bacterial peroxidase superfamily and most likely belong to a different gene family. The relationship between animal and non-animal peroxidases probably represents an example of convergent evolution to a common enzymatic mechanism.     

Molecular nuclear therapies for thyroid carcinoma

Thyroid cancer, divided in the subvarieties of papillary and follicular carcinoma, together also called differentiated thyroid carcinoma (DTC), medullary thyroid carcinoma (MTC) and anaplastic thyroid carcinoma (ATC), is the most common endocrine malignancy. Over the course of the last seven decades multiple molecular nuclear therapies have been tried to treat the various varieties of thyroid cancer. The sodium iodine symporter (NIS) substrate I-131 is a well known and extremely successful agent to treat DTC, but is not successful in treating other thyroid cancer varieties and some de-differentiated DTC tumors. An alternative to I-131 are radioactively labeled somatostatin analogues, which have predominantly been used to target MTC, but may also be effective in some DTC cases. In experimental preclinical studies the re-induction of NIS expression or transfection with recombinant NIS shows some promise for the treatment of ATC and dedifferentiated DTC. Furthermore, several other potential radioactive NIS substrates are developed. In this review, we will extensively discuss the aforementioned established therapeutic modalities and promising new concepts in molecular nuclear therapy of thyroid carcinoma.     

Molecular basis of epithelial thyroid tumorigenesis

The results of experiments carried out in different laboratories (including ours) during the last 10 years have enabled us to propose the hypothesis that there are different initiators able to start the epithelial thyroid tumorigenic process via different pathways:--gsp and TSHR genes: at the origin of hyperfunctioning tumors (toxic nodules and adenomas);--ras and probably gsp genes (in a minority of samples): via a vesicular adenoma progressing eventually to a vesicular carcinoma. This could be also the case for ret but only in radiation-associated tumours;--ras, ret, trk and probably gsp and met: starting from small papillary lesions ('spontaneous' or radiation-induced) and progressing to a clinically evident papillary carcinoma;--the p53 gene playing a role only in the final dedifferentiation process. Simultaneous alteration in the same sample of combinations of ras, gsp, ret, trk and TSHR was found in only a minority of the approximately 150 tumours studied. These data suggest an interchangeable role for these genes in the initiation of 'spontaneous' or radiation-associated epithelial thyroid tumorigenesis. The requirement of one of the genes cited above to interact with other genes must not be neglected. Ras is the most frequently altered gene in 'spontaneous' thyroid tumours and ret in radiation-associated thyroid tumours.     

Molecular changes in protoplast-derived rice plants

Summary To determine whether regeneration of rice plants from protoplast culture induces DNA polymorphisms, progeny plants from direct regenerants of such cultures were examined for restriction fragment length polymorphisms (RFLP analysis). Significantly increased levels of DNA polymorphism were found compared with those in non-tissue culture control plants. Analysis with gene sequences representative of different functional domains, revealed that such polymorphisms are apparently widespread and not associated with any particular region. Analysis by comparative digestion with both methylation-sensitive and insensitive restriction enzymes revealed that methylation changes cannot be regarded as a major factor in the induction of these DNA polymorphisms.     

Serum changes in trace elements during thyroid cancers

The objective was to examine changes in trace elements due to thyroid cancer in humans. Serum levels and tissue contents of trace elements (Zn, Cu, Mn, Mg, Fe and Se) were measured in 43 patients with thyroid cancer before and 4 days after surgery were compared to normal values. The serum levels of zinc in cancer patients were lower than those of normal subjects. Surgical removal of the cancer resulted in the restoration of these levels. Although serum Cu levels in patients were not different from normal, but post-operatively these levels rose significantly (p < 0.001). Levels of Fe, Mg and Mn were significantly lower (p < 0.001) post-operatively. There was no significant change in Serum Se levels. The thyroid tissue contents of these trace elements did not show a difference between the normal (Juxta-tumor) thyroid tissue and the cancerous lesion. Out of the six trace elements examined, the decrease of serum levels of zinc in cancer patients may be linked to the disease condition. It is suggested that this change: (a) may be used to demonstrate successful cancer surgery and (b) may have implications for a long-term follow-up of thyroid cancer patients.     

Serum changes in trace elements during thyroid cancers

The objective was to examine changes in trace elements due to thyroid cancer in humans. Serum levels and tissue contents of trace elements (Zn, Cu, Mn, Mg, Fe and Se) were measured in 43 patients with thyroid cancer before and 4 days after surgery were compared to normal values. The serum levels of zinc in cancer patients were lower than those of normal subjects. Surgical removal of the cancer resulted in the restoration of these levels. Although serum Cu levels in patients were not different from normal, but post-operatively these levels rose significantly (p < 0.001). Levels of Fe, Mg and Mn were significantly lower (p < 0.001) post-operatively. There was no significant change in Serum Se levels. The thyroid tissue contents of these trace elements did not show a difference between the normal (Juxta-tumor) thyroid tissue and the cancerous lesion. Out of the six trace elements examined, the decrease of serum levels of zinc in cancer patients may be linked to the disease condition. It is suggested that this change: (a) may be used to demonstrate successful cancer surgery and (b) may have implications for a long-term follow-up of thyroid cancer patients.     

Low-protein diet changes thyroid function in lactating rats

Lactating rats were fed with free access to an 8% protein-restricted diet (PR); the control group was fed a 23% protein diet (C). An energy-restricted (pair-fed) group was given the same food as the animals in the control group, but the amounts of food consumed by both PF and PR were about the same. The body weight and serum albumin concentration of PR and PF dams were significantly (P < 0. 05) lower than that of the controls. The PR group had a significant increase in serum-free triiodothyronine (FT3) concentration, 24-hr mammary gland and milk radioiodine (I131) uptake (67%, 278%, and 200%, respectively) as compared with the controls. On the other hand, those animals had a significantly lower serum-free thyroxine (FT4) concentration and 2- and 24-hr thyroid I131 uptake (67%, 64%, and 74%, respectively). Protein malnutrition during lactation did not alter thyroid or liver 5'-deiodinase activity significantly. However, PF dams had a significantly lower (25%) thyroid 5'-deiodinase activity. These data suggest that protein-restricted lactating dams had an adaptive change in the thyroid function, which could be important to increase the transference of iodine or triiodothyronine through the milk to their pups and prevent sequelae of neonatal hypothyroidism.     

Polyclonal origin of medullary carcinoma of the thyroid in multiple endocrine neoplasia type 2

Multiple endocrine neoplasia type 2 (MEN 2) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma (MTC) and other tumors. Since MTC can also occur in a sporadic form and as familial medullary thyroid carcinoma, this neoplasm offers a unique opportunity to investigate the difference of origin, if any, between the sporadic and the hereditary forms of a tumor. While sporadic malignancies have usually been found to result from a mutational event occurring at the single-cell level and are therefore monoclonal, studies on hereditary neoplasms have been scarce and often produced conflicting results. In order to determine the clonal origin of sporadic MTCs and of those occurring in MEN 2 syndromes we used a clonality assay based on a polymorphic trinucleotide repeat of the X-linked human androgen-receptor gene. We found that 10 out of 11 MTCs expressed a polyclonal pattern of X inactivation, including a significant percentage of the cases clinically defined as sporadic. Received: 21 May 1996 / Revised: 14 August 1996