Immunoreactive gastrin-releasing peptide in medullary thyroid carcinoma.

We have previously shown that immunoassayable concentration of somatostatin (SRIH) was elevated in 70% of 34 consecutive medullary thyroid carcinoma (MTC) tissue samples. In the present study gastrin releasing peptide (GRP)-like immunoreactivity was measured in tissue extracts from these 34 MTC (25 inherited, 7 sporadic, 2 unclassified) and in 7 normal thyroid tissue. Plasma SRIH, calcitonin (CT) and carcinoembryonic antigen were assayed in all patients. Normal thyroid tissue contained less than 61 pmol GRP per g wet weight; in contrast GRP concentration was elevated (62-7800 pmol/g) in 32/34 tumor extracts. The distribution of tissue GRP values were similar in sporadic as well as in familial MTC. We found no significant correlation between tumor GRP concentration and plasma SRIH (r = -0.05), plasma CT (r = -0.24), or plasma carcinoembryonic antigen levels (r = -0.21). Tumor concentrations of immunoreactive GRP and SRIH were positively correlated when logarithmic transformation was used (P less than 0.01). Thus GRP, as well as SRIH, is a major product of tumoral C cells in human MTC when systematically evaluated in a large number of cases.

Anaplastic carcinoma of the thyroid. A clinicopathologic study of 121 cases.

One hundred twenty-one cases of anaplastic carcinoma of the thyroid treated at M.D. Anderson Cancer Center, Houston, were reviewed. Anaplastic carcinoma is a rapidly growing neoplasm with a dismal prognosis. The mean survival of our patients was 7.2 +/- 10 months. A significant percentage of our patients (35%) had areas of well-differentiated thyroid carcinoma elsewhere, supporting the hypothesis that anaplastic thyroid carcinoma arises from preexisting well-differentiated thyroid carcinoma. Twenty-four of 30 tumors analyzed (84%) stained for keratin, 28 (93.3%) stained for vimentin, and ten (33%) stained for epithelial membrane antigen. Younger patients lived longer than older patients, and patients whose disease was earlier-stage at presentation responded better than patients with metastases at presentation. Radical surgery alone did not significantly increase survival duration over less radical surgery. The role of multimodality therapy needs further evaluation.

Establishment and characterization of continuous cell line MTC-SK derived from a human medullary thyroid carcinoma.

Tumor cells of a human medullary thyroid carcinoma were isolated and propagated in tissue culture. Several cell lines with different morphology developed from the primary culture, among others a fibroblast-like growing cell line (MTC-F) and a cell line growing as a suspension of single cells and spherical cell clusters (MTC-SK). The MTC-SK cell line was serially propagated for 90 passages, over 3 years. When examined at different times throughout the in vitro period, MTC-SK exhibited properties characteristic of medullary thyroid carcinomas: the cells maintained their epithelioid morphology; endocrine granules were demonstrated in the cytoplasm by electron microscopy; in situ hybridization confirmed the production of calcitonin- and bombesin-mRNA (gastrin releasing peptide); the cells revealed positive immunoreactivity with antibodies to calcitonin, calcitonin gene-related peptide, and bombesin. The in vitro properties of the MTC-SK cells corresponded to the results obtained from the tissue of origin. Cytogenetic studies of the MTC-F cell line revealed a supernumerary metacentric chromosome (20?). In the MTC-SK cell line the predominant findings were terminal chromosomal rearrangements most frequently concerning chromosome 11p, i.e., the locus of the calcitonin and calcitonin gene-related peptide genes and the H-ras oncogene, and a characteristic instability of the centromeric region of chromosome 16 and somatic pairing of the homologous chromosomes 16.

TSH subunit gene promoters from a murine alpha-subunit producing tumor function normally.

The murine thyrotropic MGH101A tumor is characterized by absent thyrotropin (TSH) beta gene expression and altered thyroid hormone (T3) regulation of the alpha-subunit. Comparison of the promoter structures of both alpha and TSH beta subunit genes from MGH101A with the promoter in expressing TtT-97 thyrotropes revealed no detectable differences. Transfection of the TSH beta promoter from MGH101A linked to luciferase showed minimal expression in primary or cloned MGH101A cells, or L-cells. However, a 6- to 10-fold increase in expression was exhibited in transfected thyrotropes. For the alpha gene, promoter activity was highest in thyrotropes and in cloned MGH101A cells, 5-fold lower in MGH101A tumors, and 10-fold lower in L-cells. Both promoters were not substantially affected by T3 treatment in MGH101A cells. In thyrotropes, promoter activity was inhibited 62.5% and 57.7% by 10 nM T3 treatment for the TSH beta and alpha genes, respectively. DNase I protection showed that factors from TtT-97 but not from MGH101A cells interacted with regions in the TSH beta promoter, while nuclear extracts from each tumor demonstrated at least one protein-DNA interaction with the alpha-subunit promoter. These studies suggest that the molecular defects in the MGH101A tumor are related to the absence of trans-acting factors and are not a result of altered primary gene structure.

Distinct patterns of chromogranin A-related species can be demonstrated in endocrine cells.

We have studied the pattern of chromogranin A (CgA)-related species in different human endocrine cells that produce CgA and also express the calcitonin gene. Antibodies against CgA peptides that span its linear sequence were used in Western analysis of cell lines derived from medullary thyroid carcinoma (MTC), small cell lung cancers (SCLC), epidermoid cell lung cancer (ECLC) and a pulmonary carcinoid tumor (CRND). Each of the cell lines demonstrated a distinct pattern of CgA-related species. Gel filtration studies also revealed multiple and different forms of immunoreactive CgA in the cell lines. Although proteolysis may contribute to our results, these observations suggest that native CgA is processed to smaller species in a tissue-specific pattern by different endocrine cells. More conclusive studies, however, are necessary to establish that cell processing leads to the specific CgA moieties that we have observed.

[Immunohistological coexpression of keratin and vimentin in the epithelial neoplastic cells].

Filed formalin-fixed paraffin blocks of 128 cases of epithelial neoplasms were selected for immunohistochemical study of keratin and vimentin expression. The results showed that 35.1% (45/128) of different carcinomas expressed vimentin. The immuno-positivity of vimentin in thyroid carcinomas, ovarian carcinomas, prostatic adenocarcinomas, pulmonary carcinomas and malignant mesotheliomas were 81.8%, 42.8%, 66.7%, 30.5% and 53.4%, respectively. Carcinomas of breast, kidneys, salivary glands, adrenal glands and nasopharyngeal carcinomas also showed various degrees of positive reaction. The results suggest that an immunohistochemical positive vimentin reaction does not exclude histopathological diagnosis of carcinomas. The significance and noticeable aspects of immunohistochemical methods in histopathological diagnosis are also discussed.

[Immunohistochemical study and lectin distribution of thyroid carcinoma originated from follicular epithelium].

97 cases of thyroid carcinoma originated from follicular epithelium were investigated by using histological and immunohistochemical techniques with special reference to lectin distribution. According to the WHO histological typing of thyroid tumours, these cases were divided into three categories as follows: papillary carcinoma of thyroid (PCT) 56, follicular carcinoma of thyroid (FCT) 31 and undifferentiated carcinoma of thyroid (UCT) 10. Results showed that three different kinds of thyroid carcinoma presented various hormone function and distribution of lectins. The positive rate of Tg immunoreactivity was significantly different between these three kinds of tumour, i.e. PCT greater than FCT greater than UCT. Additionally, the positive rate of T4 and T3 immunoreactivity was lower than that of Tg. Some Gastrin, SS and calcitonin positive cells were also recognized in carcinoma of thyroid. Lectin--binding rate of WGA, PNA, SBA and UEA to 97 cases of thyroid carcinoma and 9 cases of normal thyroid tissue revealed that different lectin had a selective binding activity to various types of thyroid carcinoma and normal thyroid cells. From the data obtained, it seemed that the morphological differentiation of thyroid carcinoma was in correspondence with difference of function, and the extent of cell differentiation may be closely related to the biological behavior of the tumour.

Rectal carcinoid tumor metastasizing to the thyroid and pancreas. An autopsy case exploiting immunohistochemistry for differentiation from tumors involving multiple endocrine organs.

A 58-year-old male patient with rectal carcinoid tumor is presented. The tumor extensively involved the lymph nodes and liver, and multiple tumors were also recognized in the pancreas and thyroid. Grossly, it was uncertain whether the latter were metastases from the rectal carcinoid or all were coincident primary tumors involving multiple endocrine organs, so-called multiple endocrine neoplasia (MEN) syndrome. Histologic, histochemical and electron microscopic examinations of the tumors in both the pancreas and thyroid showed similar features to those of the rectal carcinoid. The neoplastic cells in all involved organs commonly expressed positive immunoreactivity for somatostatin, but negativity for carcinoembryonic antigen, calcitonin, calcitonin gene-related peptide, thyroglobulin, insulin, glucagon and pancreatic polypeptide. These immunohistochemical results confirmed that the tumors observed in multiple endocrine organs were indeed metastatic from the rectal carcinoid, rather than being a new combination of MEN syndrome. Some neuroendocrine tumors may develop widespread metastasis, sometimes creating problems with differentiation from multiple primary endocrine tumors. Immunohistochemistry may be of great help in setting this issue.

[Flow cytometric DNA analysis of papillary carcinoma of the thyroid using paraffin-embedded specimens].

A flow cytometric DNA analysis of papillary thyroid carcinomas has been performed on paraffin-embedded specimens, in order to determine the clinical usefulness of this method by defining the degree of the biological malignancy. Fifty-eight patients with papillary carcinomas were followed from 2 to 8 years. They were classified according to their prognosis into three groups: non-recurrent (47 patients), recurrent (5 patients), and a deceased group (6 patients). Aneuploidy was found in 6 (10.3%) of the total 58 patients. It was more frequently detected in the recurrent (40%) and in the deceased (33.3%) groups than in the non-recurrent group (4.3%). Higher proliferating index values were observed in the deceased group than in the recurrent and non-recurrent groups. These results indicate that a DNA analysis, using routine paraffin-embedded materials, is a useful adjunct in the determination of the degree of the biological malignancy of a papillary carcinoma.

Non-sulphated cholecystokinin in human medullary thyroid carcinomas.

The expression of gastrin/cholecystokinin (CCK) peptides and their precursors was examined in 16 medullary carcinomas of the human thyroid. Measurements with libraries of sequence-specific radioimmunoassays before and after enzymatic cleavage of extracts and chromatographic fractions showed that the carcinomas contained 1.7 pmol carboxyamidated CCK/g tissue (median; range 0.6-21.8 pmol/g), 0.9 pmol glycine-extended precursor/g (median; range less than 0.2-2.3 pmol/g) and 2.3 pmol further COOH-terminal-extended proCCK/g (median; range 0.9-6.2 pmol/g). Neither carboxyamidated gastrins nor any progastrins could be measured. Gel and reverse-phase chromatography revealed only small molecular forms, i.e. greater than 90% of the amidated immunoreactivity eluted like non-sulphated CCK-8 or CCK-7. The results show that human medullary thyroid carcinomas synthesize CCK peptides. The predominance of non-sulphated CCK is unusual. Taken together with earlier observations from dogs and pigs, our results raise the possibility that small non-sulphated CCK peptides modulate thyroid C-cell secretion in an autocrine manner.

Immunoreactive atrial natriuretic peptide in the thyroid gland.

Human thyroid follicles and primary cell cultures derived from them demonstrated atrial natriuretic peptide (ANP)-like immunoreactivity when stained with a monoclonal antibody raised against rat alpha-ANP (ANP 1-28). In thyroid sections the staining was most intense in the tall cuboidal epithelium of small follicles. The intracellular distribution of immunoreactive (ir)-ANP in primary cultures of thyroid follicular cells consisted of discrete granules with a largely perinuclear distribution. The granule density increased with time in culture but was unaffected by exogenous ANP, suggesting an intrinsic synthesis of the immunoreactivity. Thyroid stimulating hormone (TSH; thyrotropin) failed to alter the distribution of ir-ANP after either short-term (6 h) or long-term (1-12 day) exposure. Epinephrine or norepinephrine treatment, however, caused a reduction in the ir-ANP granularity compared with controls in what might represent a stimulated release of the immunoreactivity. The present results suggest that the peptide ANP coexists with thyroid hormones in follicular cells and that the two endocrine activities might be under separate control mechanisms.

[An immunohistochemical study of estrogen receptors in thyroid carcinoma].

Estrogen receptors immunoreactivity (ER-IR) has been examined in 64 cases of human thyroid carcinomas. ER-IR was found to be located in the nucleus of cancer cells and was detected in 30 (57.7%) of 52 papillary carcinomas that were examined. Eleven cases showing a good number of ER-IR positive cancer cells revealed a lowered EORTC index of under 65, and no ER-IR was found in 12 anaplastic carcinomas. These findings indicate that ER-IR might generally bear a similar reciprocal relationship to the malignancy of a thyroid carcinoma.

An immunohistochemical study of calcitonin-containing cells in benign and malignant thyroid lesions.

Parafollicular cells (C-cells) in benign and malignant thyroid lesions were studied immunohistochemically with a polyclonal anti-calcitonin (CT) antibody. The C-cells were seen most frequently in the middle third of the lateral lobes in the thyroid gland of normal individuals and patients with Graves' disease and chronic thyroiditis, although in the latter the number of such cells was significantly decreased (p less than 0.05). In adenomatous goiter, C-cells were present in nodular lesions from an early stage of nodule development (frequency about 19%), whereas in the later stage these cells were rarely observed inside type 1 nodules, which were generally characterized by an admixture of follicles with considerably different sizes. However, C-cells were not observed inside type 2 nodules, which were composed of similar-sized follicles, or in the parenchyma of 56 cases of benign and malignant thyroid tumors. These findings suggest that since C-cells are present in nodular lesions, the histogenesis of adenomatous goiter is quite different from that of follicular adenoma; thyroid neoplasms generally contain no C-cells in the parenchyma.

Karyometric marker features in fine needle aspirates of invasive follicular carcinoma of the thyroid.

Karyometric measurements were performed on fine needle aspirates of clearly identifiable tumor areas and adjacent normal-appearing areas in the surgical specimens from ten patients with invasive follicular carcinoma of the thyroid. Similar measurements were performed on aspirates from nine patients free of thyroid disease (controls). A total of 95 karyometric features were evaluated for each nucleus. Analysis of variance of optical density values indicated (1) a similarity between tumor and normal-appearing nuclei from carcinoma cases, (2) a significant difference between those nuclei and control nuclei and (3) that most of the differences were due to the differences of tissue origin. Stepwise linear discriminant analysis selected ten features that produced a statistically highly significant separation of tumor nuclei from control nuclei. A similar analysis selected six features that produced a statistically highly significant discrimination of normal-appearing nuclei from control nuclei; the validity of those karyometric features as markers of malignancy in normal-appearing nuclei from tissues adjacent to invasive follicular carcinomas of the thyroid was demonstrated by analysis in further training and control sets of nuclei. This analysis in thyroid aspirates identified more marker features than did a previous similar analysis using tissue sections.

Karyometric marker features in fine needle aspirates of microinvasive follicular carcinoma of the thyroid.

Karyometric measurements were performed on fine needle aspirates of clearly identifiable tumor areas and adjacent normal-appearing areas in the surgical specimens from ten patients with microinvasive follicular carcinoma of the thyroid. Similar measurements were performed on aspirates from nine patients free of thyroid disease (controls). A total of 95 karyometric features were evaluated for each nucleus. As compared with the control nuclei, the normal-appearing nuclei showed a 6% increase in total nuclear optical density (OD) while the tumor nuclei showed a 14% increase. Analysis of variance indicated a significant difference between the normal-appearing nuclei and the control nuclei, with most of the difference due to the differences of tissue origin. Discriminant analysis selected nine features that produced a statistically highly significant separation of tumor nuclei from control nuclei. A similar analysis selected five features that produced a statistically highly significant discrimination of normal-appearing nuclei from control nuclei; the validity of those karyometric features as markers of malignancy in normal-appearing nuclei from tissues adjacent to microinvasive follicular carcinomas of the thyroid was demonstrated by analysis in further training and test sets of nuclei.

Hürthle-cell neoplasms of the thyroid gland.

The difference in biologic behavior of Hürthle-cell neoplasms as reported in several series may be explained by the use of different diagnostic pathologic criteria, and the selection of patients with neoplasms of varying clinical stages (treatment at the time of initial diagnosis versus treatment for advanced disease, or initial diagnosis at an advanced stage). On the basis of all of the available evidence, it appears that Hürthle-cell neoplasms exhibit a biologic behavior similar to that of corresponding follicular neoplasms although these latter tumors may have a slightly higher propensity to metastasize. Survival and cure rates are lower than those for papillary cancer and are comparable to those for follicular carcinoma. DNA analysis may be of some help in predicting clinical behavior; aneuploid neoplasms are more often associated with the pathologic features of malignant tumors and may follow a more aggressive clinical course. At present, however, we are unaware of specific surgical strategies that are based upon DNA analysis. Tumor size and morphometric analysis of cytological features have not been particularly valuable for estimating the clinical course of these tumors.

Expression of keratin 19 distinguishes papillary thyroid carcinoma from follicular carcinomas and follicular thyroid adenoma.

Keratin expression with the use of chain-specific monoclonal antikeratin antibodies was investigated in normal thyroid tissue (n = 4), colloid nodules (n = 19), follicular thyroid adenomas (n = 18), follicular carcinomas (n = 10), and papillary carcinomas (n = 12). Frozen sections were stained with monoclonal antibodies M20 (keratin 8), M9 (keratin 18), and LP2K (keratin 19) with the use of the indirect immunoperoxidase technique. The immunohistochemical findings showed that the expression of keratins 8 and 18 was equally extensive in all normal, benign, and malignant lesions tested. In contrast, different staining patterns were observed with the use of monoclonal antibody to keratin 19. Follicular carcinomas were only focally stained with this antibody or were not reactive at all. Keratin 19, however, was present in all the tumor cells of papillary tissues and in a moderate amount of cells of nonneoplastic thyroid lesions and follicular adenomas. In papillary carcinoma, an identical homogeneous expression of keratin 19 was observed in both papillary and follicular structures, which suggests a common cellular origin. These results show that immunohistochemical staining with the use of monoclonal antibody against keratin 19 is useful to distinguish papillary thyroid carcinomas from follicular adenomas and follicular thyroid carcinomas.

Impact of diagnostic immunohistochemistry on the recognition and management of two cases of thyroid cancer with protracted courses.

This report presents two cancer cases with protracted courses in which diagnostic immunohistochemistry for thyroglobulin and/or calcitonin was performed several years after the original light microscopic interpretation. In both cases, diagnostic immunohistochemistry suggested significant changes in tumor classification. In light of current controversies and interpretive problems in this area, confirmatory tests for serum calcitonin and serum thyroglobulin and scans for iodine 131 uptake were performed. These confirmed the immunohistochemical evidence, and led to major changes in patient management. Several similar cases were found in the literature. In cancer cases with a protracted course, but with atypical or discordant clinical and/or pathologic features, diagnostic immunohistochemistry for thyroid markers may merit consideration because of the potential for meaningful changes in clinical management.

The significance of immunochemically staining calcitonin and CEA in fine-needle aspiration biopsy materials from medullary carcinoma of the thyroid.

A review of ten surgical cases of medullary carcinoma of the thyroid (MCT) demonstrates the usefulness of establishing a preoperative cytological diagnosis through the immunochemical staining of calcitonin (CT) and CEA in fine-needle aspiration (FNA) biopsy smears. If MCT is suspected after routinely performing May-Grünwald-Giemsa stained cytology of an FNA biopsy in the outpatient clinic, then CT and CEA staining is recommended for confirmation of MCT. The advantages of this immunochemical staining include that it can be carried out in the outpatient clinic within one day and also that it is much less costly than measuring serum CT and CEA.