Calcium infusion and pentagastrin injection in diagnosis of medullary thyroid carcinoma.

Calcium infusion and pentagastrin injection were compared as tests to stimulate calcitonin secretion for the detection of medullary carcinoma of the thyroid. Plasma concentrations of immunoreactive calcitonin were measured by radioimmunoassay before and during both stimulation tests in 2 persons who had been found at operation to have medullary thyroid carcinoma, 1 relative in whom a cervical lymph node biopsy had shown medullary thyroid carcinoma and 36 asymptomatic relatives. The tests were carried out on separate days by intravenous infusion of calcium gluconate for 2 hours, to provide 3.75 mg/kg of elemental calcium per hour, and rapid intravenous injection of 0.5 microgram/kg of pentagastrin. Before stimulation immunoreactive calcitonin was undetectable in the plasma of 34 of the 36 asymptomatic persons; the 2 with elevated baseline concentrations of the hormone had a positive response to both tests. Seven others showed an increase in plasma immunoreactive calcitonin concentration only after pentagastrin injection. The two persons with initially elevated values and three of the seven with increased values after pentagastrin injection were found at subsequent operation to have focal medullary carcinoma and parafollicular cell hyperplasia; after the operation immunoreactive calcitonin was undetectable in the plasma, even after stimulation. Rapid injection of pentagastrin is more reliable than slow infusion of calcium as a stimulation test for the early detection of medullary thyroid carcinoma.     

Plasma immunoreactive calcitonin in lung cancer.

We have measured plasma calcitonin in 135 untreated eucalemic men with lung cancer and a control/smoker population. Calcitonin levels were determined by radioimmunoassay and validated by immunoextraction. Plasma immunoreactive calcitonin moieties were purified by immunoadsorbent chromatography, treated with mercaptoethanol and urea, and characterized by gel filtration. Artifacts in human calcitonin radioimmunoassays of cancer-patient plasmas were detected by parallel plasma incubations in a salmon calcitonin radioimmunoassay system which does not detect human calcitonin and by immunoprecipitation of tracer at the end of radioimmunoassay incubations. Heating fresh plasmas to 65 degrees C for 1.5 hours reduced radioimmunoassay artifacts without loss of calcitonin moieties. Such characterization of hypercalcitoninemia in each of the histopathological types of lung cancer has raised some important questions about the interpretation of plasma calcitonin radioimmunoassay measurements in lung cancer. Based on inhibition of tracer-antibody binding, plasma calcitonin seemed to be elevated in 18% (14/80) of basal plasma samples obtained from patients with epidermoid or with anaplastic lung cancer. Unequivocal hypercalcitoninemia (heat stable, causing no inhibition of antibody-tracer binding in the salmon calcitonin radioimmunoassays, and immunoextractable with human calcitonin antibodies) was not found in any of the apparently hypercalcitoninemic plasmas from persons with epidermoid or anaplastic lung cancer. By contrast, unequivocal hypercalcitoninemia was found in 27% (15/55) of plasmas from patients with small cell carcinoma or adenocarcinoma. Most of the immunoreactive calcitonin recovered from small cell and adenocarcinoma lung cancer plasmas with unequivocally elevated calcitonin is much larger than calcitonin monomer.     

Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

Immunoperoxidase staining of fine needle aspiration specimens previously stained by the Papanicolaou technique

The use of immunoperoxidase techniques was investigated in 21 fine needle aspiration (FNA) cytology smears that had been previously stained by the Papanicolaou technique. The retrospectively selected slides were destained before applying the immunostain, utilizing antisera to calcitonin, prostatic acid phosphatase (PrAP), prostate-specific antigen (PSA), alpha-lactalbumin (AL), S-100 protein (S-100), carcinoembryonic antigen (CEA), common leukocyte antigen (LA), epithelial membrane antigen (EMA) and alpha-fetoprotein (AFP). Positive results were obtained with six of nine small-cell carcinomas of the lung stained with EMA, all three colonic carcinomas stained with CEA, one of two prostatic carcinomas stained with PSA and PrAP, one of two lymphomas stained with LA and the one medullary thyroid carcinoma stained with calcitonin. Negative staining results were observed in the one melanoma stained with S-100, the two breast carcinomas stained with AL and the one hepatocellular carcinoma stained with AFP. These results indicate that immunostaining can be a helpful diagnostic tool in diagnosing some fine needle aspirates using smears previously stained with the Papanicolaou stain.     

Immunocytochemical localization of insulin- and somatostatin-like material in human breast tumors

Four types of human breast lesions and C3H mouse mammary adenocarcinomas (type A) were examined for the immunocytochemical localization of cells containing hormone-like substances. Insulin- or somatostatin-like immunoreactive material was observed in scattered single cells and nests of tumor cells in seven of eight infiltrating duct carcinomas, and in the majority of tumor cells from an anaplastic carcinoma. A few somatostatin-immunoreactive cells were observed in only one of seven fibroadenomas studied. No immunoreactive cells were observed in mouse adenocarcinomas or in human breast dysplasias. These results suggest that cells with hormone-like immunoreactivity may be a common feature in two types of malignant human breast tumors.     

Basal and stimulated calcitonin secretion in primary hyperparathyroidism before and after parathyroidectomy

To investigate calcitonin secretion in primary hyperparathyroidism, basal and stimulated (3 mg Ca++/kg body weight/10 min) immunoreactive calcitonin plasma levels were studied before parathyroidectomy. Plasma calcitonin levels were raised in 50% of patients regardless of sex, but a significant correlation between basal plasma calcium and calcitonin was found only in males. A reduced calcitonin response to calcium infusion was observed in all patients. Parathyroidectomy invariably induced a normalization of calcitonin basal levels. Our findings confirm the existence of a decreased parafollicular cell reserve probably as a consequence of the persistent hypercalcemic state in hyperparathyroid patients and suggest that it is more frequent in females.     

Heterogeneity of immunoreactive calcitonin in extracts of normal human thyroid

Immunoreactive calcitonin in serum of patients with medullary carcinoma of the thyroid and in extracts of this tumor has been found to exhibit heterogeneity when analysed by gel-chromatography and radioimmunoassay. In this study normal human thyroid tissue was homogenized in buffered saline a neutral ph and a soluble fraction was obtained after ultracentrifugation. Extracts were analysed by gel-filtration before and after treatment with detergents, with denaturing agents and after reduction and alkylation. The stability in vitro of the extracted immunoreactive calcitonin was determined at different temperatures and the changes in heterogeneity analysed by gel-filtration. The results suggest that at least part of the largest form of immunoreactive calcitonin is an artefact of the chromatographic procedure. Moieties with a molecular weight below 12000 Dalton were present and could be identical with prehormones described by others. However, no indication for a sequential degradation releasing calcitonin monomer from larger precursors was found in normal thyroid.     

Production of immunoreactive calcitonin and parathyroid hormone by embryonal carcinoma cells: alteration with retinoic acid-induced differentiation

To determine possible ectopic production of, and altered responsiveness to, specific hormones and growth factors which may be involved in mediating embryonic differentiation and development embryonal carcinoma cells in culture have been employed to serve as an in vitro system of embryogenesis. Exposure of F9 embryonal carcinoma cells to all-trans-retinoic acid previously has been shown to induce differentiation of these undifferentiated stem cells to parietal endoderm and to markedly alter the ability of calcitonin and parathyroid hormone to stimulate adenylate cyclase activity. Evidence is presented that F9 cells secrete immunoreactive calcitonin into the culture medium (200 pg/12 hr/10(7) cells) while parietal yolk sac (PYS) cells secrete immunoreactive parathyroid hormone (800 pg/12 hr/10(7) cells). Retinoic-induced differentiation of F9 cells to endoderm results in a progressive reduction in immunoreactive calcitonin production, while there is an increase in the level of immunoreactive parathyroid hormone found in the conditioned medium. After exposure of F9 cells to retinoic acid for 5 days, little calcitonin is detectable in 12-hr conditioned medium. Changes in the intracellular levels of immunoreactive calcitonin and PTH follow a pattern similar to that noted for changes in the amount of secreted hormones. Thus, immunoreactive calcitonin is produced by undifferentiated F9 cells which possess a calcitonin responsive adenylate cyclase system, while parathyroid hormone is produced by parietal endoderm cells which respond to parathyroid hormone with increased cyclic AMP synthesis. Sephadex G50 gel filtration of F9-conditioned medium shows two peaks of immunoreactive calcitonin with Mr of 3500 and 20,000. Immunoprecipitation of calcitonin from 35S-labeled F9 cells reveals a specific band of 20,000 Mr. Likewise, two peaks of parathyroid hormone immunoreactive material of Mr 8000 and 39,000 are noted after gel filtration of PYS cell-conditioned medium, whereas parathyroid hormone immunoprecipitation from the same cells reveals a specific band of 39,000 Mr. These results raise the possibility that embryo production of these two hormones at specific stages in development may contribute to the regulation of subsequent steps of differentiation.     

Tumour calcitonin. Interaction with specific calcitonin receptors.

The human epidermoid bronchial carcinoma (BEN) cell line has been shown to have specific membrane binding sites for calcitonin and to secrete high-molecular-weight forms (ranging from 40000 to 10000) of immunoreactive calcitonin. Synthetic salmon and human calcitonins and a thyroid extract of porcine calcitonin have been shown to displace 125I-labelled salmon calcitonin from the receptors in a dose-related fashion. The binding to these receptors of calcitonins derived from the BEN cell line and a medullary thyroid carcinoma with molecular weights ranging from 28000 to 3500 (both separated by gel-filtration chromatography) has been investigated. Neither major peaks of BEN-cell-line calcitonin showed receptor binding activity. Only one form of medullary thyroid carcinoma calcitonin, that which co-eluted with synthetic calcitonin monomer on gel-filtration chromatography, caused any significant displacement of labelled hormone from the receptors.     

Neurophysins as markers of vasopressin and oxytocin release. A study in carcinoma of the lung

Vasopressin-neurophysin (hNpI), oxytocin-neurophysin (hNpII) and blood osmolality were assayed before any treatment in basal conditions in 35 patients suffering from lung carcinoma (20 oat cell, 6 undifferentiated and 9 well-differentiated epidermoid cell carcinomas). Plasma vasopressin (antidiuretic hormone, ADH) was also assayed in 7 of the 20 patients suffering from oat cell carcinoma. We found a close correlation (r = 0.98) between plasma ADH and hNpI levels in the 7 patients. Further, hNpI was elevated in 13 out of the 20 oat cell carcinoma patients and in none of the epidermoid-cell carcinoma group; however, searching for an abnormality of ADH secretion as reflected by a detectable plasma hNpI level together with subnormal plasma osmolality revealed 2 additional positive results in the oat cell carcinoma group, and 2 out of the 6 in the undifferentiated-cell carcinoma group. hNpII was increased together with an increase in hNpI in 6 oat cell carcinoma patients; it was specifically increased without hNpI increment in 2 additional oat cell carcinoma patients and in 2 patients of the undifferentiated-cell carcinoma group (different from the 2 positive for the hNpI-osmolality ratio). hNpI and hNpII were normal in the majority of undifferentiated and all of the differentiated epidermoid-cell carcinoma group. Hence, our results show that simultaneous measurements of hNpI, hNpII, and blood osmolality could detect abnormalities in 17 out of 20 oat cell carcinoma patients, in 4 of the 9 undifferentiated-cell carcinoma patients, but in none of the differentiated epidermoid-cell carcinoma patients, suggesting that the neurophysin assay can be used for the early detection of oat cell- and possibly other neuroendocrine-derived carcinomas.     

Immunoreactive calcitonin: a tumor marker for myelogenous leukemias

In a series of 59 patients with chronic or acute myelogenous leukemia (CML, AML) we investigated whether circulating immunoreactive human calcitonin (i-hCT) levels correlate with diagnosis, response to therapy and clinical course. I-hCT was detectable in plasma samples of 88% of patients with CML in the chronic phase and in 100% of patients with CML in blastic transformation. In the AML patients, a significant relation was observed between the cytological subtype and i-hCT levels at diagnosis. In sequentially studied patients the i-hCT plasma concentration was related to the overall mass of leukemic cells, being lower when complete remission was achieved than at diagnosis and increasing at time of recurrence. These data suggest that circulating i-hCT levels can serve as a "tumor marker" in human myelogenous leukemias.     

Qualitative difference of subcellular localization of tumor-associated carbohydrate (Le(x)) antigens in renal cell carcinoma and normal kidney.

Renal cell carcinomas are immunohistochemically positive for oligosaccharides with the Le(x) determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) and its derivatives, as oncofetal antigens, and their expression is closely related to a better prognosis of the patients. This study was designed to clarify the difference in antigen localization at the ultrastructural level between renal cell carcinoma and normal tissues. In normal kidneys, Le(x) detected by monoclonal antibody (MAb) FH 2 and sialylated extended Le(x) (sialyl Le(x)-i) by MAb FH 6 were identified along the plasma membrane of microvilli of proximal tubule epithelial cells, with occasional immunoreactivity along the basolateral plasma membranes. Intracellular localization was very sparse. Renal cell carcinoma showed localization of Le(x) and sialyl Le(x)-i antigens along the cell membrane and in the cytosol as aggregates or filaments. Immunoreactive materials were also observed in the lumen formed among carcinoma cells. The cytosolic immunoreactivity, not observed in the normal kidney, was regarded as "abnormal cytosolic accumulation" of the antigens. This pattern was more pronounced in clear-cell carcinoma. Pretreatment of specimens with chloroform-methanol, which extracts glycolipids, decreased immunoreactivity in carcinoma tissues, particularly that in the cytosol. The extracts contained substances immunoreactive for MAb FH6. Our study has demonstrated that (a) remarkable changes occur in the ultrastructural localization patterns of sialyl Le(x)-i and Le(x) in renal cell carcinoma and (b) considerable amounts of glycolipids are contained in the substances with sialyl Le(x)-i deposited in the cytosol of clear-cell carcinoma.     

Age-related increase of calcitonin gene-related peptide in rat thyroid and circulation.

Elevated calcitonin levels in thyroid gland extracts and in plasma accompanied by C-cell hyperplasia are frequently found in old rats, in particular those raised in laboratory conditions. In parallel with calcitonin, we demonstrate here that the thyroidal content and plasma levels of immunoreactive calcitonin gene-related peptide (i-CGRP) significantly increase with age in rats (p less than 0.0001). C18 Sep-Pak-extractable i-CGRP level in plasma was 35% of the total i-CGRP. Gel permeation chromatography and rp-HPLC studies revealed a number of immunoreactive molecular forms of CGRP and only 40-50% of the acid-extracted immunoreactivity was coeluted with the synthetic CGRP(1-37). The i-CGRP level measured in plasma was highly correlated with the thyroidal content of CGRP (p less than 0.001) and also with the age of the rat (p less than 0.001), suggesting an age-related increase of contribution of CGRP from thyroid gland into the circulation.     

Matricellular CCN6 (WISP3) protein: a tumor suppressor for mammary metaplastic carcinomas

Located at 6q22–23, Ccn6 (WISP3) encodes for a matrix-associated protein of the CCN family, characterized by regulatory, rather than structural, roles in development and cancer. CCN6, the least studied member of the CCN family, shares the conserved multimodular structure of CCN proteins, as well as their tissue and cell-type specific functions. In the breast, CCN6 is a critical regulator of epithelial-to-mesenchymal transitions (EMT) and tumor initiating cells. Studies using human breast cancer tissue samples demonstrated that CCN6 messenger RNA and protein are expressed in normal breast epithelia but reduced or lost in aggressive breast cancer phenotypes, especially inflammatory breast cancer and metaplastic carcinomas. Metaplastic carcinomas are mesenchymal-like triple negative breast carcinomas, enriched for markers of EMT and stemness. RNAseq analyses of the TCGA Breast Cancer cohort show reduced CCN6 expression in approximately 50% of metaplastic carcinomas compared to normal breast. Our group identified frameshift mutations of Ccn6 in a subset of human metaplastic breast carcinoma. Importantly, conditional, mammary epithelial-cell specific ccn6 (wisp3) knockout mice develop invasive high-grade mammary carcinomas that recapitulate human spindle cell metaplastic carcinomas, demonstrating a tumor suppressor function for ccn6. Our studies on CCN6 functions in metaplastic carcinoma highlight the potential of CCN6 as a novel therapeutic approach for this specific type of breast cancer.     

Reduced calcitonin reserve in young hypogonadic osteoporotic men

Calcitonin is a potent inhibitor of bone resorption and in both sexes, plasma levels progressively decrease with age: therefore, a relative deficiency of calcitonin may be involved in the pathogenesis of osteoporosis in the elderly. Calcitonin plasma levels of young hypogonadic men with osteoporosis are significantly lower than controls: the hypothesis that the decreased calcitonin plasma levels in the elderly are due to a reduced secretory capacity of the "C" cells of the thyroid gland, related to age, does not explain the low calcitonin plasma levels found in young hypogonadic osteoporotic men. Our hypothesis is that gonadal steroid deficiency may participate in the mechanisms regulating calcitonin secretion. Therefore, we studied ten males affected by hypogonadotropic hypogonadism and ten normal men, of comparable age, as controls: we measured plasma levels of testosterone, 17 beta estradiol, androstenedione and calcitonin, and the response of calcitonin to an i.v. bolus of pentagastrin, a well known "C" cells stimulatory drug. Testosterone and calcitonin plasma levels and the response of calcitonin to pentagastrin were also evaluated after 6 months of replacement therapy with testosterone. Basal levels of testosterone, 17 beta estradiol, androstenedione and calcitonin, and the response of calcitonin to pentagastrin, are significantly lower in our patients than in controls, demonstrating that hypogonadotropic hypogonadic subjects have a lower secretory reserve of calcitonin. After testosterone therapy the basal calcitonin plasma levels and its response to pentagastrin stimulus did not differ from controls, suggesting that gonadal steroids influence the calcitonin secretion and reserve. Our data cannot clarify whether osteoporosis of hypogonadotropic hypogonadic patients is related to androgen or estrogen deficiency; however, they suggest that the mechanisms by which gonadal steroid influence bone metabolism may involve calcitonin secretion.     

Algorithm for a DNA-cytophotometric diagnosis and grading of malignancy

An algorithm for processing data on nuclear DNA content obtained cytophotometrically was developed (1) to obtain an objective discrimination between benign and malignant lesions in conventional cytologic smears secondarily stained according to Feulgen and (2) to obtain an objective degree of tumor malignancy on a continuous scale of malignancy grades. Investigations in 258 malignant tumors (95 malignant lymphomas, 52 uterine cervix carcinomas, 28 prostate carcinomas, 18 breast carcinomas, 45 malignant bone tumors and 19 larynx carcinomas) and in 74 benign lesions in these organs yielded a diagnostic accuracy of no false-positive, no false-negative and 21% suspicious diagnoses. The probability that "suspicious" cases were malignant was 81%. The overall diagnostic accuracy for non-negative cases thus amounted to 100%. Results in 95 patients with different malignant lymphomas and in 16 patients with squamous-cell carcinoma of the larynx demonstrated the prognostic validity of the DNA-grading system.     

Glycosylation reduces the bioactivity of calcitonin

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.     

Purification and partial characterization of high-molecular-weight forms of ectopic calcitonin from a human bronchial carcinoma cell line.

Studies on the high-molecular-weight immunoreactive calcitonin produced ectopically in culture by an epidermoid bronchial carcinoma cell line are reported. In cell-exposed medium, the principal component has a molecular weight of 40000 and molecules of mol.wts. 13000 and 10000 also occur. Only a trace amount of material co-eluting with 35000-mol.wt. human calcitonin is detectable. None of the calcitonins show cross-reactivity with anti-corticotropin serum. The 40000-mol.wt. immunoreactive calcitonin is readily proteolysed to the 13000- and 10000-mol.wt. components, but the 10000-mol.wt. component behaves as a comparatively stable 'core' molecule. By using immunoprecipitation and high-pressure liquid chromatography (h.p.l.c.), it is possible to prepare radiochemically homogeneous 10000-mol.wt. immunoreactive calcitonin from cells grown in the presence of individual 35S- or 3H-labelled amino acids. Peptide mapping of enzymic digests of this material by h.p.l.c. shows that it contains peptides in common with synthetic human calcitonin.