Microfluoremetric analysis of DNA content changes in murine teratocarcinoma.

The multipotential stem cell of the murine tetratocarcinoma, embryonal carcinoma (EC), is capable of differentiation in vivo and in vitro to nonneoplastic progeny. Undifferentiated EC cells, spontaneously differentiating tetratocarcinoma cells, and differentiated cells derived from EC cells were analyzed for DNA content and chromosome number distributions. Flow microfluorometric and fluorescence cytophotometric analysis of DNA content showed that EC cells had a characteristic diploid (2c) distribution, whereas several differentiated cell lines derived from EC cells had 4c DNA distributions. The tetraploid cell populations studied were capable of cell division but had restricted differentiative potential and were either of low tumorigenicity or non-tumorigenic. In vivo teratocarcinomas, comprised of both EC cells and differentiated cell types, contained diploid and tetraploid populations. Chromosomally, EC cells were neardiploid (39 chromosomes) and differentiated cells were near-tetraploid (62 to 76 chromosomes). The teratocarcinoma provides a model for studying the basic mechanisms that control the growth dynamics of the rapidly and slowly proliferating cell populations present in many tumors.

Cytogenetic evidence for premeiotic transformation of human testicular cancers.

To determine the point at which transformation of the germ cell occurs during meiosis in nonseminomatous testicular cancer, the sex chromosome compositions of 15 cell lines derived from primary tumors or metastases of 12 patients with testicular cancer were analyzed by trypsin G-banding analysis and Y-body staining. The simultaneous existence of both X- and Y-chromosomes in a single cell has been confirmed in 14 cell lines. This suggests that transformation of the cell occurs before the first meiotic division because it is known that segregation of X- and Y-chromosomes occurs during the first meiotic division. An incidental finding was the presence of Barr bodies in some cell lines containing more than one X-chromosome, which is consistent with the known primitive nature of testicular cancer and its ability to differentiate independently from the male host.

Molecular properties of F9 embryoglycan recognized by a unique antibody in sera from patients with germ cell tumors.

Human antibody against an embryoglycan present on a mouse teratocarcinoma cell line F9 was found in sera from 16 of 29 patients with embryonal carcinoma, yolk sac tumor, immature teratoma, and choriocarcinoma of gonadal and extragonadal origins by Farr assay. In contrast, none of the sera from patients (77 cases) with dysgerminoma, seminoma, germinoma, and mature teratoma or from patients (118 cases) with nongerm cell types of ovarian tumors contained this antibody. The antigenic embryoglycan was of high molecular weight (Mr greater than 70,000) on Sephacryl S300 column chromatography and carried binding sites for Grifonia simplicifolia agglutinin-1. The antigenic embryoglycan was also found in F9 cell-cultured medium. Absorption of patients' sera with synthetic Blood Group B trisaccharides failed to remove the antibody against F9 embryoglycan. None of these patients' sera showed higher hemagglutination titer to rabbit erythrocytes than the normal range. In contrast, alpha-galactosyl carbohydrates obtained from Ehrlich ascites tumor cells effectively inhibited the binding of patients' sera with F9 embryoglycan. These results indicate that the human antibody against F9 embryoglycan recognizes alpha-galactosyl structures that are distinct from B blood group antigen, but are cross-reactive with alpha-galactosyl structures on Ehrlich ascites cells.

Disease relapse in patients with stage I nonseminomatous germ cell tumor of the testis on active surveillance.

Thirty-six patients with apparent stage I nonseminomatous germ cell tumor (NSGCT) of the testis were treated by inguinal orchidectomy and intensive follow-up only. Assessment included measurement of serum alpha fetoprotein (alpha FP) and beta human chorionic gonadotropin (beta HCG) (tumor markers) and chest x-ray monthly for 1 year, then twice monthly for 1 year, with computed tomographic (CT) scans of abdomen and chest repeated three times monthly for the first year and six times monthly for the second year. Median follow-up was 36 months (range, 14 to 92 months). Relapse occurred in 12 patients (33.3%) at a median of 7 months (range, 2 to 28 months). Elevated markers were of limited importance in relapse detection, confirming the need for close clinical and radiological follow-up. Of nine histological factors examined in the primary tumor only the presence of lymphatic invasion was associated with a significantly higher relapse rate. All patients were treated at relapse with cisplatin-based chemotherapy. Four underwent surgery in addition, two before and two after chemotherapy. Eleven were rendered disease-free, but four had a second relapse. One patient has died, one is alive with disease, and ten are disease-free. Chemotherapy failed to cure six patients who had relapsed but bulk of disease was not a factor. Despite the good overall result reported here, optimal postorchidectomy management of apparent stage I disease remains to be defined.

Isolation of 16,000-dalton parathyroid hormone-like proteins from two animal tumors causing humoral hypercalcemia of malignancy.

A 16K PTH-like protein with a unique primary structure has recently been isolated from several human tumors associated with the syndrome of humoral hypercalcemia of malignancy. Certain spontaneous and transplantable animal tumors also cause this syndrome. The responsible mediator in these animal tumors is not known. We report the isolation of 16K proteins from the rat H500 Leydig cell tumor and the canine apocrine cell adenocarcinoma of the anal sac. Both proteins are potent activators of PTH receptor-coupled adenylate cyclase in bone cells. Both proteins demonstrate similarities in amino acid composition to one another and to the human PTH-like protein. Limited amino-terminal sequence information from the canine protein demonstrates homology with the human PTH-like protein. Antibodies raised to a synthetic human PTH-(1-36)-like peptide cross-react with both the rat and canine proteins in an immunoradiometric assay. These data demonstrate that by physical and immunological criteria PTH-like peptides are present in these animal tumors that appear to be closely related to the human PTH-like peptide. These data further suggest that this protein is not unique to humans, but has an evolutionary origin which extends back at least 65-80 million yr.

A monoclonal antibody against a synthetic peptide is specific for the free native human chorionic gonadotropin beta-subunit.

A totally synthetic molecule (109-145 peptide) analogous to the beta-subunit carboxyl terminus was used as an antigen in the development of antibodies by the hybridoma technique. A monoclonal antibody (702 D7) specifically recognized the free native beta-human CG (beta hCG). 702 D7 was of the immunoglobulin G1 subclass and was directed against an antigenic site localized in a 10-amino acid sequence (109-118) or less. The recognition of an epitope located in the 109-118 region could explain the specific recognition of beta hCG observed with 702 D7, in contrast to monoclonal antibodies directed against a 118-145 region with a recognition of both beta hCG and whole hCG, as observed with a second monoclonal antibody (1032) to synthetic peptide. Immunohistochemical results and preliminary data obtained from the immunoradiometric assay show that 702 D7 provides a clinical tool for the detection of free beta-subunit secretion even at low concentrations, and could allow the study of this subunit or its metabolites produced by normal and tumoral cells.

Thyrotropic activity of acidic isoelectric variants of human chorionic gonadotropin from trophoblastic tumors.

Previous studies have indicated that hCG has a weak intrinsic thyroid-stimulating activity. Differences in the molecular composition and biological activity of hCG in patients with trophoblastic diseases and pregnant women occur, but are not well defined. Therefore, we have studied the effect of serum samples and purified hCG preparations from patients with trophoblastic diseases on T3 release from human and porcine thyroid slices in vitro. We examined 30 serum samples from 13 patients with nonseminomatous testicular germ cell tumors, 3 from women with choriocarcinoma, and 5 from patients with hydatidiform moles. In all but 1 serum sample from the tumor patients, but in none of 11 serum samples of pregnant women, T3-releasing activity was found. Two patients with testicular cancer and 1 patient with molar pregnancy experienced episodes of frank hyperthyroidism. Isoelectric focusing on polyacrylamide gels of tumor sera (n = 15) revealed substantial amounts of acidic isoelectric variants, pI 3.3-3.9, which were only barely detectable in pregnancy sera. The percentage of acidic hCG variants with pI 3.3-4.0 to total hCG with pI 3.3-5.2, as determined by hCG (+hCG beta) RIA of the eluted fractions of polyacrylamide gel isoelectric focussing, varied from 12-45% in sera of tumor patients and from 0-4% in pregnant sera. We purified the acidic variants of hCG with pI 3.6-3.8 (hCGav) from the urine of our patients. The beta-subunit of purified hCGav had a slightly higher mol wt (35,750) than that of hCG CR 119 (34,190) on polyacrylamide gel electrophoresis. The hCGav showed a dose-dependant stimulation of T3 release and cAMP generation from human thyroid slices, whereas the other hCG fractions on isoelectric focussing had no thyrotropic effect in similar dose levels. The TSH-like activity of hCGav could be roughly estimated as 10 mIU TSH/IU hCGav. Anti-hCG (+hCG beta) antiserum, but not anti-hTSH antiserum, neutralized the biological activity of hCGav. These findings strongly suggest that acidic hCG variants act as functional stimulators of the human thyroid in vitro. Since these molecular variants of hCG can exist in patients with trophoblastic diseases in significant amounts, they could be responsible for some cases of hyperthyroidism in trophoblastic diseases.

Measurement of circulating parathyroid hormone-related protein in rats with humoral hypercalcemia of malignancy using a two-site immunoradiometric assay.

The Rice H500 rat Leydig cell tumor is a well characterized model of humoral hypercalcemia of malignancy (HHM). Circulating concentrations of PTH-related protein (PTHRP) have not been reported in this or any other animal model of HHM. Taking advantage of the marked N-terminal amino acid homology between rodent and human PTHRPs, we have adapted a sensitive two-site immunoradiometric assay developed for measurement of human PTHRP for use in measuring rat PTHRP. Circulating calcium and PTHRP concentrations were serially measured after sc passage of the Leydig cell tumor in rats. Significant hypercalcemia and elevation of PTHRP occurred on day 9 after tumor inoculation. When grouped by tumor size, both plasma calcium and PTHRP levels were significantly elevated in animals with tumor burdens greater than 10 cc. The PTHRP concentration was strongly correlated with both serum calcium (r = 0.88) and tumor size (r = 0.80). Circulating rat PTHRP averaged 12.8 pM on day 9 and 27.5 pM on day 10 or 11. PTHRP was undetectable in the plasma of 19 control rats. In 3 rats, plasma calcium returned to normal, and PTHRP became undetectable within 24 h after tumor excision. Rat milk displayed a PTHRP concentration of 2000 pM, while acid-urea extract of the rat tumor contained 0.32 pmol/mg protein. Dilutions of rat plasma, milk, and tumor extract displayed response curves that were parallel to the human PTHRP-(1-74) standard in the assay. This two-site immunoradiometric assay is a sensitive and easily performed means of measuring rat PTHRP. It should be useful in studying this animal model of HHM and the function of PTHRP in normal tissues.

A simultaneous flow cytometric assay for c-myc oncoprotein and DNA in nuclei from paraffin embedded material.

A simultaneous flow cytometric assay for the c-myc oncoprotein and DNA in nuclei extracted from archival paraffin wax embedded clinical biopsies is presented. The nuclei were extracted by pepsin digestion after dewaxing 20 micron sections. The c-myc oncoprotein was probed with a mouse monoclonal antibody. This was raised against a synthetic peptide corresponding to a hydrophilic region of the protein predicted from the amino acid sequence. The technique is illustrated with biopsies from patients with testicular cancer and with benign and malignant neoplasms of the colon.

A rapid single step staining technique for DNA analysis by flow microfluorimetry.

A new procedure is reported for the staining of DNA, for flow microfluorimetry. It allows the production of stained cell nuclei in a single step by incorporating the DNA stain with a solution of the nonionic detergent Triton-X-100. This method has been found to be applicable to all DNA fluorochromes tested (ethidium bromide, propidium iodide, mithramycin, DAPI, Hoechst 33342). DNA histograms obtained in this way are comparable to those using conventional staining techniques, e.g., ethanol fixation followed by staining. Using this procedure the DNA content distribution of solid tissue or cells from suspension or monolayer cultures can be generated in less than 5 min.

DNA flow cytometry in human testicular cancer.

DNA flow cytometry revealed aneuploid tumour stemlines in 19 of 20 primary testicular cancers without significant difference of the ploidy values between seminomas and non-seminomas. In 7 of 8 analyzable histograms the S-phase activity was 22-51%. A metastatic mature teratoma had 6% cells in S-phase. These results support the clinical observation that testicular cancer is usually a rapidly growing human tumour. The high percentage of aneuploidy in testicular cancer may be of clinical value in the diagnosis of this malignancy.

Serum and cellular biologic tumor markers in patients with urologic cancer.

During the past several years the development of radioimmunoassay and immunocytochemical techniques to detect small amounts of marker in the sera and cancer cells of cancer patients has made a significant impact on the diagnosis and management of certain cancers. Among these markers alpha-fetoprotein, human chorionic gonadotropin, and pregnancy specific beta-1 glycoprotein have been useful in the staging, detection of recurrence, prognosis, and management of testicular cancer. In this article the recent developments and future perspectives concerning these and other newer markers are discussed.

The potential value of immunohistologic techniques in the classification of ovarian and testicular tumors.

The diagnosis of neoplasia continues to rest upon the judicious application of morphologic criteria for the identification of tumor cells. Immunohistologic techniques offer the pathologist an alternative means of cell identification according to the antigenic constitution of the cell or its products. The potential effect of these techniques upon the diagnosis and classification of neoplasia is illustrated by the application of immunoperoxidase methods to the study of alpha-fetoprotein, human chorionic gonadotrophin, and steroid hormone localization in tumors of the ovary and testis.

Malignant Sertoli cell tumour of the testis. An immunohistochemical study and a review of the literature.

The fifteenth case of malignant Sertoli cell tumour is reported and the literature is reviewed. The reported case was unilateral with lung metastases. Immunohistochemical examination showed positive staining reaction within the tumour cells for vimentin and cytokeratin, while AFP, HCG, PLAP, EMA and CEA were not found, which is in accordance with the staining pattern found in normal Sertoli cells.

Immunohistochemical demonstration of multiple neurohormonal polypeptides in a case of pure testicular carcinoid.

A primary pure testicular carcinoid from a 48-year-old man was examined with antisera raised against various neurohormonal polypeptides. Histochemically, both argyrophil and argentaffin reactions were positive. The immunohistochemical finding of immunoreactive cells for 5-hydroxytryptamine (5-HT) (serotonin), substance P, and vasoactive intestinal polypeptide (VIP) demonstrated the multihormonal nature of this tumor.

Carcinoembryonic antigen staining in choriocarcinoma.

Choriocarcinoma of the colon presented as life-threatening lower gastrointestinal bleeding and a pathologic femoral fracture in a 42-year-old man. Elevated serum carcinoembryonic antigen (CEA) levels and immunohistochemical positivity for CEA in neoplastic syncytiotrophoblasts were noted, as well as the expected positivity for the beta subunit of human chorionic gonadotropin (beta HCG). Additional cases, including one of two gestational choriocarcinomas and one of four testicular choriocarcinomas studied immunocytochemically for CEA also demonstrated positivity. Although CEA staining is commonly associated with tumors derived from the surface epithelium of the gastrointestinal tract and other organs, its presence in choriocarcinomas should not be interpreted as conclusive evidence of primary origin from these sites.

The differential diagnosis of yolk sac tumor and seminoma. Usefulness of cytokeratin, alpha-fetoprotein, and alpha-1-antitrypsin immunoperoxidase reactions.

Yolk sac tumor and seminoma may have a similar appearance in focal areas. Small biopsies may therefore be difficult to interpret. The authors studied 20 yolk sac tumors and 21 seminomas to investigate the utility of immunohistochemical markers in this differential diagnosis. All yolk sac tumors stained positively for cytokeratin (CK) but so did 43% of seminomas. The CK positivity of yolk sac tumors was generally more diffuse and intense, however, there was an overlap in the spectrum of intensity of CK positivity in yolk sac tumor and seminoma. Alpha-fetoprotein (AFP) was a less sensitive (55%) marker for yolk sac tumor than CK, but AFP was quite specific in this differential diagnosis because no seminoma stained for AFP. Alpha-1-antitrypsin was not a very useful marker because of poor sensitivity and specificity. The interpretation of light microscopic patterns remains of paramount importance in the differentiation of solid yolk sac tumor from seminoma.

Detection of pregnancy specific beta1-glycoprotein in formalin-fixed tissues.

Using an enzyme-bridge immunoperoxidase method, pregnancy specific beta1-glycoprotein (PSbetaG) has been demonstrated in the cytoplasm of the trophoblast in several formalin-fixed tissues, namely, implantation sites of ovum, normal placentae, hydatidiform moles, invasive moles, and choriocarcinomata of uterus and testis. It is suggested that this technique may prove helpful in the detection of choriocarcinomatous elements in malignant tumours.

Observations on reliability of commercial assay for alpha-fetoprotein.

Thirty patients with nonseminomatous germ cell tumors of the testis were monitored with serum levels of alpha-fetoprotein (AFP). Each patient had at least one AFP determination performed in duplicate: one by the National Institutes of Health (NIH) and one by a commercially available assay. With respect to normal and elevated levels, there was agreement in 60 of the 69 duplicate AFP determinations (87 per cent). Six of the 9 discordant AFP determinations were due to increased sensitivity (a lower normal range) of the NIH assay. There was a close correlation between the quantitative levels of AFP when both the NIH and commercial levels were elevated.

Testicular plasmacytoma.

The case of a sixty-five-year-old man with multiple myeloma and a testicular plasmacytoma is described. This represents the thirty fourth reported case of testicular plasmacytoma and the first in which immunoperoxidase histochemistry has been used to demonstrate that the testicular plasma cells contain immunoglobulin of the same isotype as the patient's paraprotein. The clinical and morphologic features of previously reported testicular plasmacytoma are reviewed.