Search

Two 140 kDa collagenous glycoproteins were isolated from 5 M guanidinium chloride extracts of human uterine leiomyoma by two-dimensional preparative gel electrophoresis. The glycoproteins represented the major concanavalin A binding fraction of the extract and were also present in adult human skin. On two-dimensional gel electrophoresis the glycoproteins appeared as elongated spots, indicating variations of their isoelectric points from 5 to 6. These glycoproteins were disulfide-bonded components of high molecular mass protein and, after reduction, became sensitive to collagenase treatment that generated peptides corresponding in size to those of the noncollagenous domains of type VI collagen. Antisera raised against these purified glycoproteins reacted with either pepsin-derived alpha 1(VI) or pepsin-derived alpha 2(VI) chains but not with alpha 3(VI) chain of human type VI collagen. Reciprocally, these glycoproteins reacted with monoclonal antibodies against type VI collagen. These results indicate that the glycoproteins represent the integral alpha 1 and alpha 2 chains of type VI collagen. The globular domains of alpha 1(VI) and alpha 2(VI) chains remaining after collagenase treatment appeared on two-dimensional gel electrophoresis as elongated spots, suggesting that the noncollagenous portions determine the well known microheterogeneity of the molecule. The differences in isoelectric points between and within alpha chains may facilitate the formation of microfibrillar network.

The distribution of prekeratin, vimentin, epithelial membrane antigen (EMA), and secretory component (SC) was demonstrated immunohistochemically in 31 patients with adenomatous hyperplasia (AH), 12 patients with atypical adenomatous hyperplasia (AAH), and 39 patients with endometrial carcinoma. Prekeratin was presented in 94% of AHs, 92% of AAHs, and 87% of adenocarcinomas. Vimentin was detected in 68% of AHs, 50% of AAHs, and 37% of adenocarcinomas, showing decreased expression as the lesion progressed to malignancy (P less than 0.05). EMA was detected in 26% of AHs, 67% of AAHs, and 95% of adenocarcinomas (P less than 0.001). SC demonstrated focal and weak expression in 29% of AHs, but showed increased staining intensity in 67% of adenocarcinomas (P less than 0.01). Well-differentiated tumors expressed SC better than poorly differentiated tumors (P less than 0.01). All markers showed a heterogeneous staining pattern and, for a given histologic hyperplastic or neoplastic state, corresponded to several phenotypes. In conclusion, prekeratin seems to be a good marker for epithelial differentiation in hyperplastic endometrium, and EMA is a good marker in neoplastic endometrium. In hyperplastic lesions, the loss of vimentin expression in the absence of secretory changes gives rise to suspicions regarding their benign process. Also, EMA can help in distinguishing between hyperplastic and neoplastic states, while detection of SC may be of help in more precise grading of endometrial carcinoma.

The aim of this study was to evaluate tissue and steroid receptor heterogeneity in endometrial carcinoma specimens as a possible source of discordance between biochemically assayed receptor status and response to endocrine treatment. For this purpose the oestrogen receptor (OR) and progesterone receptor (PR) levels in specimens from 16 endometrial carcinoma patients were analysed on adjacent tissue sections using both a radiochemical and an immunohistochemical assay. With immunohistochemical receptor analysis extensive tissue and tumour cell receptor heterogeneity was observed. Many tumour samples revealed up to 75 per cent contamination with benign tissue. In the majority of cases, evaluation of immunoreactivity in normal tissue elements of the specimen could explain the apparent discordance between semiquantitative immunohistochemical receptor scoring of tumour cells and radiochemical receptor assay. Immunohistochemical analysis of OR and PR in endometrial carcinoma specimens allows a more specific determination of tumour cell receptor content and hence may yield a more accurate prediction of response to endocrine therapy than the biochemical assay.

Paraffin-embedded tissue samples from 256 patients who received primary treatment (surgical staging, reduction of tumor size, and adjuvant therapy based on surgical and pathologic risk factors) for endometrial carcinoma at the Mayo Clinic between 1979 and 1983 were analyzed by flow cytometry to determine DNA ploidy characteristics. Diploid patterns constituted 78% of the cases, whereas aneuploid and tetraploid patterns accounted for 17% and 5%, respectively. Only 10% of patients with diploid tumors had a relapse in comparison with 39% of those with nondiploid lesions (34% with aneuploid; 58% with tetraploid). Significant differences (P less than 0.001) were noted in estimated 4-year progression-free survivals--88% for patients with diploid and 57% for those with nondiploid tumors. Stage, grade, depth of myometrial invasion, histologic subtype, peritoneal cytology, and DNA ploidy all demonstrated independent prognostic significance (P less than 0.001) in this study population. When subjected to multivariate analysis, however, grade and depth of myometrial penetration failed to retain prognostic significance (P greater than 0.15) and surgical stage was marginally significant (P = 0.05), whereas histologic subtype and DNA ploidy maintained significant predictive powers (P less than 0.001 and P less than 0.01, respectively). We conclude that DNA ploidy is a major objective prognostic factor and therapeutic determinant for endometrial carcinoma.

The prognostic value of the pretreatment serum CA 125, squamous cell carcinoma antigen (SCC), and carcinoembryonic antigen (CEA) levels in relation to tumor type, vascular invasion by tumor cells, and lymph node metastases was investigated in 77 patients with cervical adenocarcinoma. In Stage IB (International Federation of Gynecology and Obstetrics [FIGO]), the five-year actuarial survival of patients with pretreatment serum CA 125 levels greater than 16 U/ml was 52.4% versus 95.6% when normal serum CA 125 levels were determined (P less than 0.01). Pretreatment serum SCC or CEA levels had no substantial prognostic value. In Stage IB (FIGO), 42% of the patients with elevated serum CA 125 levels had lymph node metastases versus 4% when normal levels were found (P = 0.012). The presence of vascular invasion (P = 0.01) or lymph node metastases (P = 0.001) was associated with an increased risk for recurrent disease. Adenosquamous tumors showed a higher incidence of vascular invasion (P = 0.05) and a higher incidence of elevated serum CA 125 levels (P = 0.03). Particularly in Stage II, adenosquamous tumors were found to have a poorer prognosis than adenocarcinomas (P = 0.0566). We conclude that in cervical adenocarcinoma serum CA 125 is an important prognostic factor and an implicit indicator of tumor virulence.

Primary and metastatic tumor tissues of serous papillary adenocarcinoma of the endometrium were examined for the following: (1) amplification of int-2, c-erbB-2 and c-myc proto-oncogenes by Southern blot hybridization; (2) DNA ploidy by flow cytometric study; (3) and expression of specific proteins, such as estrogen and progesterone receptors, keratin, vimentin, and carcinoembryonic antigen (CEA) using immunohistochemical and biochemical techniques. Amplification of c-myc was observed in the specimens from the endometrium (ten-fold) and from omental metastasis (five-fold). Both int-2 and c-erbB-2 amplification were not observed. The tumor showed aneuploidy, with the specimens from the endometrium and omental metastasis exhibiting multiple populations of aneuploid tumor cells. Estrogen and progesterone receptors could not be detected biochemically; however, immunohistochemically, estrogen receptors were observed in tumor cells forming papillary structures but not in the tumor cells of the solid, more poorly differentiated areas. A similar distribution was observed for both low and high molecular weight keratin. The findings of c-myc amplification and aneuploidy in the serous papillary adenocarcinoma of the endometrium are consistent with its aggressive behavior observed clinically and emphasize the importance of distinguishing this lesion from other types of endometrial carcinoma.

In view of advancements in treatment of certain hormone-dependent cancers with analogues of luteinizing hormone-releasing hormone (LH-RH), this study was undertaken to establish the presence and characteristics of receptors for [D-Trp6]LH-RH on the membranes of human endometrial cancer. Specific binding of [125I,D-Trp6]LH-RH was demonstrated in membrane preparations from 24 of 31 (77%) endometrial carcinomas and from 3 of 13 (23.1%) nonmalignant human endometrial specimens. Ligand binding was dependent on temperature, time, and plasma membrane concentration in a fashion expected of a peptide hormone. Mathematical analysis of the binding data showed that interaction of [125I,D-Trp6]LH-RH with the binding sites was consistent with the presence of a single class of high affinity, noncooperative receptors (Kd 9.88 +/- 4.59 x 10(-9) M; Bmax 0.70 +/- 0.14 x 10(-12) mol/mg membrane protein). The rates of association and dissociation were calculated to be 6.5 x 10(6) M-1 min-1 and 0.021 min-1, respectively. [125I,D-Trp6]LH-RH binding was not displaced by either unlabeled somatostatin or epidermal growth factor, but was displaced completely by native LH-RH. Using 125I-epidermal growth factor, specific, high-affinity receptors were also detected in membranes from 22 of 26 (85%) endometrial cancers and in all of 6 nonmalignant endometrial specimens (Kd 0.42 +/- 0.12 x 10(-9) M; Bmax 0.30 +/- 0.15 x 10(-12) mol/mg membrane protein). The potential functional role of the receptors for [D-Trp6]LH-RH in human endometrial carcinoma is not clear, but this finding provides a rationale for the use of therapeutic approaches based on LH-RH analogues in this malignancy.

Estrogen-inducible pS2 mRNA was previously detected in human cancer cell lines the growth of which was sensitive to estrogen. In the present study, the expression of the pS2 gene was analyzed in 111 gynecological carcinomas. The pS2 message was detected in greatest abundance in 6 primary carcinomas of the ovary (6 of 29), 4 of these being mucinous cystadenocarcinomas. A secondary carcinoma of the ovary, and another of the omentum (1 of 4), also contained detectable levels of pS2 mRNA. Weak pS2 mRNA signals were occasionally observed in endometrial (2 of 55) and cervical carcinomas (2 of 33) as well. There was a poor correlation between estrogen receptor and pS2 mRNA in ovarian carcinomas.

The applications of tumor markers and steroid receptors in gynecologic neoplasms are described. The value of immunohistologic techniques in the histogenetic assessment of gynecologic neoplasms is examined. Approaches to the diagnosis of similar-appearing lesions are presented.

In leiomyoma and normal myometrium estrogen receptors act independently at low or high levels of the normal serum steroid range in the menstrual cycle. It might be an inherent characteristic of leiomyomas, which results in their progressive growth in the absence of any abnormal stimulation. In the secretory phase of the menstrual cycle, serum progesterone suppresses estrogen receptor concentrations in leiomyoma. In the present study serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) showed direct as well as inverse correlations with estrogen and progesterone receptors in different phases of the menstrual cycle.

Endometrial carcinoma, now the most frequent female genital tract malignancy in Denmark, is generally accepted as an endocrine-related neoplasm. Like normal endometrium, many endometrial carcinomas contain estrogen receptors as well as progesterone receptors. The receptor content appears to correlate with several histopathological features, in particular with tumour differentiation. Well-differentiated lesions are more frequently estrogen and progesterone receptor "positive" than poorly differentiated lesions. Several studies suggest that a high content of estrogen/progesterone receptors in primary endometrial carcinomas, regardless of other prognostic factors, affects the prognosis favourably. Furthermore, receptor status seems to correlate with response of the tumour to progestin therapy. It may be of particular importance, that nearly half of poorly differentiated endometrial carcinomas contain estrogen/progesterone receptors and thus might benefit from progestin therapy. Immunohistochemical receptor analysis directly on cryostat or formalin-fixed tissue sections increases the possibility to explore the histological and biological features, which are determinative for the receptor profile of endometrial carcinomas.

We have previously shown that pregnancy-associated alpha 1-globulin, a small molecular weight (32 kDa) insulin-like growth factor-binding protein (IGF-BP), is quantitatively the major secretory protein product of the decidualized endometrium during human pregnancy and is localized principally in the decidual cell. In the present study, employing monoclonal antibodies in immunohistological techniques, the distribution and localization of IGF-BP has been examined in normal and pathological tissues of the adult and first trimester fetus. In the adult, most intense reactivity was associated with endometrial stroma and their derived decidual cells in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle, the appearance of IGF-BP in endometriotic tissue was linked with its appearance in uterine endometrial tissue. The only other adult cells where significant staining was detected was in the luteal cells of the corpus luteum. Production of the protein was not a feature of carcinomas. In the fetus, the protein was localized in lymphoid-myeloid progenitor cells and hepatocytes of the liver and at lower levels in testicular Leydig cells and adenocortical cells. These observations suggest highly specific tissue expression of this protein and support a specialized role for this protein in progenitor cells of the lymphomyeloid system, in certain steroid hormone-producing cells and in the decidual cell in pregnancy.

We report a case of primary endodermal sinus tumor (EST) of the endometrium in a 42-year-old female. Although numerous extragonadal EST have been reported, primary EST of the endometrium is exceedingly rare. To our knowledge this is the fourth documented case of this nature. The tumor had the typical microscopic features of EST, with papillary, tubular, reticular, and solid growth patterns; occasional Schiller-Duval bodies and many intracellular and extracellular periodic-acid Schiff positive hyaline globules were seen. The neoplastic cells stained positively for alpha-fetoprotein (AFP), alpha-1-antitrypsin (A1AT), cytokeratin, and placental alkaline phosphatase. The globules were positive for AFP, A1AT, albumin, transferrin, and fibronectin. The tumor cells were negative for type IV collagen and the beta subunit of human chorionic gonadotropin (B hcG). Electron microscopic examination showed intracellular and extracellular basement membrane-like material, intracytoplasmic lumina with microvilli, and glycogen. The patient was treated with total abdominal hysterectomy and bilateral salpingo-oophorectomy, followed by four cycles of adjunct chemotherapy (vinblastine, bleomycin, and cisplatinum) repeated every 3 weeks. The serum AFP level was elevated significantly before the surgery and the tumor response was monitored by serial determination of serum AFP level. There was no evidence of recurrence 24 months after surgery.

The reactivity of a new monoclonal antibody (MAb), MSN-1, raised against a human endometrial cancer cell line (SNG-II), was studied in a variety of endometrial, endocervical, and ovarian carcinomas as well as normal cycling endometrium. Moderate to strong reactivity (2-3+) was seen in six of nine normal secretory endometria (67%), one of 10 normal proliferative endometria (10%), 18 of 18 endometrial adenocarcinomas (100%), 10 of 11 endometrioid ovarian adenocarcinomas (91%), seven of nine clear cell ovarian adenocarcinomas (78%), one of 12 endometrial hyperplasias without atypia (9%), two of four endometrial hyperplasias with atypia (50%), zero of five endometrial serous adenocarcinomas, two of 17 serous ovarian adenocarcinomas (12%), zero of 10 intestinal-type mucinous ovarian adenocarcinomas, and zero of nine metastatic adenocarcinomas in ovary. Endocervical adenocarcinomas showed moderate to strong staining in 75% (six of eight). It is concluded that MSN-1 can be used to confirm endometrioid/clear cell differentiation in ovarian and endometrial tumors, cannot be used to discriminate endocervical from endometrial differentiation, cannot be used to discriminate atypical hyperplasia from carcinoma, and may be useful to distinguish between atypical (premalignant) endometrial hyperplasias and those without atypia.

Sometimes widely diverging results have been reported as regards the nuclear DNA ploidy pattern of adenocarcinomas of the endometrium. Since such discrepancies might be due to differences in the techniques applied, it seemed worthwhile to investigate this possibility in conventional uterine curetted specimens. In order to obtain a high incidence of tumours with cancer cell nuclei showing "aneuploid" DNA distribution pattern, a selection was made, so that only those adenocarcinomas that had led to a fetal outcome of the neoplastic disease were examined. The results of two image cytometric (ICM) techniques for cytochemical nuclear DNA assessments were compared. One was direct photographic cytometric measurements on Feulgen-stained sections; the other was densitometric assessments on isolated tumour cell nuclei of deparaffinised and disintegrated specimens. In 39 cases out of 43 the DNA ploidy pattern was the same by means of the two techniques. However, about half the numbers of the specimens (40 out of 83) were lost during the deparaffinisation and disintegration procedure. As far as could be found from a limited study on 20 (out of the 43) selected cases, these losses of specimens became even greater when the flow-cytometric (FCM) technique was applied on the deparaffinised specimens; about one third of these specimens were not possible to evaluate. In addition, in those where assessments by means of FCM could be made, the DNA ploidy pattern obtained differed from that of the two ICM techniques in not less than 80% of the cases. Broad peaks and high amounts of counts in the background in the DNA histograms indicated that most of the DNA assessments made by means of FCM on archival material of the present kind of curetted specimens of endometrial adenocarcinomas gave no reliable results. Consequently, differences in the techniques applied in cytochemical assessments of the nuclear DNA distribution pattern in endometrial carcinomas can explain the more or less controversial results reported from different laboratories.

Sixty-five patients with Stage I and II endometrial carcinoma were investigated. After a short-time follow-up (17-24 months) significant differences in frequency of relapses were observed between patients with tumors containing low amounts of estrogen receptor (ER less than 60 fmol/mg DNA) in the nuclear pellet, and those with tumors containing greater than 60 fmol ER/mg DNA (p = 0.01). Other prognostic factors showed no differences in frequency of relapses. In this small patient material with a short-time follow-up we therefore suggest that ER in the nuclear pellet may be an important prognostic factor.

Using an immunoenzymatic assay, cathepsin-D concentrations were measured in the cytosol of human endometrium biopsies. The level of cathepsin-D was higher in the luteal phase than in the follicular phase (P less than 0.01), suggesting increased accumulation by progesterone. Induction by progestin was confirmed by immunoprecipitation of cathepsin-D from a lysate of epithelial endometrial cells previously treated in primary culture with R5020 (10 nM); estradiol (10 nM) had no effect. Immunohistochemistry showed that cathepsin-D is mainly localized in the epithelium and that its level is higher in the luteal phase. The plasma level of cathepsin-D was stable during the menstrual cycle, ranging between 2.5-10 pmol/mL, but increased slightly during pregnancy. The mean level of cathepsin-D was higher in 19 endometrial carcinoma than in 20 normal endometrium, but was not correlated with steroid receptor status. However, using 15 pmol/mg protein as a cut-off level, the cathepsin-D status (high or low) was correlated with the degree of myometrial invasion (greater than or equal to one third) by adenocarcinoma cells, whereas steroid receptor status was not. We conclude that cathepsin-D is induced by progesterone in human endometrium, as it is in normal rat uterus, and we suggest that a low concentration of cathepsin-D in the cytosol of endometrial adenocarcinoma may indicate a favorable prognosis, since it is correlated with low myometrial invasion.

Estrogen receptor (ER) content in myoma and myometrium was determined by radio-receptor assay (RRA), and ER staining was performed by immunocytochemical staining, i.e. the peroxidase-antiperoxidase (PAP) method, in uterine samples from eight patients with uterine leiomyoma. ER content in myoma tended to be hither than that in myometrium, but the difference was not significant. The distribution pattern of ER staining was thought to be an important criterion. When the data were compared on the basis of the number of cells in the site where the largest number of positive cells aggregated, the number for myoma was significantly higher than that for myometrium (P less than 0.05). These results suggest that myoma contains more ER positive cells to obtain estrogen effects than myometrium.

HCG of chorionic tissues of hydatidiform mole was purified using ammonium sulfate fractionation, ion exchange chromatography and gel filtration. The purified hCG has a molecular weight of 1.5x10(5). Three subunits of molecular weights 11,000, 17,000 and 31,000 were obtained after treatment with sodium dodecyl sulfate and beta-mercaptoethanol. Amino acid analysis indicates a high content of Asp. and Glu., but a low content of Pro. Carbohydrate content is only 15.2%, and the amount of amino sugars and sialic acid are also comparatively low. The biologic activity of the purified hCG's of chorionic tissues is much lower than that from urine.

Eighteen (18) previously untreated patients with grade I (9) and II (9) endometrial adenocarcinoma provided endometrial samples for biochemical analysis of estrogen and progesterone receptors. The same samples were evaluated by histology and ultrastructure. Progesterone receptor negative samples (PR less than 1000 fmole/g tissue) showed more ultrastructural characteristics of malignancy and a wider range of changes than the progesterone positive specimens (PR greater than 1000 fmole/g tissue). These findings appeared to be independent of estrogen receptor status. No significant ultrastructural changes were seen when grades I and II were compared.

alpha 2-Macroglobulin (alpha 2M) was demonstrated in normal syncytiotrophoblasts of both early and full-term human placentas using immunocytological staining. alpha 2M was also detected in hydatidiform moles, the benign tumors of proliferating syncytiotrophoblasts. In contrast, no alpha 2M was detected in invasive moles or choriocarcinomas. In culture conditions, both normal syncytiotrophoblasts and choriocarcinoma cells, identified by production of human chorionic gonadotropin, were negative when stained for alpha 2M or when studied using metabolic labeling and immunoprecipitation or radioimmunoassay. However, alpha 2M was taken up from added human serum by the cultured syncytiotrophoblasts, whereas choriocarcinoma cells remained negative also under these conditions. The possible role of alpha 2M in the regulation of proteolysis in cell invasion is considered.

Extracts from uterine cervical and corpus cancers, but not from benign tumor or intact tissues tested, were found to contain a growth-promoting activity which induced the proliferation of human endometrial fibroblasts. Exposure of cultured fibroblasts to the cancer extracts increased the rate of [3H]thymidine incorporation in a dose-dependent manner. The activity was heat-labil, and not inacticated by removal lipid-soluble fraction, suggesting that the activity is associated with a protein. However, the cancer extract failed to stimulate phosphoinositide turnover. The substance(s) present in the uterine cancer extracts may activate endometrial fibroblasts proliferation through the transmembrane signaling mechanisms other than phosphoinositide turnover. The bindings of previously identified growth factors including somatomedine C, thrombin, insulin, fibroblast growth factor were not inhibited by the extracts. This is the first report to provide direct evidence that malignant uterine tumor may produce and secrete a putative growth factor-like peptide.

Sixteen tumor markers are reviewed, and measured to the ideal: produced by the tumor cell alone absent in health and in benign disease present in all patients with a given malignancy level in the blood representative of tumor mass detectable in occult disease. The only marker that approaches the ideal is human chorionic gonadotropin (HCG) in gestational trophoblastic tumors. In this malignancy, the HCG level suggests the diagnosis and stage, confirms response to therapy, and predicts relapse. The three most widely used and intensely studied tumor markers are carcinoembryonic antigen (CEA), alphafetoprotein (AFP), and HCG. CEA cannot be used in screening for cancer, but in carcinoma of the colon its elevation preoperatively increases the likelihood of advanced disease and postoperative recurrence. Postoperatively, elevated titers are often but not invariably associated with recurrent disease. AFP and HCG are useful in the management of nonseminomatous germ cell testicular tumors. Like CEA, they cannot be used for screening. They are more likely to be increased with advancing stage, and after therapy rising levels almost always mean recurrent disease. Some markers are valuable in specific circumstances, such as calcitonin in screening for familial medullary carcinoma of the thyroid. In multiple myeloma, immunoglobulins are useful in determining the tumor mass and response to therapy. In neuroblastoma, catecholamine metabolites are useful primarily in making the diagnosis. In some malignancies, the absence of effective therapy lowers the value of the marker, as for AFP in hepatoma. The remaining markers are too unreliable or too little studied to be useful in the management of an individual patient with cancer. The purpose of this paper is to provide the clinician with an understanding of the limitations of the present tumor markers that will lead to wiser use of the tests, and to provide standards to which future tumor markers should be measured.

We have compared a new enzyme immunoassay for progesterone receptors (Abbott PgR-EIA, monoclonal) with our standard radioligand binding-assay (PgR-RBA). For both assays cytosols were freshly prepared from human breast cancer specimens that had been stored frozen for up to 1 year, and predominantly tissues were used which contained critically low amounts of progesterone receptors. A highly significant correlation was observed between the PgR-EIA and PgR-RBA (Spearman: Rs = 0.85, n = 100). Using a cut-off point of 10 fmol PgR/mg protein, 87% of the values were in accordance with each other (52% negative, and 35% positive in both assays), whereas 13% scored positive (median: 16, range 11-38 fmol PgR/mg protein) in one assay and negative (median: 7, range 0-10 fmol PgR/mg protein) in the other. Also in cytosols from human ovarian and endometrial carcinoma tissues immunoreactive PgR could be detected, and significant correlations with PgR-RBA were observed (Rs = 0.94, n = 6) for both tissues.

Combined comparative study of anatomo-histological features, proliferative activity, content of X-chromatin, and ploidy of the nuclei of epithelial structures of 7 adenoacanthomas, 8 glandular-squamous cell carcinomas, and 7 adenocarcinomas (control group) of the endometrium was carried out. Microspectrophotometry of DNA revealed heterogeneity in DNA content and polyploidy typical of malignant growth in all cell populations, including squamous cell component of adenoacanthomas. The correlation between reduced degree of histological differentiation, depth of invasion, and decreased number of nuclei containing X-chromatin bodies as well as the occurrence of histological transitions suggest that adenoacanthomas and glandular-squamous cell carcinomas should be regarded as one group of two-component endometrial tumors and the terms used for their designations as synonyms. Within this group, highly and poorly differentiated variants may be distinguished which is of prognostic importance.

A nude mouse model for the growth of human endometrial carcinoma and hormonal modulation of the progesterone receptor (PR) was established previously. This study describes the effect of 17 beta-estradiol and tamoxifen (TAM) on growth rate and PR concentration in a hormonally responsive human endometrial tumor (EnCa 101) grown in this experimental system and presents the first characterization of human endometrial carcinoma PR. EnCa 101 was transplanted subcutaneously into ovariectomized, BALB/c, nu/nu athymic mice and grown under 17 beta-estradiol-stimulated, TAM-stimulated, and control conditions. Both 17 beta-estradiol and TAM increased the growth rate of EnCa 101 in nude mice, and a parallel increase in the cytosol PR concentration was observed, from 130 +/- 55 (SD) fmol/mg protein to 1,311 +/- 598 fmol/mg protein and 710 +/- 310 fmol/mg protein, respectively. PR was partially purified by phosphocellulose and DEAE cellulose chromatography, and the DEAE eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with [17 alpha-methyl-3H]promegestone ([3H]R5020). Two PR-negative tumors (EnCa K and EnCa V) were also examined in parallel. Coomassie blue staining of gels revealed that the protein patterns of all of the partially purified preparations from EnCa 101, EnCa K, and EnCa V were essentially identical. In contrast, photolabeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of EnCa 101 grown in the presence of 17 beta-estradiol or TAM revealed incorporation of [3H]R5020 into proteins of molecular weight approximately 116,000 and 85,000. Labeled proteins of molecular weight 66,000, 45,000, and 35,000 were also observed. In each case, the labeling was competable with excess non-radioactive R5020. No incorporation of [3H]R5020 was observed in EnCa 101 grown in the absence of estrogen, nor was any observed in EnCa K or EnCa V.

Protein maps of in vitro hormone-modulated adenocarcinoma cells from an endometrial tumor are described. It is noted that mapping of tumor tissues by means of the two-dimensional analysis described by O'Farrell and associates of 35S-methionine-labeled proteins can provide a characteristic map for each tumor and that the methionine-containing proteins of tumor cells can be independently modulated by the in vitro additions of estradiol and progesterone. Such protein modulation could be indicative of hormonal therapeutic responsiveness of individual endometrial adenocarcinomas and other hormone receptor-positive tumors. Tumor maps may further provide data for the identification and isolation of new tumor markers which might be correlated with the radiotherapeutic and chemotherapeutic sensitivity of the malignancy.

The cellular localization of a 24,000 (24K) mol wt protein was evaluated by monoclonal antibody immunocytochemistry in atrophic and persistent proliferative endometrium as well as in various forms of hyperplastic and neoplastic human endometria. 24K was chiefly located in the cytoplasm of ciliated cells and their precursor forms, clear cells. It was absent in atrophic, resting, and regressive forms of cystic glandular hyperplasia. Persistent proliferative endometria with a low degree of proliferation has occasional 24K immunostained cells, whereas those with active proliferation contained intense 24K immunostaining. A similar pattern characterized the active forms of cystic glandular hyperplasia. The immunostaining reaction decreased in adenomatous and atypical adenomatous hyperplasia as well as in well differentiated adenocarcinomas. In poorly differentiated carcinomas, 24K protein was virtually absent. These findings suggest that 24K protein is estrogen and ciliated cell related, and endometrial ciliogenesis and 24K protein are morphological and biochemical markers, respectively, of the estrogenic endometrial response. Their increase and decrease in hyperplastic and neoplastic endometria support the concept that early hyperplasia is highly sensitive to estrogenic stimulation, whereas with increasing architectural and cytologic atypia, the estrogenic response decreases as a reflection of growing cell populations that are independent of estrogenic influence. Immunostaining with 24K is not sensitive enough to discriminate estrogen-independent cells of atypical adenomatous hyperplasia from those of early, well differentiated adenocarcinoma.

Like breast carcinomas, endometrial carcinomas are derived from sex steroid target tissue. Progress and research directed into the clinicopathologic relationship of steroid receptors and endometrial carcinomas have been hampered by many factors, including: limited numbers of patients with advanced-stage disease compared to the number with breast carcinoma; contamination of specimens with surrounding benign endometrial components which may contribute to total steroid binding; and amount of tissue required for standard biochemical assays. Nevertheless, several clinicopathological associations have been made for steroid receptor content of endometrial carcinomas. Receptor content appears to correlate with histological differentiation in that well-differentiated lesions have higher mean levels of receptor and more receptor 'positive' lesions than do poorly differentiated lesions. Furthermore, receptor levels and status appear to correlate with prognosis of primary endometrial carcinomas and response to hormonal therapy of advanced endometrial carcinoma. Newer techniques utilizing monoclonal antibodies to directly localize receptor in tissue specimens may lead to a greater understanding of the dynamics of receptor physiology in endometrial carcinomas, and may make possible more accurate predictions of clinical behavior by allowing the direct analysis of the receptor content of the malignant component within a tissue specimen.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.

Sixteen endometrial carcinomas were studied by an immunoperoxidase method with anti-human calcitonin, 8 with and 8 without argyrophil cells. Calcitonin was demonstrated in 7 with and 2 without argyrophil cells. Calcitonin production was not associated with any specific histology of the endometrial carcinomas. Regardless of the existence of argyrophil cells, calcitonin was located in the apical portion or in the entire cytoplasm of the glandular tumor cells in the well differentiated adenocarcinomas. In a poorly differentiated adenocarcinoma and the adenosquamous carcinomas, it was found in many immature and squamous tumor cells as well as in some glandular tumor cells. The comparative studies between calcitonin and argyrophil granules in the endometrial carcinomas with argyrophil cells revealed that calcitonin was located in many tumor cells other than argyrophil cells. The extreme examples were 2 calcitonin-producing tumors without argyrophil cells. Calcitonin production of the endometrial carcinomas is a novel finding, but the biological significance of argyrophil cells remains to be further investigated.

Endometrial carcinoma is the most common genital malignancy in North America. However its pathogenesis, in particular its relationship with hyperplasia is not clear. To understand steroid hormonal interactions in the genesis and growth of human endometrial hyperplasia and carcinoma, we have assayed progesterone receptors in hyperplasia and neoplastic human endometrium by immunocytochemistry. The presence of progesterone receptors in target tissues is known to be a marker of both estrogen and progesterone action. The receptors were identified in fresh-frozen sections using a mouse monoclonal antiprogesterone receptor antibody (alpha PR6). The progesterone receptor content was high in the epithelium of hyperplasia without cytological atypia and low in the epithelium of endometrial intraepithelial neoplasia (hyperplasia with cytological atypia). In carcinomas there was a heterogenous distribution of progesterone receptors in the epithelium but low as compared to hyperplastic endometria without cytological atypia. The stroma contained relatively high progesterone receptor levels irrespective of whether the epithelium was hyperplastic or neoplastic.

A case is presented in which concentration of Tc-99m-iron ascorbate-DTPA has been demonstrated in a benign leiomyoma of the uterus. Increased vascularity in the tumor might be the cause of the Tc-99m-DTPA uptake.

The estrogen receptor was assayed, using the 2,000g supernatant and dextran-coated charcoal method, in 243 tissue samples from human breast cancer, benign breast diseases, macroscopically normal breast tissues, normal uterine myometrium, and uterine myoma. The estrogen receptor was found to be positive in 52.1% of 98 primary breast cancer and in 54.1% of 24 metastatic tumors. The receptor in the breast cancer was found to be similar to that in normal uterine myometrium in the binding character; that is, the dissociation constant of 10(-9) approximately 10(-11) M and number of binding sites of 0 approximately 2,800 fmol/mg protein. There was no correlation between the presence of the receptor and some clinical factors such as menopausal status, age of the patient, urinary 17-ketosteroid excretion, clinical stage of cancer, tumor size, positive or negative axillary lymph node metastasis, histological type, metastatic site of the cancer, or disease-free interval. The estrogen receptor appeared to be retained by metastasis of cancer, and this may lead to the use of the receptor assay with mastectomy specimens for the prediction of response to hormonal therapy in future recurrence of malignancy. Furthermore, it may be possible by this assay to select patients suitable for adjuvant therapy with hormones at the time of mastectomy. A good correlation was found between the presence of the receptor and response to the major endocrine ablation therapy in patients with advanced or metastatic breast cancer. When the receptor was negative in the cancer tissue, the change of response to the endocrine therapy was minimum. On the other hand, if the cancer contained the receptor, approximately 60% of the patients with metastatic or advanced breast cancer responded well to the major endocrine ablation therapy. Thus, the estrogen receptor of breast cancer in Japanese patients appears to bear a close resemblance to that reported in Western patients in its incidence and the correlation to some biological characteristics of the cancer.

Concentrations of luteinizing hormone-releasing factor (LRF) and human chorionic gonadotrophin (HCG) were measured by specific radioimmunoassay in homogenates of molar tissue evacuated from four patients at eight to 16 weeks of pregnancy (by menstrual dates), and in normal trophoblastic tissue of equivalent gestational age. In normal trophoblastic tissue HCG concentrations decreased, while LRF concentrations increased, with advancing gestational age. Molar tissue contained high quantities of HCG (2292 +/- 1415 s.e.(mean) iu/g wet weight vs 355 +/- 129 in the placenta) and very low, but consistently measureable, quantities of LRF (23.4 +/- 6.7 pg/g vs 12,808 +/- 2931 in the placenta). The significance of our finding of high HCG concentrations in the presence of low concentrations of LRF in neoplastic trophoblastic tissue is being further investigated.

For the evaluation of histochemical procedures for detection of androgen receptors, three human tumor cell lines have been used: PC-93 and NHIK-3025, both biochemically characterized as androgen receptor-positive, and EB-33, biochemically characterized as androgen receptor-negative. The binding of three fluorescent ligands, testosterone-17 beta-hemisuccinate-bovine serum albumin-fluorescein isothiocyanate, testosterone-17 beta-hemisuccinate-fluoresceinamine, and 5 alpha-dihydrotestosterone-17 beta-hemisuccinate-fluoresceinamine, to the cells was evaluated. The relative binding affinities of the ligands for the androgen receptors were low (less than 5% when compared to methyltrienolone). Treatment of the cells with the androgen-fluoresceinamine derivatives resulted in a fluorescent labeling of the cytoplasm in both intact and "freeze-damaged" cells of the three cell lines. This staining was independent of the presence of receptors. Nuclei were not stained. Incubation of intact cels with the protein-linked conjugate did not result in significant cellular fluorescence. Only cells with damaged membranes showed a positive histochemical reaction, both in nucleus and cytoplasm, irrespective of the receptor content of the cells. The fluorescence intensity was not suppressed with excess 5 alpha-dihydrotestosterone or methyltrienolone, which are known to prevent binding of low affinity ligands to androgen receptors. From these results it is concluded that androgen receptors cannot be detected by these fluorescent ligands with low affinity for the receptor. The observed fluorescence of the cells is therefore due to binding of the ligands to other binding sites. The visualization/histochemical demonstration of these binding sites does not appear to be related to the presence of androgen receptors.

This paper describes a new experimental model system for the induction of endometrial adenocarcinoma in hamster uterus following diethylstilbestrol (DES) treatment of the newborn female. We propose that DES acts as an initiator during early development and that other estrogens act as promoters to stimulate tumor development in the adult uterus. DES directly affects the uterus as was shown by the failure of neonatal ovariectomy to prevent early DES-induced uterine growth. Subsequently, ovarian estrogen secretion from anovulatory, polyfollicular ovaries modifies the DES-altered uterus starting between 20 and 30 days of age and continuing into adult life. Early DES effects on the uterus include stimulation of endometrial cellular differentiation and progesterone receptor production. Permanent changes in uterine collagen, DNA and progesterone receptor content were noted, but the responsiveness of the DES-altered uterus to estrogen and progestin action was not impaired. Morphogenetic changes included an increase in extracellular connective tissue elements and striking alterations in endometrial cell composition such as hyperplasia of luminal and glandular epithelia and a massive inflammatory response in the stroma. Endometrial adenocarcinomas occurred in DES-treated animals in association with exposure to either endogenous estrogen from anovulatory ovaries or exogenous estrogen treatment of the ovariectomized animal. Endometrial tumors had relatively high concentrations of estrogen and progesterone receptors, suggesting a sensitivity to hormone action. Thus, these studies (a) demonstrate the utility of this animal model for the preparation of experimental endometrial tumors, and (b) suggest that DES acts as an initiator to transform uterine cells during early development, and estrogen exposure later in life acts as a promotor to stimulate growth and proliferation of DES-transformed cells.

Human chorionic gonadotropin (hCG) in normal pregnancy and hydatidiform mole did not bind to the peanut agglutinin (PNA)-Sepharose column, whereas significant amount of hCG in choriocarcinoma adsorbed to the column. Therefore, PNA-Sepharose affinity chromatography is an effective tool for the detection of desialylated hCG and may be useful also for the diagnosis of choriocarcinoma.

Comparative study of the asparagine-linked sugar chains of hCGs purified from urine of pregnant women and of choriocarcinoma patients revealed that extensive and structural changes occur common in the sugar chains of tumor hCGs. The changes can be ascribed to enhancement of an alpha-fucosyltransferase that forms the Fuc alpha 1----6GlcNAc group in the sugar chains and the appearance of N-acetylglucosaminyltransferase IV.

Ferritin levels in extracts of tumor tissue and serum of patients with gynecological malignancies were assayed by immunodiffusion methods using a standard system of testing for ferritin. A high blood-ferritin level (500-1,000 ng/ml) was found in 8 out 21 patients with ovarian cancer, 8 out of 24 patients with endometrial carcinoma. 2 out of 8 cases of mucinous cystadenoma and in 2 out of 4 cases of pyoovarium. It was lower in other malignancies of the female genital tract. There was no correlation between the ferritin level of blood serum and that of tumor tissue extracts. Although blood serum ferritin was found in very few healthy subjects (2.5%), it is suggested that blood-ferritin level test should be carried out in the course of examination of slowly-progressing purulent adnexitis and mass screening for tumors of female genitals.