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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 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.

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.

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.

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.

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.

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.

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.

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.

The incidence and levels of cytoplasmic steroid receptors were studied in 155 endometrial carcinomas: oestrogen receptors (ER) were measured in all samples, progesterone receptors (PR) in 148 and androgen receptors (AR) in 118. Well-differentiated adenocarcinomas were significantly more likely to contain ER, PR and AR than were poorly-differentiated tumours, and mean values of ER and PR content were significantly higher in well-differentiated adenocarcinomas than in less-differentiated tumours. Receptor profiles in adenosquamous carcinomas and clear-cell carcinomas were similar to those in adenocarcinomas. Deeply invasive tumours were significantly less likely to be ER, PR or AR positive and, even when positive, such tumours had significantly lower mean levels of ER and PR. Poorly-differentiated tumours were significantly more likely to be ER-negative PR-positive than well-differentiated adenocarcinomas and adenosquamous carcinomas.

Fourteen of fifty-three cases of endometrial carcinoma (26 per cent) contained varying, usually small numbers of argyrophil cells, as demonstrated by Grimelius silver nitrate staining of formalin-fixed paraffin-embedded sections. In eight cases the argyrophilia was present in the apical region of glandular cells (type 1 cells) or throughout the cytoplasm of glandular or squamous cells (type 2 cells). The distribution of argyrophilia in these cells closely paralleled that of mucin or glycogen, and pretreatment of the sections with disease resulted in a loss of argyrophilia in the glycogen-rich tumors. In six cases individual round, ovoid, and flask-shaped argyrophilic cells were present also within the glandular epithelium (type 3 cells). In all six cases, similarly distributed cells were positive immunohistochemically for serotonin. Immunohistochemical staining for a battery of polypeptide hormones (calcitonin, gastrin, somatostatin, adrenocorticotropin [ACTH], and neurotensin) revealed positive staining for ACTH in one of the six tumors that contained type 3 cells and positive staining for somatostatin in another. Ultrastructural examination of the ACTH- and serotonin-positive tumor disclosed cells with granules 80 nm in diameter. Types 1 and 2 argyrophil cells were found in small numbers in several specimens of normal proliferative and secretory endometrium, but type 3 argyrophil cells were not identified in these specimens. Although focal argyrophilia is a frequent feature of endometrial carcinomas (26 per cent), the presence of type 3 argyrophil cells containing hormones, as evidenced by the immunohistochemical demonstration of serotonin and occasionally polypeptide hormones, is much less common (11 per cent).

Electron spin resonance (ESR) studies at -130 degrees have been made on frozen samples of normal human cervix and uterus and on frozen samples of various pathological conditions of the cervix and uterus including fibroleiomyoma and carcinoma. Fifty-five samples of normal cervix and endometrium, 40 samples of nonmalignant disturbances, 15 benign tumor samples, and 20 malignant samples were studied. Very strong ESR signals were seen in frozen powders and frozen intact samples of normal cervix and endometrium and in nonmalignant gynecological conditions. In many cases, the ESR signal was greatly decreased or even undetectable in cancer samples. The substance(s) responsible for the ESR signal in frozen intact tissue (g = 2.11 to 2.15) is decreased in concentration when the sample is ground to powder under liquid nitrogen, and an anisotropic signal (g = 2.002 to 2.035) then becomes much more evident. The ESR signals in intact and in powder samples are sensitive to temperature variations; the signals disappear around 0 degrees, and only the intact samples show significant recovery of signal on recooling. The anisotropic g values and temperature sensitivity in the powders may result from an organic peroxy radical that is more strongly associated with a metal ion in intact samples.

Epithelial membrane antigen (EMA) is a component of the human milk fat globule membrane. The production of EMA is increased in both squamous and non-squamous epithelium in a variety of diseases including many neoplasms. Using antihuman EMA monoclonal antibody, an immunoperoxidase stain for EMA was done in normal or neoplastic cervical epithelia and endometrium. Formalin-fixed paraffin-embedded tissues were obtained by either colposcopic biopsy or surgical excision of the uterus from 61 patients. Ethanol fixed imprint smears were obtained from surgically removed uteri from 84 patients. The difference between normal cervical squamous epithelium and dysplasia or carcinoma of the cervix in intensity or patterns of staining for EMA reflected the progression of the neoplastic change in the cervix. The different EMA localization in well differentiated adenocarcinoma and moderately or poorly differentiated adenocarcinoma of the cervix and the endometrium was correlated with histological differentiation.

During progestogen treatment, nickel ions were detected in the human endometrial tissue of patients with endometrial carcinoma by the dimethyloxime cytochemical method. In this paper, the mechanism of action of nickel is dealt with and, because nickel ions have earlier not be detected in human endometrial carcinomatous tissues, the question arises as a result of what kind of cell biological processes, nickel ions appear in the cells of endometrial carcinoma. It is still to be answered whether the progestogen treatment applied can be correlated with the appearance of the trace element in the carcinomatous cell.

Human endometrium, endometrial hyperplasia and endometrial carcinoma were observed by light microscope. Endometrial carcinoma was histologically divided into Grade I (53 cases), Grade II (10 cases), and Grade III (7 cases). According to the results of electron microscopical analysis, cancer cells in GIII were smaller and showed a higher N/C rate and less mitochondria in the cytoplasma than those of GI and GII. Rough endoplasmic reticula were well developed in GI and GII compared with GIII. Immunohistochemically, ER localized in 54% cases of endometrial carcinoma, and decreased in positive rates of undifferentiated carcinoma. The metaplastic area in carcinoma showed the localization of ER and CEA. A close correlation between ER and CEA was demonstrated in endometrial carcinoma. Ultrastructurally, rough endoplasmic reticula were well developed in the cytoplasma of cancer cells in the strong positive cases of ER and CEA. It has been proved that histological detection of ER and CEA in endometrial carcinoma is very important in deciding the diagnosis, prognosis and therapy.

Estrogen receptors and progesterone receptors were measured in tumors from patients with previously untreated endometrial carcinoma before and after a 5-day course of tamoxifen citrate. On initial biopsy, 13 of 25 tumors (52%) were progesterone receptor-positive, whereas 21 of 25 tumors (84%) were progesterone receptor-positive after tamoxifen. Grades 1 and 2 tumors were more likely to demonstrate this increased incidence of measurable progesterone receptors. Considering these results, and the work of others who have shown that progesterone receptor-positive metastatic endometrial cancer is more responsive to progestin therapy than are progesterone receptor-negative tumors, we instituted a phase II clinical trial of tamoxifen plus progestin for patients with recurrent endometrial carcinoma. Thus far, however, the 33% total response rate achieved with the combination therapy has not been superior to standard progestin therapy.

The presence of cytosol estrogen receptor (ER) and the ER of nuclear extracts in normal uterine endometrium and endometrial adenocarcinoma tissues were determined by radioreceptor assay (RRA) and enzyme immunoassay (EIA). In addition the intracellular localization of ER and tissue distribution of ER positive cells was demonstrated by immunocytochemical assay (ICA). The levels of cytosol ER measured by EIA and RRA had a significant correlation in normal endometrium (r = 0.90) (P less than 0.01) and endometrial adenocarcinoma (r = 0.96) (P less than 0.01) as well as in breast cancer (r = 0.93) (P less than 0.01). The ER measured by immunocytochemical method using the monoclonal anti-ER antibody obtained from breast cancer cells was applicable to the detection of the ER in uterine endometrial tissues. In endometrial adenocarcinoma, the levels of cytosol ER in G1 (Highly differentiated adenomatous carcinoma) tissues (187.6 +/- 189.0 fmoles/mg protein) were significantly higher than those in G2 (Moderately differentiated adenomatous carcinoma with partly solid areas) tissues (15.0 +/- 31.9 fmoles/mg protein) (P less than 0.05). The levels of cytosol ER peaked in the late follicular phase (432.0 fmoles/mg protein) and remained low in the other phases. Epithelial cells in the normal endometrium were stained uniformly by ICA, in contrast, ICA stained and non-stained cells existed concurrently in the same endometrial adenocarcinoma tissues. A similar finding was also shown in breast cancer tissues and may be related to the efficacy of anti-estrogenic drugs on the growth of whole cancer tissues.

Seven cases of human endometrial carcinoma were transplanted into nude mice. Two of these cases, histologically classed as undifferentiated adenocarcinoma, were succeeded in the initial transplantation, but only 1, designated JEA-1, was established as serially transplanted tumor line in nude mice, which contained no estrogen and progesterone receptors assayed by sucrose density gradient centrifugation. JEA-1 tumor cells transplanted serially maintained the morphological and the functional characteristics of the original human tumor. Consequently, the JEA-1 tumor line will be useful for the experimental model of cancer treatments and the analysis of sensitivity for sex steroid hormones. Effects of sex steroid hormones on JEA-1 tumor cells had been examined and the electron microscopic observation and in receptor level, alteration of the tumor tissue were found. Ultrastructurally, the tumor cells which had been administrated estradiol and progesterone revealed various morphological changes resembling that of normal endometrial epithelium in the proliferative and the secretory phase, respectively. In addition, in the tumor tissue which had been administrated estradiol, the low, but positive, estrogen receptor sites were detectable. These findings may suggest that means of estradiol exposure will be able to induce the estrogen and progesterone receptors in endometrial carcinoma initially without these hormone receptors.

This study demonstrates a significantly increased survival time for women with estrogen receptor-positive adenocarcinoma of the endometrium when compared with that of those with estrogen receptor-negative tumors (p less than 0.05). This prognostic factor provides information which is additional to, and independent of, that provided by the histologic grade and myometrial penetration of the tumor.

In a prospective study of 283 women with endometrial carcinoma, 29 of whom were premenopausal and 254 postmenopausal, the DNA content and S-phase rate were measured by flow cytometry. Over 90% of the tumors in premenopausal women were peri-diploid and the remaining tumors were tetraploid, whereas 23% of the tumors in post-menopausal women were grossly aneuploid (p less than 0.01). Similar differences were found between women younger or older than 50 years. The results indicate that the hormonal status and/or age are of importance for the development of genetically different forms of endometrial carcinomas, as judged from the DNA-distribution.

The case reported concerns a 76-year-old woman under treatment for a previously diagnosed "poorly differentiated endocervical adenocarcinoma". New biopsies revealed an adenocarcinomatous tumor with unexpected melanotic pigmentation. The patient underwent cesium therapy followed by colpohysterectomy with lymphadenectomy. As there were no metastases, external complementary radiotherapy was not used. Four months after surgery, a large recurrence was detected; surgical excision proved impossible but revealed a grossly pigmented tumor from which several samples were taken. The patient died 11 months after the first consultation. No autopsy was performed. Morphological study was done on the initial biopsy, on the uterine tumor and on the recurrent tumor, using histological, cytological, ultrastructural and immunohistochemical techniques. Flow cytometry and biochemical study were also carried out on the recurrent tumor. All the samples studied histologically revealed uniform tumor morphology showing a poorly differentiated adenocarcinoma with an irregular distribution of melanin pigmentation (Fontana +). Electron microscopy confirmed the epithelial nature of the tumor, showing differentiated apical poles with villosities, linked by desmosomes. Basement membranes were irregularly present. Electron microscopy also demonstrated the melanotic nature of the pigmentation with melanosomes and premelanosomes. A few membrane-bound neurosecretory granules were seen. Immunohistochemistry showed that the tumor contains no S 100 protein and that no staining was obtained with monoclonal antibodies against malignant melanoma. Hormonal secretion and chromogranin were not detected. Tumor cells contained neither GFAP nor neurofilaments. Positive staining was obtained for neuron specific enolase and synaptophysin. Tumor cells contained three types of intermediate filament proteins = Vimentin, cytokeratins and peripherin (peripherin is an intermediate filament protein identified in 1984 by Portier, of the college of France, who very kindly supplied the antiserum and was good enough to do most of the biochemical study. Peripherin is considered to be characteristic of the peripheral nervous system. This case is the first example of demonstration of peripherin in a tumor). The biochemical study gave the following results: Cytosol assays for estrogen and progesterone receptors were negative. Vimentin, cytokeratins and peripherin were demonstrated by a study carried out in the Collège de France. No GFAP was found. A study of the metabolism of melanin derivatives showed high levels of urinary dopamine, serum and cytosol L. dopa.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical localization of estrogen receptor (ER) using specific monoclonal anti-human estrogen receptor antibody, H222, with an immunoperoxidase technique was performed on fresh frozen tissue derived from 100 endometrial adenocarcinomas. Immunohistochemical evaluation incorporated both intensity and distribution of staining. In all cases, H222 localized in the nucleus of target cells. A significant quantitative relationship was shown between histological score (H-Score) and the biochemical analysis of ER content in tissue homogenates (r = 0.65, P = 0.00001). Excellent sensitivity (92%) and specificity (93%) were observed for the comparison of H-Score to the biochemical assay. Significant ER localization was present in stromal and myometrial elements, component H-Score of which correlated weakly with component H-Scores of malignant epithelial elements. Divergent receptor localization in stromal and myometrial versus malignant epithelial elements suggests that biochemical assays of endometrial carcinoma specimens may not reflect cancer-relevant receptor content. The data presented here suggest that the immunoassay of ER using H222 monoclonal antibody provides additional histochemical information to complement conventional analyses of endometrial adenocarcinomas.

Eight cases of endometrial adenocarcinoma, including 7 well differentiated cases (stage Ia three, stage Ib three, stage II one) and I poorly differentiated case (stage IVb), were treated with Tamoxifen (20 mg/day) for 7-14 days. The average age of these patients was 59.1 +/- 10.9 years old and they were all postmenopausal for between 6 months and 30 years. The endometrial tissue was biopsied before Tamoxifen administration and half of it was investigated for progestin receptor (PR) and estrogen receptor (ER) while the remainder was used for histopathological examination. After surgical treatment, some tissue fragments of the endometrium were taken for investigation of PR and ER. The effect of Tamoxifen was then studied for the amount of PR, ER and histopathology. The result was that the amount of PR was increased in well differentiated cases whereas it was unchanged in poorly differentiated ones, and the amount of ER was decreased in all cases. Meanwhile, obvious histopathological changes were noted as follows decrease of glandular epithelial stratification, increased secretory activity, increased epithelial cell size with columnar cell type, increase of transparent, vacuolated cytoplasm, increase of nuclear size with variable shape and prominent nucleoli with marginal aggregation of chromatin. However, no stromal change was noted. Furthermore, the greater the increase in the amount of PR, the more conspicuous histopathological change was noted. It was concluded that Tamoxifen could produce both functional (PR, ER) and morphological (histopathology) changes in endometrial cancers.

Endometrial carcinomas from 58 patients were graded histologically, and the histologic grade was compared with the estrogen and progesterone receptor content of the tumor tissues. Receptor content was determined by multiple concentration saturation analysis and sucrose density gradient analysis. A positive correlation was found between the presence of estrogen and progesterone receptors and the degree of tumor differentiation. The majority (85%) of well-differentiated lesions contained significant amounts of both steroid receptor proteins. In contrast, only 13% of the poorly differentiated lesions were characterized by the presence of detectable levels of both estrogen and progesterone receptor proteins. The relationship of receptor content and degree of differentiation to prognosis and potential for response to hormonal therapy is discussed.

The aim of this study was to investigate the effects of estradiol and tamoxifen (TAM) on the growth of human endometrial carcinomas in athymic mice. Tissues from primary tumors were implanted into estradiol-treated mice. In passage 2, animals were treated with (a) placebo, (b) estradiol, (c) estradiol plus TAM, and (d) TAM alone. The size of the tumors was measured weekly. Estrogen receptors (ER) were determined with the dextran-coated charcoal method and/or ER enzyme-linked immunoassay. Progesterone receptors were measured with the dextran-coated charcoal technique. Of 16 primary tumors, 2 grew in the athymic mice and were studied further. Tumor EL was positive for ER (145 fmol/mg protein) and progesterone receptors (993 fmol/mg protein). Tumor EL in passage 2 was not significantly stimulated by estradiol, but was stimulated by a combination of estradiol and TAM. Treatments (estradiol, estradiol plus TAM, or TAM) all increased tumor growth in passage 3. Tumor BR and a metastasis BR-MET were ER and progesterone receptor negative, applying dextran-coated charcoal, ER enzyme-linked immunoassay, and immunocytochemistry. The BR and BR-MET cells contain the complete ER gene but do not express any measurable amounts of ER mRNA as quantitated by Northern blot analysis, using a complete ER complementary DNA probe. In all animal passages the growth rate was significantly higher in estradiol-treated mice compared with the control. TAM alone had some growth stimulatory effect, but much smaller than observed in the estradiol group. TAM inhibited estradiol-stimulated growth. These results suggest that estradiol and possibly TAM are capable of stimulating tumor growth in the athymic mice independently from ER, potentially through a host-mediated mechanism.

Unlabelled: Immunohistochemical staining was performed for carcinoembryonic antigen (CEA) in tissues taken from 41 cases of cervical and 67 cases of endometrial adenocarcinomas. Utilizing this method, smear specimens from 21 cases with cervical and 25 cases with endometrial adenocarcinomas were investigated at the cell level., The Results: 1) The investigation at the tissue level indicated that CEA staining was positive in 80% of cases of cervical adenocarcinoma. On the other hand, the positive rate was as low as 55% of the endometrial adenocarcinoma cases. It further declined to 35% when limited to pure adenocarcinoma without squamous elements. In addition, a difference was recognized between the cervical and endometrial groups in their localization. It was apparent that while even the cytoplasm in most of the cervical adenocarcinoma appeared to be markedly stained, such a tendency was observed and was weak in only a part of the cell membrane in the endometrial adenocarcinoma cases. 2) The studies with smear specimens indicated that while 57% (13/21) of the cervical adenocarcinoma cases were CEA positive, only 12% (3/25) were positive in endometrial adenocarcinoma cases. Therefore, a difference was recognized between the two at the cell level in their CEA staining characteristics. 3) Our observation revealed that CEA was more prominent in the poorly differentiated type than in the well differentiated one, in both cervical and endometrial adenocarcinoma. This seems to indicate a relationship between the histological differentiation and production of CEA. On the basis of the above findings, it was inferred that although both cervical and endometrial adenocarcinoma occur in the common Müllerian duct organ, they might have different biological characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined the distribution of high- and low-molecular-weight cytokeratins, vimentin, and carcinoembryonic antigen (CEA) in normal endometrial glands and endocervical glands (20 cases each) and in endometrial and endocervical adenocarcinomas (29 cases and 15 cases respectively). Low- and high-molecular-weight cytokeratin staining was present in normal endometrial and endocervical epithelium and in carcinomas. Coexpression of vimentin and cytokeratin was universally present in normal proliferative endometrial glands, with marked decrease or absence of vimentin staining in secretory phase patterns. Vimentin staining had a perinuclear distribution within the cells, in contrast to the cytokeratins, which stained diffusely. Vimentin was found in only 65% of endometrial adenocarcinomas. Staining was typically focal as well as regional in portions of the tumors. Vimentin was never observed in normal or neoplastic endocervical epithelium. Ultrastructural studies corroborate the perinuclear vimentin immunostaining pattern we observed in endometrial adenocarcinomas. CEA staining results were similar to those previously reported. These data indicate that the presence of vimentin may readily distinguish endometrial from endocervical carcinoma and is diagnostically useful in the study of metastatic adenocarcinomas.

Progesterone receptor content was measured in tissue samples from 175 patients with endometrial adenocarcinoma by use of the dextran-charcoal method. The estradiol receptor content was determined in 138 of these samples. Ninety-two tumors (52.6%) tested positive for progesterone receptors (greater than 50 fmol/mg cytosol protein) and 111 (80.4%) tested positive for estradiol receptors (greater than 6 fmol/mg). Median follow-up was 27.3 months (range 1 to 152 months). Progesterone receptor status correlated significantly with grade, histology, adnexal spread, age, and recurrence rate in stage I cancer. There was no correlation between progesterone receptor status and clinical stage, myometrial invasion, peritoneal cytology, retroperitoneal lymph node involvement, or spread to the cervix. Estradiol receptor status correlated with adnexal spread and recurrence rate. Recurrence in patients with stage I disease was significantly more common if tumors were negative for progesterone receptor (16 of 43, 37.2%) than if they were positive (four of 57, 7%; p less than 0.001). Recurrence was also more common if tumors were negative for estradiol receptor (seven of 17, 41.2%) than if they were positive (eight of 63, 12.7%; p = 0.02). In recurrent or advanced disease, response to progestin was independent of estradiol receptor content, but tumors positive for progesterone receptors responded significantly more often than those lacking progesterone receptors. Overall survival was superior for patients with progesterone receptor-positive tumors (p = 0.001). Although survival in clinical stages I and II was also superior in patients with lesions positive for progesterone receptors (p = 0.13), there was no statistical difference in survival between patients with progesterone receptor-positive or -negative cancers and surgical stages I and II disease (p = 0.12). Estradiol receptor status had no apparent correlation with survival.

Endometrial adenocarcinomas from 96 patients were studied for their biochemical estrogen receptor content as well as immunohistochemical localization of estrogen receptor. A well-characterized specific monoclonal antihuman estrogen receptor antibody (H222 sp gamma) as amplified by peroxidase/antiperoxidase techniques was used for immunohistologic localization. Immunohistochemical evaluation incorporated both the intensity and distribution of staining into a semiquantitative analysis (HSCORE). The total HSCORE was the sum of the relative contributions of the four histologic components: benign epithelium, malignant epithelium, stroma, and myometrium. These results were compared with clinicopathologic features of the tumors. Excellent sensitivity (91.5%) and specificity (93.1%) were observed for immunohistochemical analyses as compared with the biochemical analysis of estrogen receptor for these tissues. The cancer component HSCORE correlated better with grade than did the biochemical estrogen receptor determination. Correlation was also observed between surgical stage and estrogen receptor content. Decreasing biochemical estrogen receptor content predicted advanced surgical stage (p = 0.0003), as did the total HSCORE (p = 0.003); however, the HSCORE of the cancer component only did not correlate with advanced surgical stage (p = 0.11). Although immunohistochemical analyses predicted biologic differentiation better than did the biochemical technique, stage correlated better with biochemical estrogen receptor analysis and total HSCORE than did the cancer component HSCORE. The HSCORE of the cancer component may better predict biologic behavior and therefore identify cancers more likely to respond to hormonal therapy.

Recent experimental results from our laboratories revealed the following facts: Addition of GMP to homogenates or cytosol prepared from endometrial tissue or cultured endometrial adenocarcinoma cells during the assay for specific estrogen binders markedly increases specific binding levels. The effect is completed in about 15 min at 4 C (Fleming et al, 1983). Cyclic AMP has the opposite effect and in many cases lowers the number of binding sites to undetectable levels. ATP, a nucleotide that stimulates a particulate form of guanylate cyclase, Na2MoO4, a compound that can elevate cGMP levels (Fleming and Blumenthal, unpublished) and GTP, a metabolic precursor of cGMP, increase specific estradiol binding in the presence of plasma membranes and soluble factors. Cyclic AMP reduces the levels of estrogen binding when added to cell homogenates or to cytosol and counteracts the effects of cGMP, MoO4, ATP and GTP. ATP is required for the expression of cGMP and cAMP effects on estradiol binding. It is therefore likely that phosphorylations are involved in the generation and inactivation of estrogen binding sites. Divalent cation requirements for these effects also suggest participation of protein kinases in these processes. The reported effects of nucleotides and molybdate have been observed in specimens of histologically normal endometrium, in specimens of endometrial carcinoma, in two endometrial adenocarcinoma cell lines, HEC-1 and HEC-50 (Suzuki et al, 1980), and in two breast cancer cell lines, CG-5, a variant of MCF-7 obtained in Iacobelli's laboratory (Natoli et al, 1983), and in T47D) (Fleming et al, in press) Rapid changes in the levels of estrogen binding capacity observed in endometrial cells in culture can be associated with changes in cGMP/cAMP ratios shown, to vary during the cell cycle. Although it has not yet been demonstrated that cGMP-induced increases in specific estrogen binding can enhance responses to available estrogens, such possibility is of potential importance. Reduction of estrogen receptor levels in patients with cancers of estrogen sensitive tissues may inhibit tumor growth promoted by endogenous estrogen. Cho-Chung et al have recently reported that cholera toxin causes a reduction in estrogen receptor levels and arrests hormone dependent growth of DMBA-induced mammary carcinoma in rats (Cho-Chung et al, 1983). They postulated that the effect of cholera toxin is mediated by a cAMP effect on the estrogen receptor, an hypothesis supported by the observation that only tumors containing receptor responded to treatment. Conversely, cGMP-induced increases in specific estrogen binders may be useful in promoting a response of tumors to estr

It has been reported that the response of target cells to steroid hormone (SH) stimulation may depend on their position in the cell cycle. The DNA and RNA contents of malignant cells of the endometrium cultured in vitro were measured using flow cytometry (FCM). We also measured estrogen receptor (ER) and progesterone receptor (PR) levels of cells at different positions in the cell cycle. The G1 and S phases of the cell cycle were investigated using cells synchronized by sodium n-butyrate (G1 block), methotrexate (S block), and excess thymidine (S block). For DNA measurements, the cells were stained with propidium iodide following RNase treatment. For RNA measurements (double-stranded RNA) the cells were treated with DNase. We found that S phase synchronization by methotrexate was 136.2% of control (100%). Using the excess thymidine block and release procedure, the S phase fraction was 185.1% of control. G1 phase synchronization by sodium n-butyrate was 134% of control. The estrogen receptor level in G1 phase synchronized cells increased to 5.94 fmol/micrograms DNA in the cytosol and 12.35 fmol/micrograms DNA in the nuclear fraction. These levels represent a sevenfold total increase over that of the control estrogen receptor level. Cells in S phase showed no significant increase in estrogen receptor levels over control cells. Based on this study, the functional increase of the steroid receptor was most significant in the G1 phase.

Flow cytometric analyses of the DNA content of neoplastic cells were performed in 60 cases of endometrial carcinoma using formalin-fixed, paraffin-embedded tissues. Each tumor was graded histologically prior to flow cytometry. Aneuploidy was found in almost half of the tumors, including those of moderate and high degrees of differentiation, although the poorly differentiated tumors were more commonly aneuploid. The cytometric and histologic results were then compared with prognosis (5-year survival). In general, survival was greater with diploid than with aneuploid tumors. With well-differentiated tumors, patients with both diploid and aneuploid lesions had a favorable prognosis, but with moderately and poorly differentiated tumors those with diploidy had a more favorable prognosis than those with aneuploidy. We conclude that flow cytometry is of principal prognostic value in poorly differentiated endometrial carcinomas.

The clinicopathologic associations and effect on prognosis of cytoplasmic steroid receptor content were studied in 168 patients with clinical Stage I and II endometrial carcinoma. Cytoplasmic estrogen receptor status was associated (p less than 0.01) with histologic differentiation, nuclear differentiation, and histologic documentation of extrauterine metastases. Progesterone receptor status was related (p less than 0.05) to histologic differentiation and histologic cell type, and combined estrogen receptor/progesterone receptor status was associated (p less than 0.05) with histologic differentiation, peritoneal cytology, extrauterine metastases, and histologic cell type among the 105 patients who had determination of both estrogen and progesterone receptors. Single-factor analysis revealed significant (p less than 0.05) effects of estrogen receptor status, progesterone receptor status, and estrogen receptor/progesterone receptor status on disease-free survival. All other clinicopathologic features significantly (p less than 0.05) affected prognosis, except for peritoneal cytology. With use of stepwise regression analysis of proportional hazards, estrogen receptor, progesterone receptor, and combined estrogen receptor/progesterone receptor status were significant independent prognostic factors, replacing histologic assessment of glandular or nuclear differentiation in the models. These data suggest that receptor status of primary endometrial carcinomas provides important information relevant to tumor behavior which complements the information provided by conventional clinicopathologic analysis.

The progesterone receptor (PR) content was determined in the cytosol and in the nuclear fraction in 28 cases of endometrial adenocarcinoma after in vitro incubation of tissue slices in the presence of progesterone. In 17 instances besides the malignant, nontransformed tissue from the same uterus was taken for analysis. The level of PR was estimated by Scatchard plot analysis or by one-point assay, whenever not enough tissue was available. The distribution of PR between the cytosol and the nuclear fraction was studied. Positive correlation between the concentrations of total and of nuclear progesterone binding sites was found in all samples of nontransformed tissue whereas in cancerous tissue this was true only in about 40% of cases. In about 60% of neoplastic tissue samples the PR were namely found predominantly in the cytosol. Since we observe that in many malignant endometria the PR are poorly transferred from the cytoplasm to the nucleus even under optimal conditions for in vitro activation/translocation, we conclude that in these cases the action of progesterone is for some reason altered either at the level of receptor activation or at the level of translocation of the hormone-receptor complex from the cytoplasm to the nucleus.

The presence of an estrogen-regulated protein with 24,000 molecular weight has been studied in 47 patients with endometrial carcinomas and in 29 patients with cervical carcinomas in order to correlate its presence with that of estrogen receptors (ERs) and progesterone receptors (PgRs). In the cytosol tumor samples the Mr 24,000 protein was detected by the Western blot technique using a monoclonal antibody (C11), while the presence of ER and PgR was studied by the one-point dextran-coated charcoal assay. In the tumor tissue sections immunohistochemistry was applied to detect Mr 24,000 protein, ER, and PgR; in these cases monoclonal antireceptor antibodies (H222 and mPRI) were used to localize the receptor proteins. In endometrial and endocervical adenocarcinomas the presence of Mr 24,000 protein correlated significantly with that of ER (P less than or equal to 0.05) in the cytosol samples; when the evaluation was performed in the tumor sections, the presence of Mr 24,000 protein correlated with that of ER (P less than or equal to 0.005) and PgR (P less than or equal to 0.05) as well. The study also showed that almost 70% of the well-differentiated adenocarcinomas had ER, PgR, and Mr 24,000 protein. In 25% of the endometrial adenocarcinomas examined the tumors were associated with normal, proliferative, and hyperplastic endometrium; in these cases the presence of ER, PgR, and Mr 24,000 protein was evaluated by immunohistochemistry in the malignant and nonmalignant endometrium. On the other hand, there was a lack of correlation between Mr 24,000 protein, ER, and PgR in the squamous carcinomas of the uterine cervix and in the endometrial adenocarcinomas with squamous cells. In most of these cases the tumors lacked ER and PgR although 80% of them contained the Mr 24,000 protein to a variable degree. It is suggested that Mr 24,000 protein is involved in growth and differentiation (the Mr 24,000 protein is a heat shock protein) and that the gene coding of this protein is under hormonal control only in those tissues where growth and differentiation are strongly hormonally controlled (breast and endometrium).

Estradiol receptor (ER) and progesterone receptor (PR) levels were measured in 216 endometrial adenocarcinomas. Tumors containing more than 10 fmol/mg protein (for ER) and 20 fmol/mg (for PR) were considered receptor-positive. Tumors were both ER- and PR-positive in 75%. In stage I and II tumors, ER and PR levels were significantly higher than in stages III and IV. A similar relationship was established for minor (not deeper than 0.5 cm) and considerable involvement (deeper than 0.5 cm). There was an inverse correlation between ER and PR levels and ER- and PR-positive tumors incidence, on the one hand, and anaplasia, on the other. Adjuvant progestin therapy proved ineffective in tumors with a PR level under 100 mol/mg protein.

Thirty-two samples from adenocarcinoma of the uterine body were analyzed for estrogen receptor (ER). ER was measured with two techniques: isoelectric focusing (IF) in polyacrylamide gels and enzyme immuno assay (EIA) with monoclonal antibodies. When comparing, a very good correlation between IF and EIA was found. ER recovery was, however, increased twice when using the latter technique. The advantages of EIA in terms of its rapidity and simplicity should be considered against its, at present, greater expenses compared with methods based on radiolabeled ligands.

The steroid receptor content of the primary endometrial cancer of 22 patients who were treated for recurrent or advanced disease has been measured and correlated with response to medroxyprogesterone acetate. No patient with a progesterone receptor (PR)-negative tumor responded and only 2 patients with PR-positive tumors responded, perhaps related to the low levels of PR in the tumors. It waits to be assessed whether receptor status is as good a guide to response to hormone therapy as tumor differentiation, site of recurrence, or disease-free interval.