Human endometrial adenocarcinoma transplanted into nude mice: growth regulation by estradiol.

A model for studying the growth of primary tumors of human endometrium and its regulation by 17 beta-estradiol has been developed in which ovariectomized nude mice are used as recipients. The receptors for sex steroids are maintained during serial transplantation of the tumor in this system. Although the rate of growth of receptor-negative endometrial tumors transplanted into ovariectomized nude mice is unaffected by the sustained presence or absence of estradiol, the growth of receptor-positive tumors is significantly increased by estradiol. Receptor-positive tumors treated with estradiol produced elevated concentrations of progesterone receptor. That the progesterone receptor is functional in this tumor is evident from the induction of estradiol 17 beta-dehydrogenase activity upon progestin administration. These findings are consistent with receptor-mediated regulation of growth of endometrial carcinoma.

Hormonal therapy of human endometrial adenocarcinoma in a nude mouse model.

The hypothesis that 17 beta-estradiol or tamoxifen (TAM) can potentiate clinical response of endometrial cancer treated with progestin was tested in an ovariectomized nude mouse system, using a sex steroid receptor-positive and a receptor-negative human endometrial carcinoma. Animals were divided into three groups: control; 17 beta-estradiol-treated; and TAM-treated. When tumors of a group reached about 1 cm in diameter, subgroups were given either 0.9% NaCl solution (saline) or medroxyprogesterone acetate (MPA). The receptor-negative tumor grew rapidly in all three groups, and several animals were dead before or during progestin treatment. The growth rate of receptor-containing carcinoma was significantly increased in TAM-treated mice compared to controls (p less than 0.02) but significantly less than that in 17 beta-estradiol-treated animals (p less than 0.01). Endometrial carcinoma in 17 beta-estradiol-saline-treated animals continued to grow rapidly, and all animals were dead by 11 weeks. The growth of tumors in the 17 beta-estradiol-progestin group was suppressed at 11 weeks, and some of these animals lived 20 weeks. Administration of progestin to TAM-exposed animals resulted in a remarkable regression of the tumor compared to TAM-saline-treated group. The growth rate of tumors in control animals (no implants) was unaffected by progestin treatment. We conclude that, in this nude mouse model, treatment with TAM and MPA is superior to MPA alone, or 17 beta-estradiol plus MPA for sex steroid receptor-positive endometrial carcinoma.

Estrogen-like effects of tamoxifen on human endometrial carcinoma transplanted into nude mice.

The effect of tamoxifen (TAM) on human endometrial carcinoma was investigated in nude mice bearing an estrogen receptor-positive or estrogen receptor-negative tumor. The receptor-negative tumor grew rapidly, and the rates of tumor growth of 17 beta-estradiol or TAM-treated animals were identical to the rate of controls. The estradiol receptor and progesterone receptor (PR) concentrations in the tumor cytosol remained undetectable under all experimental conditions. In contrast, the rate of growth of steroid receptor positive tumor was significantly accelerated in the presence of TAM compared to controls (p less than 0.02). The increased tumor growth rate was, however, significantly lower (p less than 0.01) than that observed in animals receiving 17 beta-estradiol. The PR concentration in these tumors was elevated in response to TAM treatment. That the TAM-induced PR was indeed functional was evident from (a) increased activities of the progestin-sensitive enzyme, 17 beta-estradiol hydroxysteroid dehydrogenase and (b) histological appearance of subnuclear vacuolization in these tumors after progestin administration. These studies indicate that continuous, short-term administration of TAM to nude mice results in an estrogen-like effect on endometrial carcinoma. Based on the finding that TAM induces functional PR, we predict that steroid receptor-positive endometrial carcinoma may show a greater response rate to combined, long-term treatment with TAM and progestin.

Heterogeneity of progesterone receptor distribution in human endometrial adenocarcinoma.

The clinical response of advanced endometrial adenocarcinoma to progestin therapy does not correlate perfectly with biochemically assayed progesterone receptor status of the tumor. We have previously suggested that heterogeneity of progesterone receptor at the cellular, tumor, and tissue levels, not detectable by the biochemical assay, might contribute to this discrepancy. A monoclonal antibody, hPRa-1, generated against human progesterone receptor, was used in the present study to immunohistologically define the heterogeneity of progesterone receptor distribution in primary endometrial carcinomas. Twenty-four hysterectomy specimens removed for the treatment of endometrial adenocarcinoma were examined by biochemical assay of progesterone receptor and immunohistochemistry. In two cases, in which tumor occupied almost all of the endometrial lining, more extensive sampling was performed with removal of four noncontiguous sites. Each site was subdivided for immunohistochemistry and biochemical assay of progesterone receptor. When present, progesterone receptor localization was confined to the nuclei of target cells. Variability in the distribution and intensity of staining was consistently observed within the tumors. Of 24 tumors 15 were determined to be progesterone receptor positive by biochemical assay, while 12 of 24 tumors displayed immunolocalization for progesterone receptor. The correlation of the results by the two methods was high (20 of 24 cases, 83%), and the discrepancies in three cases appeared to reflect tissue and tumor heterogeneity. Immunolocalization has demonstrated that heterogeneity is present at the tumor, tissue, and cellular level within endometrial carcinomas, and the failure of some progesterone receptor-positive (by biochemical assay) tumors to respond to progestin therapy may reflect false positive results due to contamination of progesterone receptor-negative tumors by adjacent benign endometrium or myometrium.

Differential distribution of estrogen and progesterone receptors in rabbit uterus detected by dual immunofluorescence.

The action of sex steroids on the growth and differentiation of target tissues requires the presence of specific intracellular receptors. We compared the distribution of cells containing estrogen receptor (ER) and/or progesterone receptor (PR) in rabbit uterus by immunohistochemistry using monoclonal antibodies directed against these receptors. Initial experiments using serial cryostat sections surprisingly revealed the intensity of staining for ER to be inversely proportional to that of PR, as follows: ER, luminal and glandular epithelium greater than myometrium greater than stroma; PR, stroma greater than myometrium greater than glands greater than luminal epithelium. Localization was strictly confined to the nuclei of target cells. Single and dual immunofluorescent labeling of ER and PR in cryostat sections was accomplished using fluorochromes with differing emission spectra. Individual fields of dual labeled sections were examined for red [phycoerythrin (ER)] and green [fluorescein (PR)] fluorescence, with the same distribution as noted by single antibody immunohistochemistry. Myometrial nuclei displayed fluorescence of equivalent relative intensity for both antibodies. Further, sequential exposure photomicrography (exposure first in the spectrum of phycoerythrin emission, followed by exposure in the spectrum of fluorescein emission) revealed the presence of occasional stromal cells staining only for PR and some luminal cells staining only for ER. This differential distribution of ER and PR within various cell populations of rabbit is a novel observation and challenges current concepts of receptor regulation. Dual immunofluorescent localization of both ER and PR within individual cells provides a unique perspective from which to investigate the interactive influences of these sex steroid receptors at the cellular level.

Monoclonal antibodies to human progesterone receptor: characterization by biochemical and immunohistochemical techniques.

Progesterone receptor (PR) from a human endometrial carcinoma (EnCa 101) grown in nude mice consists of two hormone-binding proteins with mol wt around 116,000 and 85,000. To generate monoclonal antibodies against this receptor, PR was partially purified from EnCa 101 and used to immunize Robertsonian mice. Immune mouse spleens were fused with HL-1 Friendly myeloma-653 cells, and hybridomas were screened by solid phase dot-blot assay and double antibody precipitation. Seven stable hybridomas were obtained, designated hPRa 1-7. Subisotyping revealed that hPRa 1 and 6 were immunoglobulin G2b, while the remainder were immunoglobulin G1. Ultracentrifugation in high salt sucrose gradients showed that six of the seven antibodies effected a shift of [3H]progestin-labeled PR from EnCa 101; only hPRa 4 was ineffective in this regard. Protein blots of EnCa 101 cytosols and DEAE eluates revealed that hPRa 1, 3, 4, 5, and 7 recognized both PR proteins equally. hPRa 2 recognized principally the 116,000 mol wt PR protein; it recognized the lower mol wt PR protein very poorly if at all, whereas hPRa 6 recognized only the 116,000 mol wt protein. Interestingly, the latter was consistently detected as a closely migrating triplet. Immunolocalization of PR by hPRa 1-7 in tissue sections was confined to nuclei of target tissues and varied in intensity: hPRa 7 greater than 3 = 5 greater than 6 = 2 greater than 1 greater than 4. In proliferative phase uterus, the intensity of staining was ranked: endometrial gland nuclei (3+) greater than myometrial cell nuclei (2-3+) greater than endometrial stromal cell nuclei (0-1+). Thus, seven monoclonal antibodies directed against human PR have been prepared, and their suitability for the study of PR by biochemical and immunohistochemical techniques has been demonstrated.

Villoglandular adenocarcinoma of the endometrium: a clinicopathologic study of 61 cases: a gynecologic oncology group study.

Papillary endometrioid or villoglandular adenocarcinoma (VGA) is a relatively common type of endometrial adenocarcinoma, but studies describing its behavior have yielded conflicting results. Patients with a component of VGA were identified in a review of 819 women entered in a Gynecology Oncology Group Study (Protocol 33) of clinical stages I and II endometrial adenocarcinoma. Cases with coexisting foci of serous or clear cell carcinoma were excluded from further consideration. Of the 61 cases that formed the study sample, there were 24 with pure villoglandular differentiation and 37 who were admixed with typical endometrioid adenocarcinoma (EA). The general clinicopathologic features of patients with pure and mixed VGA are compared with 469 patients with pure EA. The VGAs were better differentiated (grade 1 or 2--97% of VGA versus 74% EA, p = 0.001). but they were not significantly different with respect to median age, depth of invasion, or frequency of nodal spread. Six of the 61 patients with VGA died of their tumor. The disease-specific survival rate at 3 years for VGA is 94% (95% confidence interval: 0.88-0.99) compared with 88% (95% CI: 0.86-0.91) for EA. Two of the patients who died had pure villoglandular tumors and four had mixed villoglandular and endometrioid carcinoma. In view of the frequent admixture of VGA and EA and their generally similar biological characteristics, with a prognosis similar to that of typical EA, we conclude that VGA should be considered a variant of EA.

Apparent resistance in human endometrial carcinoma during combination treatment with tamoxifen and progestin may result from desensitization following downregulation of tumor progesterone receptor.

Combined treatment with tamoxifen and progestin effectively controlled human endometrial tumor growth in the nude mouse model. However, after an initial response the tumors became 'resistant' to continuous progestin administration. Tumors excised during the growth arrest or regrowth phases, showed return of the typical growth characteristics of EnCa-101, upon serial transplantation. The characteristic progesterone receptor proteins were observed by Western blot analysis in tamoxifen treated tumors, while tumors treated with both tamoxifen and progestin were devoid of receptor, beginning at 7 days after initiation of progestin therapy. Thus, downregulation of endometrial tumor PR resulting from continuous progestin administration presumably leads to desensitization to progestin, after an initial growth inhibitory response.

Paget's disease in a retroperitoneal teratoma.

Extragonadal, mature teratomas of the retroperitoneum are rare, with fewer than 100 cases having been reported. The frequency of malignancy arising from one of the benign elements in this location is unknown, but it appears to be low. A unique feature of this case is the presence of Paget's disease in the squamous cyst lining of a mature teratoma that harbored an adenocarcinoma.

The immunohistochemical discrimination of endometrioid adenocarcinomas.

Carcinomas of endometrioid histology frequently arise in the endometrium, ovary, and endocervix and involve the pelvic tissues in women. Adenocarcinomas of psuedoendometrioid morphology developing in the colon also frequently involve the ovary. The authors retrospectively examined 97 adenocarcinomas from the uterus, cervix, ovary, and colon to ascertain whether the site of origin could be determined by using a battery of antibodies with the immunoperoxidase method on formalin-fixed tissue. This study was restricted to tumors with endometrioid morphology. There were 27 endometrial, 16 ovarian, 23 endocervical adenocarcinomas, and 31 psuedoendometrioid colonic adenocarcinomas. The battery of antibodies included vimentin (V), monoclonal carcinoembryonic antigen (mCEA), and monoclonal CEA D-14. V-positive cells were defined by the presence of a crisp paranuclear band of staining, and CEA-positive cells showed irregular or diffuse cytoplasmic staining. V diffusely decorated 22 of 27 (81.4%) of endometrial tumors, 3 of 23 (13%) of endocervical tumors, (rare, focal staining), diffusely stained 5 of 16 (31.3%) of ovarian tumors, and was rare and focal in 2 of 31 (6.4%) of colon tumors. Both CEA antibodies were negative for cytoplasmic staining in both endometrial and ovarian tumors, but decorated from 65.2% (CEA D-14) to 95.6% (monoclonal CEA) of endocervical tumors and from 83.8% (CEA D14) to 90.3% (mCEA) of colonic tumors. The authors conclude that endometrioid adenocarcinomas developing in endometrium and ovary are most often strongly V positive and CEA negative, which greatly aids in distinguishing them from endometrioid or pseudoendometrioid tumors arising in endocervix and colon, which are only rarely, and very focally V and CEA positive. The antibodies do not allow for discrimination between endocervical and colonic tumors. CEA D-14 offered no immunodiagnostic superiority over mCEA. These results support the use of immunohistochemistry is assisting in the distinction of endometrial from endocervical primary sites in curettage specimens and in metastatic sites.

Histologic response of normal human endometrium to steroid hormones in athymic mice.

Despite the widespread use of hormonal therapy for menopausal symptoms, oral contraception, and treatment of metastatic breast carcinoma, information concerning the histologic and biologic effects of individual sex steroids on the human endometrium remains incomplete. Repetitive endometrial biopsy is impractical, and ethical constraints limit the dosage and duration of administration for some steroids. The ovariectomized athymic mouse was investigated as a host for human endometrium in which the hormonal milieu may be manipulated and the histologic response determined. Minced proliferative-phase endometrium from hysterectomy specimens of normally cycling women was inserted into the subcutis of 4- to 6-week-old mice. Proliferation of endometrial gland cells occurred in the transplanted endometrium of estradiol-treated mice, while the complete sequence of secretory-phase events, including subnuclear vacuolization, luminal secretion, and decidualization of stroma, was observed during a 14-day period of treatment with estradiol and progestin (medroxyprogesterone acetate). Progestin treatment alone also caused secretory-phase changes, but the response was delayed and appeared weaker. The transplanted endometrium in control mice appeared to be inactive. These observations provide support for the use of this model to study the histologic response of human endometrium to sex steroids.

Variance in the interpretation of cervical biopsy specimens obtained for atypical squamous cells of undetermined significance.

We sought to determine whether the variability in dysplasia rates in cases of atypical squamous cells of undetermined significance (ASCUS) reflects variability in interpretation of cervical biopsy specimens. In phase 1, 124 biopsy specimens obtained because of a cytologic diagnosis of ASCUS were reviewed independently by 5 experienced pathologists. Diagnostic choices were normal, squamous metaplasia, reactive, indeterminate, low-grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion (HSIL). The rate of dysplasia ranged from 23% to 51%. All pathologists agreed in 28% of cases. In 52% of cases, the diagnoses ranged from benign to dysplasia. The overall interobserver agreement was poor. In phase 2, 60 cervical biopsy specimens (21 obtained for ASCUS, 22 for LSIL, and 17 for HSIL) were evaluated using the same diagnostic choices. Agreement was better in biopsies performed for HSIL and LSIL compared to those for ASCUS. Intraobserver reproducibility in the interpretation of biopsies performed for ASCUS ranged from poor to excellent. We conclude that variability in the interpretation of biopsy specimens plays an important role in the differences in rates of dysplasia reported for the follow-up of ASCUS.

Clomiphene citrate stimulation as an adjunct in locating ovarian tissue in ovarian remnant syndrome.

Ovarian remnant syndrome results from residual ovarian tissue after bilateral oophorectomy. The syndrome is associated with chronic pelvic pain and is suspected when premenopausal levels of FSH and LH are present in a patient with documented bilateral oophorectomy. Histologic demonstration of ovarian tissue at operation confirms the diagnosis. We treated a patient with ovarian remnant syndrome with a 10-day course of clomiphene citrate, 100 mg daily, to stimulate the residual ovarian tissue and facilitate localization. Preoperative ultrasound revealed a 5.0 x 3.5 x 6.2-cm cystic mass in the right adnexal region. Exploratory laparotomy easily localized the mass, and it was removed intact. Histologic slides demonstrated normal ovarian tissue with multiple follicles in various stages of development and a corpus luteum cyst. Clomiphene citrate is capable of stimulating an ovarian remnant, producing an enlarged, cystic structure easily localized by ultrasound. The increased size and preoperative knowledge of the location facilitated surgical removal.

Virilizing paraovarian tumors: a consequence of Nelson's syndrome?

In a patient with Nelson's syndrome, elevated peripheral concentrations of androgens usually associated with neoplasm prompted ovarian vein catheterization. Androgen excess was limited to the right ovary. However, laparotomy revealed bilateral multiple paraovarian nodules with histologic appearance of adrenocortical tissue. The occurrence of virilization from ectopic adrenal tissue with markedly elevated ACTH concentrations is exceedingly rare.

Glassy cell carcinoma of the uterine cervix. An ultrastructural study and review.

Glassy cell carcinoma is an unusual neoplasm of the uterine cervix that accounts for 1% to 2% of all cervical malignant neoplasms. Although it seems to have a distinct histologic appearance, it has generally not been recognized, as indicated by the extremely small number of cases reported in the literature. Previous studies have indicated an aggressive behavior, a poor response to irradiation and surgery, and a tendency to widespread metastases. It occurs in a younger age group (mean age, 41 years) than other cervical neoplasms and often has been associated with pregnancy. The histogenesis is not known, but electron microscopic examination of one case we studied, and review of one previously described case, demonstrate that it is an adenosquamous carcinoma. The need to define this neoplasm better will be satisfied only by additional, thorough histologic and clinical studies.

Morphology of human uterine cancer in nude mice. Effects of hormone and antihormone treatment.

We have recently described an experimental model for studying the hormonal modulation of growth and receptor content of human endometrial adenocarcinoma in ovariectomized athymic mice. The morphologic features of endometrial carcinomas can be maintained during serial transplantation in control animals. Further, administration of estradiol, tamoxifen citrate, and medroxyprogesterone acetate to animals with tumors containing estradiol and progesterone receptors resulted in histologic and ultrastructural changes in most foci resembling the response of normal human endometrium to these agents. No morphologic change was detected in the steroid-receptor-negative tumors of hormone-treated mice. These results demonstrate that histologic changes parallel the changes in the growth rate and hormone receptor content of tumors in this model system, and they provide support for the concept of receptor-mediated alteration in the morphologic features of endometrial adenocarcinoma.

Squamous differentiation in carcinoma of the endometrium: a critical appraisal of adenoacanthoma and adenosquamous carcinoma.

Although squamous differentiation within endometrial carcinomas has long been recognized by pathologists, its biologic significance has been the subject of continued debate. While some authors have found a worsened prognosis for women who have tumors with squamous elements, others have reported the prognosis to be better than conventional endometrial adenocarcinomas. Persisting confusion and disagreement about the use of the terms adenoacanthoma and adenosquamous carcinoma have complicated the issue. In this article we review the literature on the pathogenesis of squamous differentiation in the endometrium and discuss the histologic features, prevalence, and biologic behavior of adenocarcinoma with squamous differentiation. We conclude that keratin is a constituent of normal and neoplastic endometrial epithelial cells, and that overt squamous differentiation occurs by a mechanism that is currently unknown. Squamous differentiation is present in about 25% of endometrial adenocarcinomas, a frequency that appears to have been constant for the past 50 years. The squamous component of endometrial carcinomas may histologically appear benign, malignant, or indeterminant, and in the majority of instances closely parallels the differentiation of the glandular component. Endometrial adenocarcinomas with malignant-appearing squamous elements usually have poorly differentiated glandular components and have a prognosis identical to that of poorly differentiated adenocarcinoma without squamous differentiation. Endometrial adenocarcinomas with benign-appearing squamous elements are usually associated with well-differentiated glandular components and have a prognosis identical to that of typical well-differentiated adenocarcinoma. Some endometrial adenocarcinomas contain foci of squamous differentiation that appear neither clearly benign nor malignant. These often are associated with moderately differentiated glandular components; the prognosis for these women is not yet clearly defined. At the present time we are unable to attribute any prognostic significance to the presence of squamous differentiation in endometrial carcinomas. Because of the confusion and semantic arguments that revolve about the use of the terms adenoacanthoma and adenosquamous carcinomas, we recommend that those terms be abandoned and be replaced by the single term adenocarcinoma with squamous differentiation. As for any other endometrial tumor, the pathologist should provide information on histological grade, depth of myometrial invasion, and presence of vascular involvement or spread to the cervix in order to guide the gynecologist in determining appropriate therapy.

Hepatic granulomata. Presenting with prolonged fever. Resolution with anti-inflammatory treatment.

A patient is described who presented with a 1-month history of daily fever to 38.8 degrees C. There was no sign of joint pain or swelling and no skin rash. The patient had impressive hepatomegaly without splenomegaly. The only abnormal laboratory test was a sedimentation rate of 120 mm/hr. Ultrasound examination showed hypoechoic foci throughout the liver. These foci were confirmed by CT scan, which showed multiple well-marginated lesions of decreased attenuation and variable size throughout the right and left lobes of the liver. A liver biopsy specimen showed large nodules that were yellow and gritty in texture. Microscopic examination of biopsy specimens of these nodules showed extensive areas of necrotizing granulomatous inflammation with palisading histiocytes and occasional giant cells surrounded by necrotic foci. There was an associated fibroinflammatory infiltrate. The patient was treated with a nonsteroidal anti-inflammatory agent with prompt cessation of fever. A repeat CT examination of the liver after 14 months of treatment showed only mild hepatomegaly and a normal liver parenchyma. The focal lesions had disappeared. This is a case of hepatic granulomata in a child showing features of necrotizing inflammation.

Sex steroid receptors in normal and malignant endometrium.

Estradiol and progesterone receptor concentrations have been measured and their hormonal regulation extensively studied in normal human endometrium. However, in endometrial cancer, the biochemical assays presently used face the complex problem of tumor and tissue heterogeneity. This problem may be circumvented by immunocytochemistry on tissue sections proven to be histologically malignant. The in vivo experimental model developed in our laboratory is an ideal source of tissue necessary for purification of the progesterone receptor and antibody production. Hopefully, the physiology of the receptor may be studied under ideal conditions in this experimental system.