Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas.

Although the link between the BRCA1 tumour-suppressor gene and hereditary breast and ovarian cancer is established, the role, if any, of BRCA1 in non-familial cancers is unclear. BRCA1 mutations are rare in sporadic cancers, but loss of BRCA1 resulting from reduced expression or incorrect subcellular localization is postulated to be important in non-familial breast and ovarian cancers. Epigenetic loss, however, has not received general acceptance due to controversy regarding the subcellular localization of BRCA1 proteins, reports of which have ranged from exclusively nuclear, to conditionally nuclear, to the ER/golgi, to cytoplasmic invaginations into the nucleus. In an attempt to resolve this issue, we have comprehensively characterized 19 anti-BRCA1 antibodies. These reagents detect a 220-kD protein localized in discrete nuclear foci in all epithelial cell lines, including those derived from breast malignancies. Immunohistochemical staining of human breast specimens also revealed BRCA1 nuclear foci in benign breast, invasive lobular cancers and low-grade ductal carcinomas. Conversely, BRCA1 expression was reduced or undetectable in the majority of high-grade, ductal carcinomas, suggesting that absence of BRCA1 may contribute to the pathogenesis of a significant percentage of sporadic breast cancers.

GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

Determinants of ovarian cancer risk. I. Reproductive experiences and family history.

Reproductive experiences and family history were assessed in 215 white females with epithelial ovarian cancer and in 215 control women matched by age, race, and residence. Pregnancy exerted a strong protective effect against ovarian cancer, which increased with the number of live-born children. After adjustment for parity, an effect of age at first live birth and breast-feeding was not apparent. Menstrual events did not differ significantly between cases and controls, although cases were more likely to have had an earlier menopause and less likely to have had a surgical menopause. Women with ovarian cancer had more frequently used menopausal hormones in cyclic fashion compared to controls. Regarding family history, women with ovarian cancer more frequently reported consanguinity in their ancestry and a highly frequency of primary relatives with cancer of the colon, lung, ovary, and prostate gland.

BRCA1, BRCA2, and Rad51 operate in a common DNA damage response pathway.

The two major hereditary breast cancer susceptibility genes, BRCA1 and BRCA2, are associated with early-onset breast and/or ovarian cancer and encode products that each interact with the product of the eukaryotic RecA homologue, hRad51. We have recently found that BRCA1 and BRCA2 coexist in a common biochemical complex. The two proteins also colocalize in subnuclear foci in somatic cells as well as on the axial elements of developing synaptonemal complexes in meiotic cells. Thus, BRCA1 and BRCA2 participate in a common DNA damage response pathway associated with the activation of homologous recombination and double-strand break repair. Dysfunction of this pathway may be a general phenomenon in the majority of cases of hereditary breast and/or ovarian cancer. The BRCA1/BRCA2 complex may function in postreplicational repair processes activated during the DNA synthesis stage of the cell cycle.

DNA polymerase stalling, sister chromatid recombination and the BRCA genes.

Heritable predisposition to breast and/or ovarian cancer is determined, in part, by germline mutation affecting one of two tumor suppressor genes, BRCA1 and BRCA2 (Miki et al., 1994; Wooster et al., 1995). These genes are required for the maintenance of genomic integrity and for control of homologous recombination in somatic and meiotic cells. Here, we explore the hypothesis that a major role of the BRCA gene products in the somatic DNA damage response centers upon the control of recombination between sister chromatids during S phase. By analogy with model organisms, we suggest that stalling of a mammalian DNA polymerase complex by its encounter with abnormal DNA structure calls forth a series of responses that collaborate to enforce appropriate recombinational outcomes, and to suppress inappropriate or 'illegitimate' recombination.

Differential cyclic AMP responses to calcitonin among human ovarian carcinoma cell lines: a calcitonin-responsive line derived from a rare tumor type.

Several human tumor cell lines have been reported to have specific receptors for calcitonin (CT) and CT-responsive adenylate cyclase. In order to correlate patterns of responsiveness to CT, parathyroid hormone (PTH) and prostaglandin E2 (PGE2) with tumor morphology and intermediate filament protein expression, we examined four human ovarian tumor cell lines (BIN-16, BIN-22, BIN-53, BIN-67) which had been cultured from cells of metastatic foci. In two cell lines (BIN-53 and -16) there were small increases in cAMP content after exposure to CT and in three cell lines (BIN-53, -16, and -22) larger increases with PGE2. There was no cAMP response in any of the cells to PTH. In BIN-67 cells, however, CT induced a striking (greater than 20-fold) increase in cAMP content. Histologically, the CT-nonresponsive tumor lines were derived from serous adenocarcinomas while the CT-responsive tumor line was from a rare small cell carcinoma. Gel electrophoretic and immunofluorescence microscopic analyses had previously disclosed that the CT-nonresponsive cell lines contained high levels of simple epithelial keratins and no or very low levels of vimentin (characteristic of ovarian surface epithelial cells), while the CT-responsive cell line contained almost exclusively vimentin. Thus, cells cultured from a rare type of ovarian tumor were CT-responsive and were distinguishable from CT-nonresponsive ovarian tumor cells by initial tumor histology and intermediate filament protein expression.

The effects of continuous androgen secretion on the hypothalamic-pituitary axis in woman: evidence from a luteinized thecoma of the ovary.

Hyperandrogenic states in women are often accompanied by disruption of gonadotropin secretion. However, the role of androgens per se in the pathogenesis of this abnormality is poorly understood. We report a woman with a virilizing ovarian tumor in whom the effects of continuous androgen secretion on the hypothalamic-pituitary axis were investigated in detail. A 29-yr-old woman with previously normal reproductive function, including prior fertility, was evaluated for amenorrhea and hirsutism. She had elevated peripheral serum levels of testosterone (T; 337-500 ng/dl) and androstenedione (A; 258-353 ng/dl). Her serum LH level was above the normal follicular phase range and was hyperresponsive to LHRH, whereas the FSH level was below normal early follicular phase levels and increased minimally in response to LHRH. A luteinized thecoma of the left ovary, shown by catherization of the ovarian venous blood to be secreting both T and A, was removed. Postoperatively, serum T and A levels returned to normal, and the patient had a normal ovulatory menstrual cycle in the 30 days after the operation, documented by daily determinations of plasma estradiol, progesterone, and gonadotropin levels. A repeat LHRH test in the follicular phase of the second postoperative menstrual cycle was completely normal. This case indicates that the characteristic abnormalities of gonadotropin secretion observed in hyperandrogenic states such as polycystic ovarian disease can result from chronic androgen secretion by an ovarian tumor and that normal folliculogenesis and gonadotropin secretion can be promptly restored by the elimination of the androgen excess.

H-Y antigen in a 46,XX female with dysgenetic ovaries.

Hypogonadism secondary to ovarian dysgenesis or resistant ovary syndrome was diagnosed in a 19-yr-old obese woman with primary amenorrhea, a 46,XX karyotype, and an H-Y+ cellular phenotype. Small ovoid gonads (1.5 X 0.6 cm) were found found bilaterally; these were encased in a dense venous network. The stroma was ovarian, and primordial follicles and some primary follicles were present, but there were no follicles at or beyond the antrum stage. There was no evidence of testicular tissue and no evidence of malignancy. Analysis of serological data indicated the possibility of residual H-Y antigen in the blood cells of the mother.

The spectrum of abnormal patterns of gonadotropin-releasing hormone secretion in men with idiopathic hypogonadotropic hypogonadism: clinical and laboratory correlations.

Several lines of evidence indicate that hypothalamic-pituitary-gonadal activity varies among men with idiopathic hypogonadotropic hypogonadism (IHH). To test the hypothesis that a spectrum of abnormalities of GnRH secretion underlies the syndrome of IHH, we characterized the patterns of GnRH-induced gonadotropin secretion during periods of frequent sampling in 50 consecutive men with IHH and contrasted them with those in 20 normal men. The largest group of IHH patients (n = 42) had no detectable LH or FSH pulsations and could be categorized into 2 subsets according to the presence or absence of evidence of spontaneous puberty. The most severely affected subset (n = 32), who recalled no history of puberty, had testes with a mean volume of 3.3 +/- 0.5 (+/- SEM) ml, with a prepubertal appearance on biopsy, and often were anosmic (n = 17). The second subset of apulsatile IHH men (n = 10) had histories of partial or complete spontaneous sexual development with subsequent isolated loss of sexual function, testes with a mean volume of 13.3 +/- 1.9 ml (P less than 0.01 compared to the first subset), a pubertal or adult appearance of the testes on biopsy, and an intact sense of smell. In a second group of IHH patients (n = 3), LH was secreted predominantly in a nighttime pattern similar to that of normal children during early puberty. These men were aged 18-24 yr, had a mean testicular volume of 10.5 +/- 2.3 ml, pubertal changes on testicular biopsy, and an intact sense of smell. A third group of IHH men (n = 4) had LH pulses of abnormally low amplitude. Only one patient in this group had a history of spontaneous sexual development. The mean testicular volume of these patients was 5.6 +/- 1.9 ml, and the testes appeared prepubertal (n = 3) or pubertal (n = 1) on biopsy. In addition to these groups, another patient had apparent LH pulsations and nearly normal amplitude, but the LH was bioinactive and appeared to consist chiefly of alpha-subunit. Testing of other anterior pituitary hormone functions did not distinguish IHH men from normal men. However, those IHH patients with some evidence of endogenous GnRH secretion had higher basal and stimulated serum PRL levels than IHH men without such evidence (P less than 0.05), suggesting an influence of GnRH on PRL secretion.

A mutation in the follicle-stimulating hormone receptor occurs frequently in human ovarian sex cord tumors.

A subset of ovarian tumors, referred to as sex cord-stromal tumors, produce endocrine manifestations due to the secretion of estrogens or androgens. Because gonadotropins induce the growth, differentiation, and function of the steroid-producing cells of the ovary, we hypothesized that mutations in the FSH receptor (FSH-R) might occur in this group of tumors. Ovarian sex cord tumors (n = 13), small cell carcinomas of the ovary (n = 3), and control DNA specimens (n = 116) were screened for mutations in the transmembrane domains of the FSH-R. A heterozygous T-->C mutation was found at nucleotide 1777 that converts codon 591 from phenylalanine to serine (F591S). This sixth transmembrane domain mutation was found in 9 of 13 (69%) sex cord tumors and 2 of 3 ovarian small cell carcinomas, but it was not present in control specimens, including 5 normal ovaries, 5 nonsex cord ovarian tumors, 16 thyroid tumors, or 90 specimens of peripheral blood leukocyte DNA, suggesting that this nucleotide change is not a polymorphism. The functional effects of identified mutations were assessed by expression of the wild-type or the F591S mutant FSH-R in COS-7 cells. The F591S mutation eliminated FSH-stimulated cAMP production, and a similar effect was observed when this mutation was introduced into the homologous location of the LH receptor. The high prevalence of the F591S mutation in the FSH-R suggests that it plays a role in the development of ovarian sex cord tumors.

Malignant melanoma metastatic to the ovary. A clinicopathologic analysis of 20 cases.

Twenty cases of malignant melanoma metastatic to the ovary are reported. The patients, whose ages ranged from 21 to 60 (average 37.5) years, typically presented because of abdominal swelling or pain. Approximately 50% of the patients also had metastatic tumor outside the ovary, usually within the pelvis and upper abdomen, at the time of presentation. Twelve patients were known to have had a cutaneous malignant melanoma 1 month to 13 years before their ovarian tumors were discovered, and pigmented lesions had been removed previously from three other patients. Most patients are known to have died within a few years of discovery of their ovarian tumors but two were alive without evidence of disease 5 and 8 years later. The ovarian tumors, which were bilateral in nine cases, ranged up to 20 (average 10.5 cm) in greatest dimension. Six of them were either entirely black or had discernible black or brown foci. The most common microscopic appearance was that of large cells with abundant eosinophilic cytoplasm growing in nodular aggregates or diffusely. Occasional tumors were characterized by small cells with scanty cytoplasm, and in five tumors spindle cells were present. Another pattern was growth in the form of discrete rounded aggregates having a nevoid appearance. Eight tumors contained folliclelike spaces. Major cytologic features of the tumors included prominent nucleoli in 13, cytoplasmic pseudoinclusions in many nuclei in five, and intracytoplasmic melanin pigment in nine cases. In the 10 cases studied immunohistochemically, most of the tumor cells were strongly positive for S-100 protein and fewer cells were positive for HMB-45 in the seven tumors that were stained for this antigen. Melanosomes were identified in the three tumors examined ultrastructurally. These neoplasms often were difficult to differentiate from many other types of tumors, including juvenile granulosa cell tumor and small cell carcinoma, because of the presence of folliclelike spaces.

Atypical forms of microglandular hyperplasia of the cervix simulating carcinoma. A report of five cases and review of the literature.

We report five cases of microglandular hyperplasia with unusual histologic features. The diagnosis of carcinoma--usually clear cell adenocarcinoma--was seriously entertained in all the cases. Three of the patients were premenopausal; two were postmenopausal. Two of them were on oral contraceptives; one was on estrogen and progesterone replacement therapy. The histologic features that caused diagnostic difficulty were: a solid, sheet-like proliferation of cells (four cases), a pseudoinfiltrative pattern (one case), abundant stromal hyalinization (one case), signet ring cells (two cases), hobnail cells (two cases), and the presence of moderate degrees of nuclear atypicality (two cases) and occasional mitotic figures (two cases). However, foci with these features usually merged with areas of typical microglandular hyperplasia. After conservative therapy in four cases, follow-up of up to 12 years has been unremarkable. It is important for pathologists to be aware of these unusual histologic appearances of microglandular hyperplasia to avoid its misinterpretation as a malignant tumor.

Nephrogenic adenoma. A report of 15 cases, review of the literature, and comparison with clear cell adenocarcinoma of the urinary tract.

The clinical and pathological features of 15 previously unreported and 98 reported cases of nephrogenic adenoma were reviewed and compared with those of the 19 reported clear cell adenocarcinomas of the urinary bladder and urethra. Nephrogenic adenoma has occurred in patients from 4 to 83 (average 41) years of age. Two thirds of the patients were male, and most had a history of a genitourinary operation or procedure, genitourinary trauma, urinary calculi, or a renal transplant. Nephrogenic adenomas are typically small but have measured up to 7 cm. They may be papillary, polypoid, or sessile, and approximately 18% have been multiple. Microscopic examination shows, singly or in combination, tubules, cysts, and papillae lined by cells that typically have scanty cytoplasm and exhibit little cytological atypia and no or only very rare mitotic figures. Clear cell adenocarcinomas, in contrast, occur in a generally older age group, have a strong predilection for females, and typically lack the clinical features associated with nephrogenic adenomas. The carcinomas are almost always solitary and usually large. The microscopic findings of a diffuse growth pattern, clear cells with a high cytoplasmic glycogen content, and significant nuclear atypia and mitotic activity are characteristic of carcinomas in contrast to nephrogenic adenomas.

Ovarian steroid cell tumors (not otherwise specified). A clinicopathological analysis of 63 cases.

The clinical and pathological features of 63 steroid cell tumors, not otherwise specified, were reviewed. The patients ranged in age from 2 1/2 to 80 years. The most common initial manifestation was virilization (41%); four patients had estrogenic manifestations, and four had hypercortisolemia with Cushing's syndrome. The tumors, 6% of which were bilateral, ranged from 1.2 to 45 cm in greatest dimension. Microscopic examination revealed two types of cells, which had overlapping features: those with abundant eosinophilic cytoplasm and those with vacuolated cytoplasm. Fat stains were positive in 75% of the 16 cases in which they were performed. Follow-up data ranging from 1 to 19 years (average 5.2 years) in duration were available for 50 patients. In 24 cases, the tumor was designated probably benign (no evidence of spread beyond the ovary within 3 or more years postoperatively). In 18 patients, the tumor was clinically malignant. The best pathological correlates of malignant behavior were: the presence of two or more mitotic figures per 10 high power fields (92% malignant); necrosis (86% malignant); a diameter of 7 cm or greater (78% malignant); hemorrhage (77% malignant); and grade 2 or 3 nuclear atypia (64% malignant).

"Adenoid cystic" carcinoma and adenoid basal carcinoma of the uterine cervix. A study of 28 cases.

Adenoid cystic carcinoma (ACC) and adenoid basal carcinoma (ABC) of the uterine cervix are rare tumors that have often been regarded as a single entity. We studied 28 cases of these neoplasms, with 14 cases in each category. Most patients were over 60 years of age, and there was a high proportion of black women. The majority of the women with ACC presented with postmenopausal bleeding and had an obvious mass on pelvic examination. Despite the tumors' architectural similarity to ACC of the salivary gland, microscopic examination of the cervical carcinomas showed necrosis, a high mitotic rate, and greater nuclear pleomorphism. In all but one of the cases, the tumor cells were negative for S-100 protein on immunoperoxidase staining--a finding that provides evidence against a myoepithelial component. However, S-100-positive dendritic cells were present in the stroma of the tumors and among the neoplastic cells. The patients with ABC were usually asymptomatic, without a gross abnormality of the cervix. Microscopic examination disclosed small nests of basaloid cells, almost always beneath, and often arising from, in situ or small invasive squamous cell carcinomas. In contrast to ABC, ACC was often complicated by local recurrence or distant metastasis. We conclude that ACC of the uterine cervix differs from ACC of salivary gland origin and is also distinct clinically and pathologically from cervical ABC.

Minimal-deviation endometrioid adenocarcinoma of the uterine cervix. A report of five cases of a distinctive neoplasm that may be misinterpreted as benign.

We describe five cervical adenocarcinomas with unusual, deceptively benign histological features that occurred in women 34 to 42 years of age and caused problems in interpretation. The tumors were incidental findings in hysterectomy or cone-biopsy specimens in four patients; the fifth patient was investigated because abnormal glandular cells were found on a Papanicolaou smear. One patient had been exposed in utero to diethylstilbestrol. The cervix is known to have been abnormal on gross evaluation in only one case. Microscopic examination disclosed a deceptively benign-appearing proliferation of glands and cysts for the most part unassociated with a stromal reaction. Cilia were present in four neoplasms and apical snouts in three. Features that indicated the neoplastic nature of the glandular proliferation in these cases, to varying extents in individual cases, included the number of glands and their distribution, the shapes of the glands, their presence deep in the cervical wall, the focal presence of a stromal reaction, and moderate cytologic atypicality with occasional mitotic figures. None of the tumors is known to have recurred or metastasized. In our opinion, these distinctive neoplasms represent minimal-deviation endometrioid adenocarcinomas of the cervix.

Uterine carcinomas simulating microglandular hyperplasia. A report of six cases.

Six uterine adenocarcinomas, one cervical and five endometrial, that simulated microglandular hyperplasia histologically are described. The cervical neoplasm occurred in a 34-year-old woman who had been taking oral contraceptives. The five endometrial cancers occurred in postmenopausal patients 57 to 69 years of age; two of them were receiving, or had been receiving, premarin and provera and two were receiving only premarin. The patient with the cervical neoplasm had an abnormality observed on pelvic examination. The five postmenopausal patients presented because of vaginal spotting, bleeding, or discharge; their carcinomas were discovered in endometrial curettage specimens. On initial pathologic examination two specimens were misinterpreted as benign and in the remaining four cases there was uncertainty as to whether the lesion was benign or malignant. All the neoplasms had conspicuous microglandular patterns, with the gland lumens typically containing eosinophilic mucinous secretion and numerous acute inflammatory cells, which were also characteristically prominent in the stroma. Subsequent hysterectomies in all the cases showed residual adenocarcinoma with more typical features. Three of the endometrial tumors were mucinous adenocarcinomas and two mixed mucinous and endometrioid adenocarcinomas; the cervical tumor was a moderately differentiated adenocarcinoma of endocervical type. These cases illustrate that microglandular hyperplasia should be diagnosed with caution in a postmenopausal patient, particularly if lesional tissue is present in an endometrial curettage specimen, and, rarely, cervical adenocarcinomas have foci that simulate microglandular hyperplasia.

Oxyphilic clear cell carcinoma of the ovary. A report of nine cases.

Nine clear cell carcinomas (CCC) of the ovary with a prominent component of cells with abundant eosinophilic cytoplasm are reported. The majority of these tumors--which we have designated "oxyphilic clear cell carcinomas"--were misinterpreted by the referring pathologists as tumors of other types. Each specimen, however, had one or more features establishing it as a clear cell carcinoma, including tubules and cysts lined by cuboidal, hobnail, or flattened cells; nests and sheets of cells with abundant clear cytoplasm containing glycogen; and an adjacent adenofibromatous component. Two tumors were bilateral. The ages of the patients (average, 55 years), their clinical presentation, and the gross appearance of the neoplasms were similar to those of clear cell carcinomas in general. The diagnosis of clear cell carcinoma should always be considered in the differential diagnosis of an ovarian tumor with oxyphil cells, particularly if the patient is postmenopausal. Thorough sampling should be undertaken in such cases to identify other, more typical foci of clear cell carcinoma in order to avoid misdiagnosis.

Testosterone-secreting adrenal ganglioneuroma containing Leydig cells.

A 58-year-old woman was virilized by a testosterone-secreting adrenal ganglioneuroma containing Leydig cells with crystalloids of Reinke. Examination of the ovaries revealed stromal hyperthecosis and hilus cell hyperplasia. The evidence from this case, as well as related cases in the literature, suggests that cells of the adrenal gland may become transformed into cells of gonadal type in both hyperplastic and neoplastic disorders.