BRCA1 expression and improved survival in ovarian cancer patients treated with intraperitoneal cisplatin and paclitaxel: a Gynecologic Oncology Group Study.

BACKGROUND: Breast cancer 1, early onset (BRCA1) is a tumour-suppressor gene associated with familial epithelial ovarian cancer (EOC). Reduced BRCA1 expression is associated with enhanced sensitivity to platinum-based chemotherapy. We sought to examine the prognostic relevance of BRCA1 expression in EOC patients treated with intraperitoneal platinum/taxane. METHODS: The GOG-172 was a phase III, multi-institutional randomised trial of intravenous paclitaxel and cisplatin (IV therapy) vs intravenous paclitaxel, intraperitoneal cisplatin plus paclitaxel (IP therapy) in patients with optimally resected stage III EOC. The BRCA1 expression was assessed with immunohistochemistry (IHC) staining blinded to clinical outcome in archival tumour specimens. Slides with 10% staining were defined as aberrant and >10% as normal. Correlations between BRCA1 expression and progression-free survival (PFS) and overall survival (OS) were analysed using Kaplan-Meier method and Cox regression analysis. RESULTS: Of the 393 patients, 189 tumours had aberrant expression, and 204 had normal BRCA1 expression. There was an interaction between BRCA1 expression and route of administration on OS (P=0.014) but not PFS (P=0.054). In tumours with normal BRCA1 expression, the median OS was 58 months for IP group vs 50 months for IV group (P=0.818). In tumours with aberrant BRCA1 expression, the median OS was 84 vs 47 months in the IP vs IV group, respectively (P=0.0002). Aberrant BRCA1 expression was an independent prognostic factor for better survival in women randomised to IP therapy (hazard ratio (HR)=0.67, 95% confidence interval (CI)=0.47-0.97, P=0.032). Similar survival was observed in the IV and IP patients with normal BRCA1 expression. Multivariate but not univariate modelling demonstrated that IV patients with aberrant vs normal BRCA1 expression had worse survival. CONCLUSION: Decreased BRCA1 expression is associated with a 36-month survival improvement in patients with EOC treated with IP chemotherapy. Although these results merit validation in future studies, the results suggest that decreased BRCA1 expression predicts for improved response to cisplatin-based IP chemotherapy with cisplatin and paclitaxel.

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer.

BACKGROUND: Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH). METHODS: Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines. RESULTS: Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10(-11)). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10(-5) and 10(-29)), and identified breast and pancreatic cell lines with BRCA defects. CONCLUSION: The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

Tumorigenic epithelial stem cells and their normal counterparts.

ABC transporters are highly conserved and represent a major protective mechanism for barrier tissues as well as adult tissue stem cells. Emerging data support the existence of a cancer stem cell that shares features of tissue stem cells, including the ability to self-renew and undergo dysregulated differentiation. Here we show that a rare population of cells coexpressing MDR transporters and stem cell markers is a common feature across therapy-naive epithelial cancers as well as normal epithelial tissue. MDR+ and MDR- candidate tumor stem and progenitor populations were all capable of generating highly anaplastic transplantable human tumors in NOD/SCID. The finding that rare cells bearing stem cell markers and having intrinsic MDR expression and activity are already present within the tumorigenic compartment before treatment with cytotoxic agents is of critical importance to cancer therapy. Just as damaged normal epithelial tissues regenerate after chemotherapy by virtue of highly protected resting tissue stem cells, the existence of malignant counterparts in therapy-naive epithelial cancers suggests a common mechanism by which normal and tumor stem cells protect themselves against toxic injury.

Normal pregnancy is associated with peripheral leukocyte activation.

PROBLEM: Normal pregnancy has been described as both a pro-inflammatory condition and a T helper (Th)2-dominated state. Deviations in the percentage of different subpopulations of circulating leukocytes have been detected, although with conflicting results. This study was designed to analyse further the phenotype of subpopulations of peripheral blood leukocytes in normal pregnant women. METHOD OF STUDY: Whole-blood flow cytometry was used to differentiate subsets of leukocytes using directly labeled monoclonal antibodies to specific cell surface antigens and to a panel of activation-associated markers in 33 normal pregnant women in their third trimester and in 26 non-pregnant controls. RESULTS: We found a significant increase in the proportion of granulocytes and of CD8+ T lymphocytes during pregnancy. Up-regulation of the expression of adhesion molecules was observed on granulocytes, monocytes and T lymphocytes. CONCLUSIONS: Pregnancy alters the representation of leukocyte subpopulations in the maternal circulation and is associated with systemic activation of leukocytes.

Physiological range of human chorionic gonadotropin for support of early human pregnancy.

OBJECTIVE: To determine the physiological range of hCG in early pregnancy. DESIGN: Retrospective study of patient charts. SETTING: Magee-Women's Hospital IVF clinic, Monroeville, Pennsylvania. PATIENT(S): Sixty patients with successful, singleton birth outcomes. INTERVENTION(S): Serum hCG measurements on days 12-16 post-oocyte retrieval (OR). MAIN OUTCOME MEASURE(S): Lowest values, highest values, mean values, quartile mean values, and 48-hour doubling times for days 12-16 post-OR. RESULT(S): The average production of hCG in successful pregnancies is roughly 4-fold greater than the lowest amount observed in successful pregnancies, suggesting that a considerable excess of hCG is normally produced. Additionally, the average doubling time is almost 2-fold greater than the slowest doubling rate. CONCLUSION(S): The data from this study provide a set of values for the minimum and maximum threshold of hCG for days 12-16 post-OR that may be physiologically required, although not entirely predictive, for a successful IVF pregnancy outcome.

Placental cellular immune response in women infected with human parvovirus B19 during pregnancy.

Human parvovirus B19 can cause congenital infection with variable morbidity and mortality in the fetus and neonate. Although much information exists on the B19-specific antibody response in pregnant women, little information is available describing the cell-mediated immune (CMI) response at the maternal-fetal interface. The focus of this study was to characterize the CMI response within placentas from women who seroconverted to B19 during their pregnancies and compare it to controls. Immunohistochemical techniques were used to identify the various immune cells and the inflammatory cytokine present within placental tissue sections. Group 1 consisted of placentas from 25 women whose pregnancies were complicated by B19 infection; 6 women with good outcome (near-term or term delivery), and 19 with poor outcome (spontaneous abortion, nonimmune hydrops fetalis, or fetal death). Group 2 consisted of placentas from 20 women whose pregnancies were complicated with nonimmune hydrops fetalis of known, noninfectious etiology. Group 3 consisted of placentas from eight women whose pregnancies ended in either term delivery or elective abortion. The results of the study revealed a statistically significant increase in the number of CD3-positive T cells present within placentas from group 1 compared to group 2 or 3 (13.3 versus 2 and 1, respectively) (P < 0.001). In addition, the inflammatory cytokine interleukin 2 was detected in every placenta within group 1 but was absent from all placentas evaluated from groups 2 and 3. Together, these findings demonstrate evidence for an inflammation-mediated cellular immune response within placentas from women whose pregnancies are complicated with B19 infection.

Stimulation of uterine cell cytokine production by ovarian hormones.

PROBLEM: Although leukocytes do not possess significant numbers of ovarian steroid hormone receptors, their numbers in the endometrium vary consistently, relative to the menstrual cycle. The possibility that cell types within the endometrium express leukocyte-attracting genes in response to ovarian hormones was investigated. METHOD OF STUDY: Endometrial biopsies were collected 10 days post-leutinizing hormone surge; the cell types were separated and cultured individually for 5 days in the presence of increasing amounts of estrogen or progesterone. Following culture, RNA was collected from cells and reverse-transcription-polymerase chain reaction was used to determine relative levels of gene expression of monocyte chemotactic proteins (MCP)-1, -2, and -3, and interleukin (IL)-12 p35 and p40. RESULTS: Although both endometrial stroma and glands were able to make MCP mRNA, steady-state levels of gene expression did not vary significantly relative to hormone treatment. The same was found for the p35 molecule of the IL-12 gene; however, differences were observed for the p40 subunit. CONCLUSIONS: Within the human endometrium, chemokines other than MCP and IL-12 are most likely responsible for cycle-related leukocyte recruitment.

Evidence for superantigen involvement in preeclampsia.

PROBLEM: Preeclampsia is the leading cause of maternal morbidity and premature fetal delivery in the United States, most likely involving the immune system in disease genesis. In this report, we tested the hypothesis that a superantigen phenomenon is an important factor in the pathogenesis of the disease. METHOD OF STUDY: A semi-quantitative polymerase chain reaction (PCR) was used to assess T-cell receptor (TCR) beta chain variable (Vbeta) regions as an indicator of T-cell expansion in both peripheral blood and basal plate of preeclamptic patients. All the subjects were also molecularly typed to identify their HLA-class II alleles. RESULTS: In peripheral blood of the majority of the patients, there was a high abundance of the Vbeta4 gene family, which was not observed in the control group. Polyclonality of this Vbeta gene family was confirmed by analysis of the Valpha chain and the complementary determining region 3 (CDR3). The majority of patients carried the Human Leukocyte Antigens (HLA)-DRB1*13 allele. CONCLUSION: We present evidence for the existence of a superantigen-like effect in at least a subset of patients with preeclampsia.

Adenovirus-directed expression of a nonphosphorylatable mutant of CREB (cAMP response element-binding protein) adversely affects the survival, but not the differentiation, of rat granulosa cells.

Although usually considered to be a constitutively expressed protein, in the primate ovary the expression of CREB (cAMP response element-binding protein) is extinguished after ovulation, and its loss is temporally associated with the cessation of proliferation of luteal cells and the ultimate commitment of the corpus luteum to undergo regression. To determine the cellular consequences of the loss of CREB expression, we expressed a nonphosphorylatable mutant of CREB (CREB M1) in primary cultures of rat granulosa cells using a replication-defective adenovirus vector. Expression of CREB M1 did not block granulosa cell differentiation as assessed by acquisition of the ability to produce estrogen and progesterone in response to FSH or forskolin. However, granulosa cells expressing CREB M1, but not adenovirus-directed beta-galactosidase or enhanced green fluorescent protein, exhibited a 35% reduction in viability that was further reduced to 65% after stimulation with 10 microM forskolin. These results demonstrate that the trophic effects of cAMP (proliferation and survival) on ovarian granulosa cells are functionally separate from the effects of cAMP on differentiation and provide novel evidence that CREB may function as a cell survival factor in the ovary. The separation of signaling pathways that govern differentiation and survival in the ovary thereby provides a mechanism by which progesterone production, which is absolutely essential for the maintenance of pregnancy, can continue despite the cessation of proliferation of luteal cells and their commitment to cell death (luteolysis).

Globoside expression within the human placenta.

This report demonstrates the presence of the neutral glycosphingolipid, globoside, on the villous trophoblast layer of human placenta. Immunoreactivity for globoside which is the receptor used by human parvovirus B19 was strongest in villous trophoblast cells of first trimester placentae, with diminished reactivity in second trimester placentae, and a near lack of staining for the antigen in those of third trimester. This relative reduction in globoside-specific immunoreactivity in placentae of increasing gestational ages was confirmed using thin-layer chromatographic analyses of extracted neutral glycolipids from the syncytiotrophoblast layer and cytotrophoblast cells of first and third trimester placental villi. The presence of globoside on the protective trophoblast layer of the villi provides a potential pathway whereby B19 may be transmitted from an infected mother to the fetus. The virus once across the placental barrier, may gain access to its erythroid precursor target cells within fetal villus capillaries. The observed change found in globoside immunoreactivity correlates well with the observation that fetal outcome is worse when maternal infection occurs during first or second trimester as compared to an infection occurring near term. The reason for this observed difference in fetal outcome may be due not only to the presence of more target cells potentially to infect during the first and second trimesters, but also to the greater number of viral receptors present on the villous trophoblast layer.

Regional specialization of the cell membrane-associated, polymorphic mucin (MUC1) in human uterine epithelia.

The cell membrane-associated, polymorphic mucin, MUC1, has been proposed to hinder implantation by virtue of its anti-adhesive properties. Consistent with this proposal is the observation of a dramatic decrease in MUC1 protein and mRNA expression in the uterine epithelium of several species at the time of implantation. In contrast, little change in glandular epithelial expression of MUC1 protein or its mRNA during the peri-implantation period has been detected in humans. However, expression in the luminal epithelium, i.e. the epithelium involved in embryo attachment, has not been reported. Using tissue samples with a clearly defined luminal epithelium and antibodies directed against the cytoplasmic domain found in all cell-associated MUC1 species (CT-1) and against two MUC1 ectodomain epitopes, HMFG-1 and HMFG-2, we demonstrate that MUC1 expression in the luminal epithelium is maintained throughout the menstrual cycle. The staining observed with CT-1 correlates with that seen with HMFG-2, but not HMFG-1. HMFG-1 reactivity was high in all regions except basal glands in the mid proliferative endometrium and fell to very low levels throughout the tissue in the mid secretory phase. In all cases, HMFG-1 reactivity could be restored by predigestion with keratanase or neuraminidase which removes keratan sulphates and sialic acids, respectively. These observations suggest that regionally restricted glycosylation generates an altered external structure of MUC1. These alterations appear to decrease accessibility to the MUC1 protein core region and are maximal in luminal epithelium at the receptive phase. Due to their large highly extended structures, MUC1 ectodomains are very likely to be among the first cell surface components encountered during human blastocyst attachment to the luminal epithelium. Thus, MUC1 either must be locally removed during the attachment process or functions actually to promote the initial steps in embryo adhesion to the apical surface of the human uterine epithelium.

Methotrexate effects on trophoblast and the corpus luteum in early pregnancy.

OBJECTIVE: Our purpose was to determine whether methotrexate affects the trophoblast or corpus luteum when administered for abortion. STUDY DESIGN: A randomized controlled trial was performed in women requesting an abortion up to 49 days' gestation. Twenty patients were treated with intramuscular methotrexate 50 mg/m2 (10 women) or 60 mg/m2 (10 women). Serum beta-human chorionic gonadotropin, progesterone, and 17-hydroxyprogesterone levels were determined at baseline and then serially after methotrexate administration for the first 24 hours, then every 24 hours for 7 days. On the seventh day misoprostol 800 microg was administered vaginally. RESULTS: Serum beta-human chorionic gonadotropin increased at a lower rate than occurs in normal pregnancy. Progesterone levels averaged 56.9 +/- 19.8 nmol/L at baseline and 45.5 +/- 20.5 nmol/L (P = .01) 1 week after methotrexate. Progesterone decreased in 16 women over the 7 days and increased in the other 4; these latter women all aborted after a single dose of misoprostol. Levels of 17-hydroxyprogesterone plateaued during the first day after methotrexate administration; both progesterone and 17-hydroxyprogesterone declined simultaneously between the third and fourth day after methotrexate. CONCLUSIONS: Methotrexate most likely primarily affects trophoblast production of human chorionic gonadotropin, as evidenced by a blunting of the expected increase in serum beta-human chorionic gonadotropin resulting in less support for the production of progesterone by the corpus luteum. However, changes in progesterone levels after methotrexate administration were inconsistent and are unlikely to represent the ultimate effect of methotrexate in abortion. The less-than-normal increase in serum beta-human chorionic gonadotropin levels after methotrexate administration is most likely a result of disruption of cytotrophoblast syncytialization. This disruption may be the true effect of methotrexate in destabilizing the implantation site of an early pregnancy.

Effects of methotrexate on trophoblast proliferation and local immune responses.

Methotrexate is a folic acid analogue that has been used successfully for the treatment of ectopic pregnancy and, in conjunction with misoprostol, for medical abortions of early intrauterine pregnancies. To administer the most efficacious treatment requires knowledge of the mechanism underlying the induction of methotrexate-induced abortion. This study was designed to ascertain trophoblast integrity, proliferation and differentiation following administration of methotrexate. In addition, to determine if methotrexate affects the local uterine immune response, we ascertained the numbers and identities of decidual leukocytes following treatment. Ten women with undesired intrauterine pregnancies of 42-49 days gestation were recruited to receive methotrexte 50 mg/m2 i.m. A suction aspiration was performed 7 days later. Tissues from gestational age-matched elective surgical abortions were used as controls. Additionally, specimens from women who received methotrexate and misoprostol for abortion in a clinical trial of oral methotrexate in combination with misoprostol, who had a suction abortion because of continued embryonic cardiac activity 14 days after the methotrexate, were evaluated. Immunoreactivity to proliferating cell nuclear antigen and cyclin D3 antibodies was used to demonstrate a marked reduction in the proliferation index of cytotrophoblasts from methotrexate-treated abortions. Methotrexate treatment failures and non-treated pregnancies had a much higher proliferation index. There was no direct destruction of the syncytiotrophoblast, as indicated by the continued presence of human placental lactogen and beta-human chorionic gonadotrophin proteins. A decrease in the total number of leukocyte cells was observed in the decidua of methotrexate-treated samples, with the large granular lymphocyte (LGL) cells showing the greatest decline in numbers. Our conclusions from this study are that methotrexate acts primarily to derail the normal developmental programme of the trophoblast stem cell population, as well as to decrease LGL cell numbers in the decidua.

Endometrial leukocytes are altered numerically and functionally in women with implantation defects.

PROBLEM: A significant cohort of women with autoimmune thyroid disease (ATD) also suffer from reduced fertility. The finding that neither exogenous hormones nor donor eggs correct the infertility suggests that the problem involves an inherent endometrial defect. METHOD OF STUDY: Endometrial leukocyte populations in women with ATD were quantitated by immunohistochemistry. A semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to examine the expression of transforming growth factor (TGF)-beta 1, interleukin (IL)-4, IL-10, and interferon (IFN)-gamma in the endometrial samples. RESULTS: A significant increase in the endometrial T-cell population was observed in women with ATD compared to controls. The relative abundance of IL-4 and IL-10 was decreased in women with ATD compared to controls, whereas IFN-gamma was increased. No difference was noted in the abundance of TGF-beta 1. The source of cytokine production for IL-4, IL-10, and IFN-gamma was the endometrial leukocytes. CONCLUSIONS: Both the leukocyte numbers and cytokine expression profile were altered significantly in a well-defined group of women with implantation defects.

Expression of beta-adrenergic receptor kinase subtypes in the pregnant rat myometrium.

OBJECTIVE: This study tested the hypothesis that the increase in uterine tachyphylaxis to beta-adrenergic stimulation during pregnancy is associated with increased expression of the beta-adrenergic receptor-inactivating protein kinases. STUDY DESIGN: Messenger ribonucleic acid was isolated from snap-frozen myometrium collected from nonpregnant and pregnant rats ranging from 10 to 22 days of gestation. Autoradiographic analysis of beta-adrenergic receptor-inactivating protein kinase messenger ribonucleic acid expression was performed after hybridization with specific complementary deoxyribonucleic acid probes for types 1 and 2 beta-adrenergic receptor-inactivating protein kinases. Probe-specific hybridization was normalized for ribosomal ribonucleic acid detected with methylene blue. Protein expression was detected by Western analysis with use of specific polyclonal antibodies. RESULTS: Myometrial beta-adrenergic receptor-inactivating protein kinase type 2 messenger ribonucleic acid and protein levels increased during the course of pregnancy and in postpartum day 1. In contrast, type 1 levels remained unchanged during the same period. Estrogen treatment resulted in a modest 20% decrease in messenger ribonucleic acid levels of both subtypes. This effect was reversed with progesterone treatment. CONCLUSION: These results suggest that the myometrium undergoes a functional remodeling late in pregnancy to a state promoting myometrial contractions. The increased myometrial expression of type 2 beta-adrenergic receptor-inactivating kinase may explain the decreased effectiveness of beta 2-adrenergic receptor-mediated contraction inhibition at the end of pregnancy and labor.

Differential expression of G1 cyclins during human placentogenesis.

Cyclins are proteins that support the progression of cell-cycle stages in proliferating cells. The purpose of this study was to determine which of the cyclin genes is involved in the regulation of normal human trophoblast proliferation. The presence and cellular localization of four G1 cyclins D1, D2, D3 and E, were determined by immunohistochemistry. This analysis indicated that cyclins E and D3 are the predominant cyclins in villous trophoblast. D2 was present only within the villous core, in fetal macrophages. Positive immunoreactivity for cyclin D1 was strongest in second and third trimester placentae, in the cells lining the intravillous vessels with additional reactivity in extravillous cytotrophoblasts. Because cyclin E protein was present in a greater percentage of cells than those that are dividing, Western blot analysis was performed to validate the fidelity of the immunohistochemistry data. The results of the Western analysis revealed that two forms of cyclin E protein of the appropriate size were present. Data collected from this study suggest that within the trophoblast lineage, cyclins D3 and E are important cell cycle regulatory proteins, and further, that cyclin E may function in trophoblast terminal differentiation as well.

Decidual leukocyte populations in ectopic pregnancies.

OBJECTIVE: To determine whether the recruitment of decidual leukocytes during pregnancy is the same in intrauterine pregnancies (IUPs) versus ectopic pregnancies (EPs). DESIGN: Intrauterine decidual samples from both EPs and IUPs were obtained for the evaluation of leukocyte populations. SETTING: In vitro experiment. PATIENT(S): Women with EPs and women with IUPs. MAIN OUTCOME MEASURE(S): Immunohistochemical identification of decidual leukocyte populations. RESULT(S): We have analyzed the decidual leukocyte populations from three women with EPs by immunohistochemical analysis. The data demonstrate a leukocyte infiltration similar to that found in decidua from normal pregnancies. CONCLUSIONS(S): These data support the hypothesis that decidual leukocyte recruitment and/or increases during pregnancy is primarily hormonally regulated.

Endometrial Th2 cytokine expression throughout the menstrual cycle and early pregnancy.

Embryonic implantation and maintenance of pregnancy is influenced by the maternal immunological response. Type 2 T-helper (Th2) cells secrete interleukins 4, 5, 6 and 10 and are associated with suppression of cell-mediated immunity. Temporal changes in the expression of these cytokines were detectable by immunohistochemistry throughout the menstrual cycle. In pregnancy, 10-fold or greater increases in interleukin 6 and 10 secretion were detectable by enzyme-linked immunoassay compared with the non-pregnant state. The pattern of expression of Th2 cytokines suggests that progesterone may influence endometrial cytokine secretion. During pregnancy, Th2 expression paralleled that of vimentin, a stromal cell marker, suggesting that these cells may be a principal source of Th2 cytokines may be a mechanism of suppressing cell-mediated immunity in the endometrium, which may in turn enhance embryonic implantation and maintenance of pregnancy.

Molecular cloning and characterization of murine cyclin E.

Using a monoclonal antibody developed to a mouse trophectodermal carcinoma stem cell line E6496D lysate, we have identified the gene that encodes murine cyclin E. The cDNA contains a consensus cyclin box and a long 3' untranslated region and shares over 75% homology with human cyclin E cDNA. We show that the gene is expressed in fetal tissues, embryonal carcinoma F9 cells and yolk sac carcinoma PYS-2 cells, but is not expressed in preimplantation stages of development. In adult tissues, cycE mRNA is detectable in the spleen and to a lesser extent in the testis and brain. These data show that cycE is developmentally regulated and unevenly expressed in fetal and adult mouse tissues.