Potential predictors of chemotherapy response in ovarian cancer--how do we define chemosensitivity?

OBJECTIVE: The aim of this study was to assess whether microvessel density (measured by CD31), vascular endothelial growth factor (VEGF) or multidrug resistance (MDR1) could determine the response to chemotherapy or act as prognostic factors in ovarian cancer. METHODS: Seventy-nine ovarian specimens were immunostained. Pearson correlation, 1-way ANOVA and chi-square were used for univariate analysis. Kaplan Meier survival curves were used, log-rank was used for univariate analysis and a Cox proportional hazards regression model was used for multivariate evaluation. Response to chemotherapy was assessed after 6 months and again after 1 year. RESULT: Quantifying VEGF proved to be a valuable independent prognostic indicator in progression-free survival (PFS) (p<0.05) and overall survival (OS) (p<0.0001). VEGF correlated with response to chemotherapy at the 6-month interval (r=0.446, p<0.001) but failed to correlate at the 1-year interval. Increased staining with CD31 was associated with decreased PFS (p<0.01) and OS (p<0.01) in univariate but not multivariate analysis. MDR1 failed to act as a prognostic marker or as a predictor of response to chemotherapy. CONCLUSION: VEGF correlates with response to chemotherapy at the 6-month but not the 12-month interval. What should our criteria be for determining sensitivity to chemotherapy? CD31, VEGF and MDR1 do play a role in some ovarian malignancies but other factors are likely to be involved and perhaps molecular profiling will determine which factors will be important for determining the response to chemotherapy.

Genome-wide analysis of deoxyribonucleic acid in endometrial cancer using comparative genomic hybridization microarrays.

The aim of this study was to identify amplified oncogenes in endometrial cancer using array-based comparative genomic hybridization (array CGH). Despite its prevalence, the molecular mechanisms of endometrial carcinogenesis are still poorly understood. The selected array CGH allows the simultaneous examination of 58 oncogenes commonly amplified in human cancers and is capable of achieving increased mapping resolution compared with conventional CGH. A subset of 8 specimens from a bank of 60 malignant and normal specimens was selected for array analysis to identify potential genes of interest. TaqMan polymerase chain reaction was carried out on the 60 specimens to examine if aberrations at the genomic level correlated with gene expression and to compare expression in normal and malignant samples. Oncogenes amplified in the endometrial cancers included AR, PIK3CA, MET, HRAS, NRAS, D17S1670, FGFR1, CTSB, RPS6KB1, LAMC2, MYC, PDGFRA, FGF4/FGF3, PAKI, and FGR. Three genes were examined at the messenger RNA level. AR and PIK3CA were higher in normal specimens, and MET was higher in malignant samples, suggesting a role for MET in endometrial cancer. Newer arrays examining more genes and larger sample numbers are necessary to elucidate the carcinogenic pathway in endometrial cancer.

Haemostatic risk factors in healthy postmenopausal women taking hormone replacement therapy.

OBJECTIVE: To compare changes in haemostatic parameters in healthy postmenopausal women taking either tibolone or 17beta-oestradiol/norethisterone acetate. METHODS: Factor VIIc, antithrombin, fibrinogen, thrombin-antithrombin complex (TAT), FDP (D-Dimer), tissue plasminogen activator (tPA) and plasminogen activator inhibitor I (PAI-1) were measured in 80 healthy postmenopausal women after 3, 6 and 12 months therapy with either 17beta-oestradiol/norethisterone acetate or tibolone. RESULTS: Both treatments significantly reduced fibrinogen, factor VIIc, antithrombin, tPA and PAI-1 antigen. Significantly lower levels of factor VIIc activity were observed on treatment with tibolone compared with 17beta-oestradiol/norethisterone acetate. TAT was unchanged with both treatments as was tPA activity. FDP (D-dimer) was increased on treatment with both preparations. CONCLUSIONS: The enhanced fibrin turnover and reduced antithrombin activity may play a role in the increased risk of venous thromboembolism in some susceptible women taking hormone replacement therapy (HRT) and could explain the lack of benefit of HRT in the secondary prevention of cardiovascular disease. The decreased levels of fibrinogen and factor VIIc found during treatment with 17beta-oestradiol/norethisterone acetate or tibolone may offer some degree of cardioprotection in healthy woman without pre-existing disease.

von Willebrand's disease: an important cause of dysfunctional uterine bleeding.

We assessed the prevalence of von Willebrand's disease (VWD) in patients with objectively confirmed dysfunctional uterine bleeding. A case-control study was designed to include 38 patients with objectively confirmed dysfunctional uterine bleeding and 38 age-matched controls with normal menstrual blood loss (MBL). Menorrhagia was defined as a mean MBL of greater than 80 ml on three consecutive menses as measured by the alkali haematin method. von Willebrand factor antigen, von Willebrand factor activity (VWF:Ac) and factor VIII:C were measured on three serial venous blood samples 1 week apart. VWD was diagnosed in five of 38 (13%) patients with menorrhagia and one of 38 (2.6%) patients with normal menstrual blood loss. The mean VWF:Ac value was significantly reduced in patients with menorrhagia (mean +/- standard deviation, 84.5 +/- 26.7 IU/dl versus 103.9 +/- 34.5 IU/dl; P < 0.01) and this effect persisted after exclusion of patients diagnosed with VWD. Failure to investigate patients for VWD will limit the potential benefits of medical therapies such as tranexamic acid or nasal desmopressin [1-desamino-8-D-arginine vasopressin, (DDAVP)] and, in addition, will lead to an increased risk associated with surgical intervention in patients with undiagnosed VWD.

Serous papillary adenocarcinomas of the ovary display heterogeneity in their response to chemotherapy.

The response of ovarian serous papillary adenocarcinomas to various cytotoxic drugs was examined using the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) cytotoxicity assay. Thirty tumors were collected and organized into four groups according to histologic grade and FIGO stage: stage III, grade 2; stage III, grade 3; stage IV, grade 2; and stage IV, grade 3. The MTS chemosensitivity assay was performed on each tumor to examine the response to cisplatin, paclitaxel, hycamtin and the combination of cisplatin and paclitaxel. Ovarian adenocarcinomas of similar stage and grade displayed varying responses to the same drug. A lower concentration of the drug was often as effective as the peak plasma concentration. For some specimens combination therapy was more effective for inhibiting tumor growth, and for others single-agent therapy gave a better response. A chemosensitivity/resistance profile is recommended before deciding on appropriate chemotherapy.

A pilot study of a thiazolidinedione, troglitazone, in nonalcoholic steatohepatitis.

OBJECTIVES: Troglitazone is a thiazolidinedione and peroxisome proliferator-activated receptor gamma (PPARgamma) ligand used to treat diabetes mellitus type II. Because hyperinsulinemia may be a factor in nonalcoholic steatohepatitis (NASH), we postulated that troglitazone could have beneficial effects in this disorder. Our study was initiated before reports of idiosyncratic hepatitis induced by this agent and was completed before its recent withdrawal from the market. METHODS: We studied 10 female patients (age 44 +/- 16) with histological NASH. All but two were obese (mean body mass index, BMI = 38 +/- 6). One had type 2 diabetes, and three had well-compensated cirrhosis with NASH. Troglitazone was given at a dose of 400 mg/day for < or = 6 months. Responders (defined as normal ALT at the end of treatment) were rebiopsied. Paired specimens were compared in blinded fashion. Mitochondria were quantitated using ultrathin electron microscopy. RESULTS: Seven of ten patients responded with normal ALT at the end of treatment. One of three nonresponders initially normalized ALT but returned to pretreatment level at 3 months. In this patient, therapy was stopped, and the ALT has remained at the baseline level with no other clinical or laboratory findings. In the responders, ALT fell from 87 +/- 38 before to 39 +/- 9 at the end of treatment (p = 0.01), and AST decreased from 77 +/- 23 to 30 +/- 8 (p = 0.002). Biopsy comparisons before and after therapy showed persistent steatohepatitis in all cases, although four of seven showed a one-point improvement in the necroinflammatory grade. Electron microscopy revealed elongation of the mitochondria after therapy. CONCLUSIONS: Normal ALT was seen in 70% of NASH patients at the end of treatment, but this biochemical response was associated with only mild histological improvement, and all follow-up biopsies had evidence of NASH. Normalization of the liver enzymes in patients with NASH who are treated with thiazolidinediones should be viewed with reservation. Follow-up biopsy is essential to evaluate the efficacy of these agents, which, at the histological level, appears to be relatively modest.

Uteroplacental hemostasis in intrauterine fetal growth retardation.

During pregnancy, extensive hemostatic changes occur in the uteroplacental circulation. Invading endovascular trophoblast cells induce physiological adaptations of uterine spiral arteries, required to accommodate the increased maternal blood flow to the intervillous space of the placenta as pregnancy advances. Much of the vascular endothelium and the underlying medial smooth muscle is replaced by trophoblasts, and fibrin or fibrinoid forms a major morphological feature of the arterial walls. Compared with endothelial cells, the trophoblast lining decidual spiral arteries have a reduced capacity to lyse fibrin, and recent studies have shown this to be caused by high levels of plasminogen activator inhibitors (PAI-1 and PAI-2). In pregnancies complicated by intrauterine fetal growth retardation (IUGR), with or without superimposed preeclampsia, a restricted physiological adaptation of uteroplacental spiral arteries is coupled with vascular lesions containing increased fibrin deposition. Significantly higher levels of PAI-1 are found in blood from the uterine vein at delivery and in tissue extracts of the placenta in these pregnancies than are found in normal pregnancy. Recent tissue culture studies have provided new information on the role of trophoblast cells in maintaining hemostatic control in the uteroplacental circulation in pregnancy. Cytotrophoblast cells isolated from the placenta and placental bed from IUGR pregnancies express significantly higher levels of PAI-1, coupled with a significant decrease in plasminogen activator activity, compared with trophoblast cells from normal pregnancy maintained in culture. This localized increased production of PAI-1 may play an important part in restricting endovascular trophoblast invasion in early pregnancy and increasing fibrin deposition and reducing uteroplacental blood flow in pregnancies complicated by IUGR.

Treatment of menorrhagia during menstruation: randomised controlled trial of ethamsylate, mefenamic acid, and tranexamic acid.

OBJECTIVE: To compare the efficacy and acceptability of ethamsylate, mefenamic acid, and tranexamic acid for treating menorrhagia. DESIGN: Randomised controlled trial. SETTING: A university department of obstetrics and gynaecology. SUBJECTS: 76 women with dysfunctional uterine bleeding. INTERVENTIONS: Treatment for five days from day 1 of menses during three consecutive menstrual periods. 27 patients were randomised to take ethamsylate 500 mg six hourly, 23 patients to take mefenamic acid 500 mg eight hourly, and 26 patients to take tranexamic acid 1 g six hourly. MAIN OUTCOMES MEASURES: Menstrual loss measured by the alkaline haematin method in three control menstrual periods and three menstrual periods during treatment; duration of bleeding; patient's estimation of blood loss; sanitary towel usage; the occurrence of dysmenorrhoea; and unwanted events. RESULTS: Ethamsylate did not reduce mean menstrual blood loss whereas mefenamic acid reduced blood loss by 20% (mean blood loss 186 ml before treatment, 148 ml during treatment) and tranexamic acid reduced blood loss by 54% (mean blood loss 164 ml before treatment, 75 ml during treatment). Sanitary towel usage was significantly reduced in patients treated with mefenamic acid and tranexamic acid. CONCLUSIONS: Tranexamic acid given during menstruation is a safe and highly effective treatment for excessive bleeding. Patients with dysfunctional uterine bleeding should be offered medical treatment with tranexamic acid before a decision is made about surgery.

The effects of hormone replacement therapy on plasma vitamin E levels in post-menopausal women.

OBJECTIVE: To measure vitamin E levels in post-menopausal women before and after HRT, compared with levels in premenopausal women. DESIGN: Post-menopausal women (n = 21) had plasma vitamin E levels measured before treatment and after 6 and 12 months treatment with HRT (2 mg 17-beta-oestradiol and 1 mg norethisterone acetate). The pre-menopausal group (n = 20) had plasma vitamin E levels measured at day 15-18 of the menstrual cycle. RESULTS: There was no significant difference in vitamin E levels between the pre-menopausal group and the post-menopausal group. Plasma vitamin E levels were not significantly altered by 12 months HRT. CONCLUSION: Post-menopausal women did not have altered levels of vitamin E compared with pre-menopausal women. Similarly HRT has no effect on plasma vitamin E levels. We conclude therefore that HRT does not reduce vitamin E levels in a similar manner to oral contraceptives and consequently post-menopausal women are unlikely to need a vitamin E supplement.

The effect of tranexamic acid on measured menstrual loss and endometrial fibrinolytic enzymes in dysfunctional uterine bleeding.

Fibrinolytic activity of the menstrual fluid is increased in dysfunctional uterine bleeding (DUB). We measured the effect of tranexamic acid on the menstrual blood loss and endometrial fibrinolytic enzymes in women with DUB (> 80 ml menstrual loss/cycle). Endometrial biopsies were taken between 24 and 36 hours after the onset of menstruation. Enzyme activity was assayed by measuring the rate of conversion of Glu-plasminogen to plasmin, using a chromogenic plasmin substrate. Antigen levels were measured using an enzyme linked immunoassay (ELISA) technique. Tranexamic acid reduced menstrual blood loss by 58% (p < 0.05). Endometrial tissue plasminogen activator activity and antigen and plasminogen activator inhibitor--type 1 antigen levels were significantly decreased by tranexamic acid. The effect of tranexamic acid on the fibrinolytic enzymes at local endometrial level may be responsible for its success in the treatment of menorrhagia.

Platelet activation in normotensive and hypertensive pregnancies complicated by intrauterine growth retardation.

OBJECTIVE: To study platelet function in normotensive and hypertensive intrauterine growth retardation (IUGR). DESIGN: ADP and collagen induced whole blood platelet aggregation, beta-thromboglobulin release and platelet count were measured in the IUGR groups at a mean gestational age of 36 weeks (28-40), and at 1, 24, 48 h and six weeks post delivery. The normal pregnancy group were studied serially at 12, 20, 28, 32 and 36 weeks of gestation and at 1, 24, 48 h and six weeks post delivery. SETTING: Trinity College Medical School, St. James's Hospital, Dublin. SUBJECTS: Twenty-nine women with a fetus with diagnosed IUGR were recruited for the study. Of these, 15 were normotensive throughout their pregnancy and the remaining 14 pregnancies were complicated by both hypertension and proteinuria. Twenty healthy primigravida acted as controls. RESULTS: In the hypertensive IUGR group, levels of collagen and ADP induced aggregation were almost 50% lower before delivery than in normal pregnancy. Platelet count in the hypertensive group was decreased by 30% compared with normal pregnancy. Levels of beta-thromboglobulin were 40 to 50% higher in both the normotensive and hypertensive IUGR groups compared with normal pregnancy. Unlike the hypertensive IUGR group, the normotensive IUGR group showed similar levels of platelet count and ADP and collagen induced aggregation to those found at 36 weeks of normal pregnancy. In the early puerperium of hypertensive pregnancies, the platelet parameters measured returned gradually to normal. The normotensive IUGR group had increased levels of ADP induced aggregation in the first 24 to 48 h post delivery. The platelet count in the normotensive but not the hypertensive group correlated with birthweight. CONCLUSIONS: Normotensive and hypertensive IUGR are accompanied by platelet activation. In normotensive IUGR, this activation appears to be confined to the uteroplacental circulation. In hypertensive IUGR, the results suggest that platelet activation also extends into the peripheral circulation resulting in a reduced platelet responsiveness and a lower platelet count. Release of vasoactive amines from activated platelets in the peripheral circulation may be responsible for the clinical syndrome of hypertension and proteinuria present in pregnancies complicated by pre-eclampsia and IUGR but absent in normotensive IUGR.

Endometrial fibrinolytic enzymes in women with normal menstruation and dysfunctional uterine bleeding.

OBJECTIVE: To study fibrinolysis in the endometrium in women with normal menstruation and dysfunctional uterine bleeding (DUB). DESIGN: Tissue plasminogen activator activity (t-PA) and antigen (t-PAAg) and plasminogen activator inhibitor Type 1 antigen (PAI-1) were measured in homogenates of endometrium sampled between 24 and 36 h after the onset of menstruation. SUBJECTS: Women complaining of menorrhagia who had negative findings at clinical examination and curettage had their menstrual blood loss (MBL) measured from the third cycle after D&C. Those with MBL greater than 80 ml per cycle formed the DUB group. MEASUREMENTS: Fibrinolytic enzyme antigen levels were measured with ELISAs. Tissue plasminogen activator activity was assayed by measuring the rate of conversion of Glu-plasminogen to plasmin, using a chromogenic plasmin substrate. CONCLUSIONS: There is a strong positive correlation between endometrial t-PA activity on the second day of menstruation and measured menstrual loss (P < 0.05). Concentrations of endometrial t-PAAg and PAI-1 antigen are higher in women with DUB compared with normal women during menstruation.

Whole blood platelet aggregation in moderate and severe pre-eclampsia.

OBJECTIVE: To compare whole blood platelet aggregation in moderate and severe pre-eclampsia with normal pregnancy. DESIGN: Whole blood platelet aggregation in response to collagen, ADP, PAF, adrenalin and arachidonic acid was measured in the pre-eclampsia group at 36 weeks gestation and at 1, 24 and 48 h and at five days and six weeks post delivery. The normal pregnancy group were studied serially at 12, 20, 28, 32, and 36 weeks gestation and at 1, 24, 48 h and six weeks post delivery. SETTING: Trinity College Medical School, St James's Hospital, Dublin. SUBJECTS: Thirty women with diagnosed pre-eclampsia were recruited for the study. Fifteen of these women had severe pre-eclampsia and the remaining 15 had moderate disease. The pre-eclampsia group were compared with 20 healthy primigravid women with uncomplicated pregnancies and deliveries. RESULTS: In women with severe pre-eclampsia, platelet aggregation in response to collagen, ADP, adrenalin and arachidonic acid was significantly lower at 36 weeks gestation compared with normal pregnancy. Lower levels of collagen induced aggregation were also found at 1 h post delivery when compared with normal pregnancy. Women with moderate pre-eclampsia showed a decreased response to aggregating agents at 36 weeks gestation but this was not significant. ADP, collagen and PAF induced aggregation was higher in women with moderate pre-eclampsia at 36 weeks gestation and during the early puerperium compared with severe pre-eclampsia. CONCLUSIONS: The clinical signs of pre-eclampsia are accompanied by a reduction in platelet responsiveness, the extent of which is related to the severity of the disease. This suggests that an abnormal platelet activation occurs early in pregnancies destined to be complicated by pre-eclampsia. This activation may be involved in the pathogenesis of pre-eclampsia since its inhibition using low dose aspirin has been shown to modify the disease in high risk pregnancies.

Increased whole blood platelet aggregation in normal pregnancy can be prevented in vitro by aspirin and dazmegrel (UK38485).

OBJECTIVE: To determine the effect of normal pregnancy and the early puerperium on whole blood platelet aggregation and to assess the role of thromboxane A2 (TXA2) in platelet aggregation in pregnancy. DESIGN: A prospective descriptive study. SETTING: TCD Medical School, St James's Hospital, Dublin. SUBJECTS: Twenty healthy primigravidae who remained normotensive during pregnancy and the puerperium. INTERVENTIONS: 20 ml blood samples were obtained serially at 12, 20, 28, 32 and 36 weeks gestation, during established labour and at 1 h, 24 h, 48 h and 6 weeks after delivery. MAIN OUTCOME MEASURES: Whole blood platelet aggregation in response to aggregating agents ADP, PAF (platelet aggregating factor) collagen, adrenaline and arachidonic acid (AA) at each stage of pregnancy and peuerperium was measured using a particle counting technique. The in vitro effect of aspirin and dazmegrel (thromboxane synthetase inhibitor UK38485) on platelet aggregation in pregnancy was also investigated. RESULTS: Platelet aggregation in response to collagen, adrenaline, ADP and AA were increased in the last trimester, during labour and at 1 h after delivery but decreased 24-48 h after delivery. Platelet aggregation in response to AA, collagen and adrenalin was reduced by both aspirin and dazmegrel. CONCLUSIONS: The earliest and most marked increases in platelet aggregation during normal pregnancy were found in response to AA and collagen. These platelet changes were prevented when whole blood was pre-incubated with either aspirin or dazmegrel. This suggests that enhanced production of TXA2 is responsible for increased platelet reactivity in normal pregnancy.

The mast cell and histamine concentration of the human post-menopausal uterus.

Mast cells and histamine concentrations have been studied in uteri removed by hysterectomy from women in their post-menopausal years. Mast cell numbers were expressed as mean numbers/mm2 following fixation in 10% formalin and staining with Azure B. The majority of mast cells, in both the endometrium and myometrium, were very densely stained. Mast cells in the myometrium showed a significant negative correlation with years post-menopausal (rs = -0.52, P less than 0.05). Extracted histamine from uterine tissue was condensed with o-phthaldialdehyde to form a fluorophore and its fluorescence was measured at 450 microns using a spectrofluorometer. No significant correlation was found between histamine concentrations in the uterine wall and years post-menopausal.

The ultrastructure of mast cells in the uterus throughout the normal menstrual cycle and the postmenopause.

During the menstrual cycle a gradation in mast cell granule ultrastructure was observed from the functional endometrium towards the myometrium of the uterus. Mast cells with particulate granules were present in the functional endometrium and those with granules containing identifiable scrolls in the basal layer of the endometrium and in the myometrium; mast cells containing very electron-dense granules were present in the deeper layers of the myometrium. The secretory activity of mast cells throughout the menstrual cycle is described. Mast cell secretion was observed to a lesser extent in the postmenopausal uterus. Mast cells with particulate granules were absent in the postmenopausal uterus and many very electron-dense granules were observed in mast cells in the myometrium.

Endometrial vascular features prior to and following exposure to levonorgestrel.

A study of endometrial vasculature, mast cell numbers and tissue levels of tissue plasminogen activator (tPA) prior to and following exposure to levonorgestrel (20 micrograms/day) administered via a vaginal ring was undertaken. Following exposure to levonorgestrel, significantly fewer arterioles were present in the endometrium. During the early secretory phase of the control cycle, a positive correlation was found between mast cell numbers and progesterone levels. Levonorgestrel-exposed biopsies had significantly higher numbers of vessels with endothelial gaps and haemostatic plugs when compared with early secretory endometrium and significantly higher numbers of haemostatic plugs when compared with mid-late secretory endometrium. During the early secretory phase, the numbers of vessels possessing haemostatic plugs positively correlated with the peripheral blood levels of oestradiol and the number of contracted endothelial cells showed a positive correlation with progesterone levels. In mid-late secretory biopsies, the numbers of vessels with contracted endothelial cells were found to correlate negatively with oestradiol levels and the difference in the levels of contracted endothelial cells between the mid-late secretory endometrium and levonorgestrel-exposed endometrium correlated positively with progesterone levels of post-treatment cycles.

Comparison between mefenamic acid and danazol in the treatment of established menorrhagia.

Forty women with established menorrhagia were treated with either mefenamic acid (500 mg thrice daily for 3-5 days in two cycles) or danazol (100 mg twice daily for 60 days) in an open parallel group randomized study. Mefenamic acid reduced mean menstrual blood loss from 160 ml to 127 ml (20%, P less than 0.01). Danazol reduced mean menstrual loss from 163 ml to 65 ml (60%, P less than 0.001). The percentage reduction in menstrual blood loss was significantly greater in the danazol group than in the mefenamic acid group, but the adverse side-effects occurred significantly more often in the danazol group (75%) than in the mefenamic acid group (30%, P less than 0.005). Overall, approximately half the women in each group were prepared to continue with the treatment they received to reduce their menstrual bleeding.

The morphologic characteristics of menstrual hemostasis in patients with unexplained menorrhagia.

The endometrium in uteri removed by hysterectomy in patients with unexplained menorrhagia was compared with the endometrium in patients with normal menstruation who had a hysterectomy for uterine prolapse. The vascular ultrastructure and hemostatic plug formation were examined from the late secretory phase throughout menstruation to day 9 of the proliferative phase of the menstrual cycle. Endothelial defects, with and without hemostatic plugs, were more common and were present longer in the endometrial blood vessels of patients with unexplained menorrhagia than in patients with normal menstruation. In normal menstruation, no vascular defects were observed after day 3 of the cycle, whereas vascular defects were observed up to day 9 in the blood vessels of patients with unexplained menorrhagia. These morphologic features are likely to play a major role in the increased menstrual bleeding in such patients without uterine pathologic findings.