Natural cytotoxicity and GnRH agonist administration in advanced endometriosis: positive modulation on natural killer activity.

OBJECTIVE: To investigate the effects of pharmacologic suppression of ovarian function on the immune system, with respect to the clinical outcome of endometriosis and the possibility of an immunoendocrine combined treatment. METHODS: After informed consent, 25 of 37 patients with revised American Fertility Society stage III and IV endometriosis who underwent postoperative medical treatment were selected and enrolled for this immunoendocrine longitudinal study. Medical treatment consisted of tryptorelinum depot injection, 3.75 mg/month for 24 weeks. Blood samples were collected before the first injection in the early follicular phase, day 2-3 of the cycle, and during medical treatment (every 4 weeks) and follow-up (every 6 months). At the end of the study, we had ten blood samples per patient to evaluate the cytotoxic activity, the number of natural killer cells, and the serum levels of estradiol. Natural killer activity was determined against the K562 cell line by target cell retention of the fluorescent dye carboxyfluorescein diacetate. RESULTS: A positive immunomodulating effect was observed during GnRH agonist administration. In particular, a significant progressive increase in natural killer cell activity was defined within the first 12 weeks of medical treatment; after three injections, we observed the highest values of cytotoxicity, with a median of 7.1 lytic units (range 0.3-14.0; P = .02). Natural cytotoxicity then decreased toward a plateau, which persisted during therapy completation and follow-up, with slight fluctuations. In patients who had recurrence, the values of natural killer cell activity were constantly lower than those in patients with disease-free follow-up, particularly within the first 12 weeks of medical treatment.

Natural killer cell activity in endometriosis: correlation between serum estradiol levels and cytotoxicity.

OBJECTIVE: To evaluate the correlation between natural killer cell activity and serum estradiol (E2) levels in patients with different stages of endometriosis. METHODS: Natural killer cell activity of peripheral blood lymphocytes and serum E2 levels were evaluated in 73 women who underwent laparoscopy for pelvic pain, infertility, and benign adnexal masses. RESULTS: The 33 patients (45%) with endometriosis showed a significant decrease in natural killer cell activity in relationship to an increase in disease stage (correlation coefficient r = -0.83, P < .001). A significant inverse relationship was observed between cytotoxicity and serum E2 levels (correlation coefficient r = -0.89, P < .001). CONCLUSION: The relationship between natural killer cell activity and serum E2 levels suggests that an immunoendocrine interaction plays an important role in the progression of endometriosis.

Altered lipid composition, increased lipid peroxidation, and altered fluidity of the membrane as evidence of platelet damage in preeclampsia.

OBJECTIVE: To assess lipid composition, lipid peroxidation, and fluidity of the membrane of platelets from preeclamptic women. METHODS: We studied 40 primigravid women at 28-32 weeks' gestation; 20 were preeclamptic and 20 were normotensive. After preparing platelet membranes, we extracted lipids, measured cholesterol and phospholipid concentrations, and calculated the proportion of unsaturated to saturated fatty acids. Lipid peroxides expressed as conjugated dienes were determined by spectrophotometry. Membrane fluidity was determined by means of fluorescent lipophilic probes. Statistical analysis was performed by the Student t test, with significance at P < .05. RESULTS: Cholesterol concentration, cholesterol-to-phospholipid ratio, the amount of unsaturated fatty acids, conjugated dienes, and membrane fluidity significantly increased in platelets from preeclamptic patients as compared with the normotensive women. CONCLUSIONS: The discrepancy between cholesterol increase and membrane fluidity increase is consistent with the increase in unsaturated fatty acid content. In the platelet membrane, unsaturated fatty acids constitute the larger substrate for lipid oxidation and can also take part in the formation of thromboxane. Therefore, platelet membrane damage in preeclampsia, through imbalance of thromboxane A2/prostacyclin production, may contribute to the onset or maintenance of vasoconstriction and hypertension.

Decrease in peripheral blood polymorphonuclear leukocyte chemotactic index in endometriosis: role of prostaglandin E2 release.

OBJECTIVE: To investigate the effect of disease on peripheral blood polymorphonuclear leukocyte chemotactic index and natural killer cell cytotoxicity and to provide additional information concerning the cell-mediated immune function in endometriosis. METHODS: Chemotactic index of peripheral blood polymorphonuclear leukocytes, natural killer cell activity, and plasma estradiol (E2) and plasma prostaglandin (PG) E2 levels were evaluated in 46 women who underwent laparoscopy or laparotomy for pelvic pain, infertility, and/or benign adnexal masses. RESULTS: The 20 women (43%) with endometriosis showed a decrease in peripheral blood polymorphonuclear leukocyte chemotactic index, related to advanced disease stage (P < .001). A significant inverse correlation was observed between plasma PGE2 levels and chemotactic index in stage III and IV endometriosis (r = -.73, P = .004). Similarly, natural cytotoxicity was decreased significantly with respect to the stage of endometriosis (P = .004) and related inversely to plasma PGE2 levels (r = -.74, P = .003). A direct relationship was observed between PGE2 and plasma E2 levels (r = .59, P = .006). CONCLUSION: Advanced endometriosis is associated with decreased peripheral blood polymorphonuclear leukocyte chemotactic index and natural killer cytotoxicity, which may be related to plasma PGE2 and E2 levels.

In vivo amifostine (WR-2721) prevents chemotherapy-induced apoptosis of peripheral blood lymphocytes from cancer patients.

Aim of this work was to evaluate whether in vivo amifostine (WR-2721, ethanethiol, 2-[(3-aminopropyl)amino]-,dihydrogen phosphate (ester), Ethyol) pretreatment was able to prevent the apoptosis of peripheral blood lymphocytes (PBLs) induced by cytotoxic drugs. The study included 19 patients with advanced gynaecological cancers who received neoadjuvant polychemotherapy consisting of three cycles of cysplatin, adriamycin, and cyclophosphamide. Five patients received randomly amifostine pretreatment (910 mg/m2). PBLs apoptosis was measured through flow-cytometry using two different methods: a) DNA fragmentation of PBLs cultured in vitro for one hour; b) measurement of early apoptotic cells through Apostain uptake by fresh PBLs. The percentage of apoptotic PBLs was increased in all patients 24 hr after the first chemotherapy cycle (27.1 +/- 15.6 vs 6.3 +/- 6.2, p<.0001). A similar increase was observed in the following chemotherapy cycle. Amifostine pretreatment prevented the apoptosis of PBLs induced by chemotherapeutic drugs. Amifostine also prevented the reduction of lymphocyte number determined by chemotherapy. The results demonstrate that amifostine protects peripheral lymphocytes from the apoptotic damage induced by chemotherapy. This effect may explain the mechanism by which amifostine prevents the chemotherapy-associated reduction of leukocyte number.

p53 immunostaining and HPV DNA detection by PCR in cervical intraepithelial neoplasia: clinical implications of a combinated evaluation.

We analyzed p53 immunoreactivity and clinical outcome in a series of cervical intraepithelial neoplasias (CIN), with respect to HPV DNA positivity. Cervical biopsy samples were obtained from 86 women who attended our Colposcopic Service from January 1993 to June 1994 due to abnormal pap-smear suspicious for CIN and/or human papillomavirus infection. Forty-one women with histologically confirmed CIN were included in the study. p53 positivity was immunohistochemically detected by monoclonal antibody anti-human p53 (pAb D0-7, Dako Denmark; dilution 1:50), and expressed as the percentage of positive cells. p53 positivity was observed in 78% of CIN lesions. In particular, all the HPV DNA-negative dysplasias expressed p53 protein while only 12 out of 21 (57%) HPV DNA-positive were p53 immunoreactive; (P = .003) the p53 immunostaining was also significantly higher in HPV DNA-negative than in positive CIN (P = .049). By analyzing p53 positivity with respect to clinical-pathologic evolution of the disease, among HPV DNA-negative cases, progressive dysplasia had significantly higher values of p53 immunostaining when compared to persistent and/or regressive lesions (P = .002). These findings imply that p53 immunostaining, when analyzed with respect to HPV DNA status, may help to understand the behavior of dysplastic lesions and define their therapeutic approach. Extensive p53 staining in HPV DNA-negative CIN is probably correlated with a high risk of progression.

Expression of p53 and apoptosis of tumor cells in locally advanced cervical carcinoma after cisplatin based neoadjuvant chemotherapy.

Various chemoantiblastic agents cause DNA damage followed by apoptotic cell death through the activation of the p53 suppressor gene. The aim of our study was to evaluate the relationship between p53 protein expression, apoptosis of autologous tumor cells, and clinical response to neoadjuvant chemotherapy in patients with cervical carcinoma. Our study included 14 women with stage II squamous cervical carcinoma who had been admitted to the Institute of Gynecology and Obstetrics, Ancona University, between January 1990 and December 1995. The patients received neoadjuvant combination chemotherapy, consisting of three cycles of cisplatin (80 mg/m2) and bleomycin (30 mg/m2). After chemotherapy, radical surgery was performed. Bioptic specimens were obtained from cervical tumors before and after chemotherapy, and processed for DNA staining and apoptosis, and immunohistochemical staining with a monoclonal antibody against p53. Ten patients (71.4%) showed a clinical response (2 complete, and 8 partial), while of the remaining 4 cases (28.6%) 3 had no change and 1 showed progression after neoadjuvant combination chemotherapy. A significant relationship was observed between the overexpression of p53 and sensitivity to chemotherapy; responder patients showed a higher frequency of p53 positive cells than non-responders (p = .05). A significant direct relationship was observed between p53 protein immunostaining and apoptosis of tumor cells both before (p = .02) and after (p = .01) chemotherapy. Our study seems to define the relationship between p53 expression and sensitivity to cisplatin based chemotherapy in locally advanced cervical carcinoma, supporting the notion that the cytotoxic action of cisplatin can activate p53 mediated apoptosis. However, the limited number of patients in our series does not permit judgement on the clinical implications of the expression of p53 in patients undergoing neoadjuvant combination chemotherapy for locally advanced cervical carcinoma.

Increased serum 72 KDa metalloproteinase in serous ovarian tumors: comparison with CA 125.

72 KDa metalloproteinase (MMP-2) is an enzyme present in neoplastic cells and also in normal fibroblasts. It specifically cleaves type IV collagen, and therefore may play a critical role in tumor invasion and metastasis mechanisms. The aim of the present study was to determine serum levels of MMP-2 in serous ovarian tumors, and compare these with serum levels of CA 125. Ten primary ovarian serous cystadenocarcinomas, 5 borderline tumors, and 10 serous cystadenomas, all treated with primary surgery, were recruited from our series of serous ovarian tumors, and studied. Patients' serum samples were obtained before surgery, and the MMP-2 levels were measured by the substrate capture enzyme-linked immunosorbet assay. The analysis of serum MMP-2, gave values significantly higher in cystadenocarcinomas than in borderline tumors and cystadenomas (one way analysis of variance, P < 0.001); in particular, serum MMP-2 was significantly correlated to the MMP-2 immunostaining of the tumor (Spearman correlation, r = 0.82, and P < 0.001). An arbitrary cutoff of the median value of normal adult female samples (0.22 units) was chosen, and all except for one patient with cystadenocarcinoma was shown to have serum MMP-2 levels above the cutoff value, with 90% sensitivity, 70% specificity, and a 75% positive predictive value (50% of Cohen's Kappa); on the other hand, CA 125 showed 80% sensitivity, and a 73% positive predictive value. The association of serum MMP-2 with CA 125 increased sensitivity to 100% in patients with cystadenocarcinoma, with 70% persisting specificity and a 77% positive predictive value (54% of Cohen's Kappa). Serum MMP-2 levels were found to be significantly increased in patients with cystadenocarcinoma in comparison with borderline tumors and cystadenomas, showing a direct relationship with tissutal MMP-2 expression in serous ovarian tumors. Although our results were preliminary, they clearly suggested that serum MMP-2 may be an interesting diagnostic marker for cystadenocarcinomas.

bcl-2 protein expression in cervical intraepithelial neoplasia: no evidence of a prognostic significance in mild and moderate lesions.

BACKGROUND: The bcl-2 proto-oncogene codes for a protein which appears to block apoptosis. In our study, we examined bcl-2 protein expression in cervical squamous metaplasia, cervical intraepithelial neoplasia (CIN) and microinvasive squamous carcinoma with the aim of identifying a relationship between bcl-2 protein expression and neoplastic development and progression. MATERIALS AND METHODS: Cervical bioptic samples were obtained from 86 white women, selected consecutively from our Colposcopic Service from January 1993 to June 1994, because of abnormal pap- smear suspicious for cervical dysplasia and/or human papilloma virus (HPV) infection. Upon histologic evaluation, 41 women had CIN, 23 cervical condyloma, and 22 squamous metaplasia. Ten patients with microinvasive squamous carcinoma, matched for age and demographic characteristics, were selected from our series of invasive cervical carcinomas and immunohistochemically analyzed. The expression of primary tumor bcl-2 protein was immunohistochemically evaluated by antihuman bcl-2 monoclonal antibody (diluted 1:100, Dako, Copenhagen, Denmark) on formalin-fixed paraffin-embedded tissue. Positive staining was expressed as a percentage of positive cells per 1000 counted dysplastic cells for each case. RESULTS: Bcl-2 immunostaining was found in all the 22 squamous metaplasias, limited to the basal layer. Nineteen of the 41 CINs (46%) were bcl-2 immunoreactive, and 2 of the 10 microinvasive carcinomas (20%). By analysing CIN lesions, the bcl-2 protein showed a striking increase in the rate of positivity with increasing severity of CIN; the bcl-2 protein expression in CINs III was significantly higher than for CINs I, CINs II or microinvasive carcinomas (P = 0.03, P = 0.02, and P = 0.03 respectively). No relationship was observed between bcl-2 immunostaining and HPV infection. bcl-2 protein expression was not useful for predicting CIN I and II evolution, although the rate of persistence/progression was higher in bcl-2 positive dysplasias (7 of 9 cases, 78%) than in negative ones (13 of 21 cases, 62%) (p = 0.88). CONCLUSIONS: Based on these results, it seems possible that the increase in bcl-2 expression in higher grade of CINs implies an increasing protection against programmed cell death, but also the induction of genetic instability in dysplastic epithelial cells and a greater capacity to evolve into invasive carcinoma.

Ras p21 immunostaining in early stage squamous cervical carcinoma: relationship with lymph nodal involvement and 72 kDa-metalloproteinase index.

BACKGROUND: Ras p21 expression seems to be associated with aggressiveness of neoplastic growth and metastatic potentially in human solid tumors. In our series of early-stage squamous cervical carcinoma, we evaluated ras p21 expression with respect to lymph nodal involvement; the aim was to analyse the ras p21 immunostaining as potential marker of lymphatic spread, and investigate the relationship between ras p21 expression and 72 kDa-metalloproteinase immunostaining. PATIENTS AND METHODS: 46 patients with FIGO stage I squamous cell cervical carcinoma, who had undergone primary radical surgery with systematic pelvic and paraaortic lymphadenectomy (Piver's type III) at the Institute of Gynecologic and Obstetrics, Ancona University, were recruited from our series of 59 consecutive cases, and included the study. Any characteristic that could be relevant for prognosis was recorded such as: histologic grade of differentiation, tumor size, lymphatic spread, or adjuvant radiotherapy. Immunohistochemical staining was performed using the avidin-biotin peroxidase complex method (LSAB, Dako, Copenhagen, Denmark). Monoclonal antibody anti-pan ras (Ab-1) (Oncogene Science) and affinity purified rabbit anti-72 kDa-metalloproteinase antibody were used. Positivity for ras p21 was evaluated by semiquantitative analysis, while 72 kDa-metalloproteinase staining was expressed as the percentage of positive cells per 10(3) counted neoplastic cells (index). RESULTS: The expression of ras p21 was observed in 31 patients (67%) with FIGO stage I squamous cervical carcinoma. No connection was found between ras p21 expression and tumor size (P = 0.2), or histologic grade (P = 0.9), while a significant relationship was observed with respect to lymph nodal status (p = 0.048). By analysing 72 kDa-metalloproteinase immunostaining, ras p21 positive carcinomas showed significantly higher 72 kDa-metalloproteinase index than the negative ones (mean + standard deviation, 23.3% + 7.7% and 13.8% + 5.1% respectively, and P < 0.001). CONCLUSIONS: Though the relatively small size of our series does not allow any definitive conclusion, a significant relationship between ras p21 expression and risk of lymphatic spread was detected in early-stage cervical carcinoma. ras p21 positivity seems to be an indicator of neoplastic aggressiveness and lymphatic spread, and is associated with significantly higher expression of 72 kDa-metalloproteinase.