Proteomic characterization of the qualitative and quantitative differences in cervical mucus composition during the menstrual cycle.

The chemical composition of the cervical mucus (CM), its physical characteristics and the volume of secretion change cyclically throughout the menstrual cycle. The aim of this study was to identify the constitutive protein composition of CM of fertile women and the changes in the CM proteome throughout the menstrual cycle. Five fertile women who had a term delivery within 1 year before the study were enrolled. Proteomic analysis was performed using an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module and coupled with an LTQ Orbitrap XL hybrid mass spectrometer; bioinformatic software was used for functional and quantitative analysis. 59, 81 and 43 proteins (mean) were respectively identified in the pre-ovulatory, ovulatory and post-ovulatory samples. 38 common proteins were identified. 42, 38 and 17 exclusive proteins were respectively identified in pre-ovulatory, ovulatory and post-ovulatory CM. The main part of CM constituents has a catalytic activity, which is mainly related to hydrolase activity. The label-free quantitative analysis of the common proteins revealed a significant reduction in the protein abundance index for antileukoproteinase, after the ovulation, and a peak of haptoglobin at ovulation. This is the first application of high-resolution MS-based proteomics for the identification of protein constituents of CM. This approach may contribute to the identification of putative biomarkers of the female reproductive tract.

Intravenous contrast ultrasound examination using contrast-tuned imaging (CnTI) and the contrast medium SonoVue for discrimination between benign and malignant adnexal masses with solid components.

OBJECTIVE: To determine whether intravenous contrast ultrasound examination is superior to gray-scale or power Doppler ultrasound for discrimination between benign and malignant adnexal masses with complex ultrasound morphology. METHODS: In an international multicenter study, 134 patients with an ovarian mass with solid components or a multilocular cyst with more than 10 cyst locules, underwent a standardized transvaginal ultrasound examination followed by contrast examination using the contrast-tuned imaging technique and intravenous injection of the contrast medium SonoVue(R). Time intensity curves were constructed, and peak intensity, area under the intensity curve, time to peak, sharpness and half wash-out time were calculated. The sensitivity and specificity with regard to malignancy were calculated and receiver-operating characteristics (ROC) curves were drawn for gray-scale, power Doppler and contrast variables and for pattern recognition (subjective assignment of a certainly benign, probably benign, uncertain or malignant diagnosis, using gray-scale and power Doppler ultrasound findings). The gold standard was the histological diagnosis of the surgically removed tumors. RESULTS: After exclusions (surgical removal of the mass > 3 months after the ultrasound examination, technical problems), 72 adnexal masses with solid components were used in our statistical analyses. The values for peak contrast signal intensity and area under the contrast signal intensity curve in malignant tumors were significantly higher than those in borderline tumors and benign tumors, while those for the benign and borderline tumors were similar. The area under the ROC curve of the best contrast variable with regard to diagnosing borderline or invasive malignancy (0.84) was larger than that of the best gray-scale (0.75) and power Doppler ultrasound variable (0.79) but smaller than that of pattern recognition (0.93). CONCLUSION: Findings on ultrasound contrast examination differed between benign and malignant tumors but there was a substantial overlap in contrast findings between benign and borderline tumors. It appears that ultrasound contrast examination is not superior to conventional ultrasound techniques, which also have difficulty in distinguishing between benign and borderline tumors, but can easily differentiate invasive malignancies from other tumors.

Preoperative local staging of endometrial cancer: transvaginal sonography vs. magnetic resonance imaging.

OBJECTIVES: To compare the accuracy of transvaginal sonography (TVS) and magnetic resonance imaging (MRI) in the preoperative staging of endometrial carcinoma. METHODS: This was a prospective study in which 74 women consecutively diagnosed with endometrial carcinoma were examined using TVS by physicians trained in gynecological sonography and MRI by radiologists with a special interest in gynecology. All patients underwent surgical-pathological staging after removal of the uterus, adnexa and pelvic lymph nodes. Sensitivity, specificity, and positive and negative predictive values were calculated for each imaging modality with regard to detection of neoplastic invasion of the outer half of the myometrium and cervical involvement. RESULTS: TVS and MRI performed equally well in the preoperative staging of endometrial cancer, with no statistically significant differences between the two techniques. The sensitivity, specificity, positive and negative predictive values, and overall diagnostic accuracy for TVS in the evaluation of myometrial infiltration were 84%, 83%, 79%, 88% and 84%, respectively. Respective values for MRI were 84%, 81%, 77%, 87% and 82%. The corresponding statistics for detection of cervical involvement were 93%, 92%, 72%, 98% and 92% for TVS; and 79%, 87%, 58%, 95% and 85% for MRI. CONCLUSIONS: When carried out by expert practitioners, TVS shows good accuracy in the local staging of endometrial carcinoma. Because of its high costs, MRI should be offered only to those in whom TVS produces images of poor quality.

The role of CnTI-SonoVue in the diagnosis of ovarian masses with papillary projections: a preliminary study.

OBJECTIVES: To describe sonographically the distribution patterns of a second-generation contrast agent in the microcirculation of unilocular and multilocular ovarian masses with papillary projections, and to investigate whether qualitative evaluation of the passage of the contrast agent can improve the performance of sonography in distinguishing between benign and malignant masses with papillary projections. METHODS: Thirty-three patients with unilocular or multilocular ovarian masses with papillary projections were enrolled into the study in three clinical centers. The contrast-enhanced transvaginal examination was performed using 'Contrast Tuned Imaging' (CnTI) technology and SonoVue ultrasound contrast agent. RESULTS: Twenty-four (73%) lesions were benign, eight (24%) were borderline ovarian tumors, and one patient presented with an endometrioid ovarian adenocarcinoma. On color and power Doppler examinations the presence of vessels was demonstrated in 17 papillary projections, while on CnTI-SonoVue examination, the presence of vessels was shown in these 17 and in six additional cases. In all cases with absent papillary perfusion after SonoVue intravenous injection, the cyst wall appeared unequivocally regular. The sensitivity and specificity of conventional color Doppler examination with regard to malignancy were 100% and 67% and the positive and negative likelihood ratios were 3.03 and 0.16, respectively. For the contrast-enhanced examination the corresponding values were 100%, 42%, 1.7 and 0.26. The difference in specificity was statistically significant (P<0.05) because 14 cases, in which papillary perfusion was detected after SonoVue injection, proved to be benign on pathological examination. CONCLUSION: Qualitative evaluation of blood circulation in papillary projections using CnTI-SonoVue examination does not improve the discrimination of benign from borderline/malignant ovarian masses with papillary projections.

Preclinical in vivo activity of a combination gemcitabine/liposomal doxorubicin against cisplatin-resistant human ovarian cancer (A2780/CDDP).

Both gemcitabine and liposomal doxorubicin are antineoplastic drugs with clinical activity in platinum-refractory ovarian cancer. The purpose of this study was to evaluate the antitumor activity of a combination gemcitabine/liposomal doxorubicin administered to athymic mice bearing cisplatin-resistant human ovarian cancer (A2780/CDDP) xenografts. Emphasis was on the use of very low doses of each drug and of different dosing schedules. Data obtained showed that combined treatment with 80 mg/kg gemcitabine and 15 mg/kg liposomal doxorubicin produced a significant enhancement of antitumor activity compared with monotherapy at the same doses of these agents. Noteworthy is the fact that the majority of xenograft-bearing animals receiving the combination therapy demonstrated a complete tumor regression at the end of the study. A similar trend was observed when doses of both drugs were reduced to 20 mg/kg gemcitabine and to 6 mg/kg liposomal doxorubicin. Again, three out of ten mice receiving the combination were tumor free at the end of the study. No significant differences were observed in antitumor activity when comparing the simultaneous vs the consecutive dosing schedule. Remarkably, no additive toxicity was observed in any experimental trials. These data encourage clinical trials to prove the advantages of this combination treatment with respect to the single-agent chemotherapy in platinum-refractory ovarian cancer patients.

Ultrasound and color power Doppler in the detection of metastatic omentum: a prospective study.

OBJECTIVES: To analyze prospectively the ability of ultrasound to detect metastatic omentum in patients with suspicious pelvic masses and to describe the sonographic features of metastatic omental disease. METHODS: One hundred and eighty-four patients were evaluated preoperatively by ultrasound examination and of these 173 were used in the analysis. We defined as an ultrasound-positive examination one which visualized intra-abdominal aperistaltic solid tissue, located above the bowel loops and below the anterior peritoneal surface. Preoperative sonographic findings were compared with pathological results. RESULTS: Sonographic detection of metastatic omentum was achieved in 104 of 173 patients (60.1%), appearing as either solid aperistaltic tissue (80.8% of cases), or as solid discrete nodules (19.2%). When considering the echostructure of the surrounding bowel loops, this tissue appeared hypoechoic in 46 (44.2%) cases and isoechoic or slightly hyperechoic in the other 58 (55.8%) cases. In the overall series, the negative and positive predictive values (NPV and PPV) and the accuracy of ultrasound examination were 92.7%, 91.3% and 91.9%, respectively. When considering only the group of ovarian tumors, the NPV, PPV and accuracy were 91.9%, 94.6%, and 93.8%, respectively. CONCLUSION: Ultrasound examination is highly accurate in detecting metastatic omental involvement in cases with suspicious pelvic masses.

Gemcitabine in epithelial ovarian cancer treatment: current role and future perspectives.

Newer agents and combinations are needed in order to improve current results in ovarian cancer treatment. Gemcitabine is a novel agent that has shown consistent activity as a single agent in the treatment of platinum-resistant ovarian cancer and a favorable toxicity profile. Because of its clinical and preclinical synergism with platinum analogs, gemcitabine has been combined with carboplatin as a convincing approach in the treatment of platinum-sensitive recurrent ovarian cancer patients. Further combination of gemcitabine and other agents, including paclitaxel, is also feasible and has been actively studied in order to establish the role of gemcitabine in the management of treated and untreated ovarian cancer patients.

Ultrasound features of different histopathological subtypes of borderline ovarian tumors.

OBJECTIVE: To describe the gray-scale sonographic and color Doppler imaging features of the most common histopathological subtypes of borderline ovarian tumors. METHODS: We analyzed retrospectively the preoperative transvaginal sonographic reports of patients with a histological diagnosis of borderline ovarian tumor. All patients were scanned consecutively by two of the investigators using transabdominal and transvaginal gray-scale imaging to assess the morphology and color Doppler to obtain indices of the blood flow. Sonographic findings were compared to histopathological data. RESULTS: A total of 113 consecutive cases were reviewed from two referral centers for gynecological oncology. At histological examination 50 tumors (44%) were classified as being serous borderline ovarian tumors (SBOT), 61 (54%) were mucinous borderline ovarian tumors (MBOT) (42 intestinal type and 19 endocervical type), and two patients (2%) presented with borderline endometrioid tumors. SBOTs and endocervical-type MBOTs had very similar sonographic features and a smaller diameter, fewer locules (usually unilocular-solid lesions) and a higher color score than intestinal-type MBOTs. Intestinal-type MBOTs were characterized by a significantly higher percentage of lesions with > 10 locules when compared with the endocervical-type MBOTs. CONCLUSION: Intestinal-type MBOTs have different sonographic features from other common borderline ovarian tumors.

Does quantitative analysis of three-dimensional power Doppler angiography have a role in the diagnosis of malignant pelvic solid tumors? A preliminary study.

OBJECTIVE: To investigate the role of a simplified method for the three-dimensional (3D) quantification of tumor vascularity in the differential diagnosis of solid pelvic masses. METHODS: Twenty-four patients with a solid pelvic mass on B-mode ultrasound evaluation underwent 3D power Doppler evaluation before surgery. The volume of interest was obtained by drawing the largest section of the mass in three orthogonal planes. The following 3D vascular parameters were calculated for all patients: relative color, average color and flow measure. Receiver-operating characteristics curve analysis was used to choose the best cut-off value. The overall agreement between the test result and the actual outcome was calculated using kappa statistics. RESULTS: Fifteen of 24 subjects were found to have pelvic malignancy. The relative color and the flow measure were significantly higher in malignant (9.7 (8.98) and 8.95 (8.78) (median (interquartile range (IQR)), respectively) than in benign masses (2 (4.42) and 1.79 (4.71), respectively; P < 0.05) but there was no difference in the average color. The best cut-off values of relative color and flow measure were 3.2 and 2.8, respectively. The use of relative color showed a sensitivity of 80% with a specificity of 67% with an overall agreement that was higher, though only marginally so, than that of qualitative 3D power Doppler (kappa = 0.47 and 0.44, respectively). CONCLUSIONS: In a small group of pelvic masses that appear malignant on B-mode ultrasonography, the use of 3D quantification of tumor vascularity yields a diagnostic accuracy that is similar to that of subjective evaluation of vascularity. We suspect, however, that the quantitative method will produce more consistent results between operators.

Sonographic features of decidualized ovarian endometriosis suspicious for malignancy.

The discovery of an ovarian mass during pregnancy is often a difficult issue because of the risk related to surgical intervention during pregnancy. Moreover, ultrasound examination is often unable to provide a definitive diagnosis. A case of decidualized ovarian endometriosis is presented to highlight the challenges in this diagnosis. We report the transvaginal ultrasound findings, including color Doppler examination, magnetic resonance characteristics and tumor marker longitudinal evaluation during the first trimester of pregnancy, as well as the final histological characteristics of the lesion. Decidualization is a rare occurrence in ovarian endometriosis and must be differentiated from malignant transformation.

Color Doppler velocimetry and three-dimensional color power angiography of cervical carcinoma.

OBJECTIVES: To investigate the blood flow within invasive cervical carcinoma by transvaginal two-dimensional (2D) color spectral Doppler and three-dimensional (3D) color power angiography and to correlate these parameters with the clinicopathological characteristics. METHODS: Seventy-four patients with invasive cervical carcinoma were enrolled for the analysis. Squamous cell carcinoma serum antigen levels (SCC) were obtained for all the patients. Sections of all malignant tissues were analyzed for tumor expression of cyclooxygenase-2 (COX-2). All patients underwent color and spectral Doppler examination and 44 patients had 3D color power angiography. Color spectral Doppler parameters (color score, lowest resistance index (RI), highest peak systolic velocity (PSV)) and 3D color power angiography indices (relative color, average color, flow measure) of FIGO I/II cervical cancers were compared with those obtained in a control group of 24 patients with a normal uterine cervix. Pulsed Doppler parameters and the 3D vascular indices were compared with clinicopathological parameters, SCC serum antigen levels and tumor COX-2 expression. RESULTS: At color Doppler analysis 72 patients (97%) showed intralesional detectable vessels. Color spectral Doppler and 3D-derived parameters were significantly different in FIGO I/II cervical cancers compared with those in women with a normal cervix. A significantly higher color score (P = 0.0008), lower RI (P = 0.032) and higher PSV (P = 0.004) were associated with a tumor diameter > or =4 cm compared with smaller tumors. The highest PSV was significantly higher in patients with FIGO stage III/IV compared with FIGO stage I/II (P = 0.0069). There was a direct correlation between PSV and SCC (r = +0.44, P = 0.003). The median relative color was significantly higher in patients with a higher color score (P = 0.0006). No statistically significant correlations were found between 3D color power angiography parameters and the clinicopathological characteristics or between the 3D vascular parameters and biological factors. CONCLUSIONS: Alterations of 3D ultrasound-derived vascular indices were found in patients with cervical cancer compared with those with a normal cervix. Moreover, some vascular indices proved to be associated with tumor size. The assessment of a possible clinical role of 2D and 3D ultrasound-derived vascular indices in cervical cancer deserves further investigation.

Tamoxifen modulates the expression of Ki67, apoptosis, and microvessel density in cervical cancer.

PURPOSE: The aim of the study was to investigate if a short-term administration of high-dose Tamoxifen (Tam) could affect the expression of biologically relevant biochemical parameters in cervical cancer tissue. EXPERIMENTAL DESIGN: The study was conducted in 24 patients with histologically confirmed cervical tumors. Biopsies were obtained by colposcopy on day 0 in all patients, who then received either 80 mg/die or 160 mg/die for 5 consecutive days until the second biopsy was obtained. Immunohistochemistry was performed with antiestrogen receptor (ER), anti-Ki67, anticaspase cleavage product of keratin 18 (M30), and anti-CD31 monoclonal antibodies. RESULTS: Eleven (45.8%) of 24 cervical tumors were ER positive. The percentage of Ki67-positive tumor cells in pre-Tam biopsies was significantly higher than the percentage in the corresponding posttreatment biopsies (z = 4.29, P = 0.0001). No difference in the pretreatment percentage of Ki67-positive cells according to ER status was found. The percentage of M30 positivity was higher in post-Tam than in pre-Tam biopsies. Microvessel density values in pre-Tam biopsies were significantly higher than corresponding values in posttreatment tissues (z = -3.72, P = 0.0002). The reduction in the percentage of Ki67-positive tumors was significantly (z = 3.58, P = 0.0003) higher in ER-positive than in ER-negative tumors, whereas no difference in Tam-induced reduction of microvessel density values according to ER status (z = -0.18, P = 0.85) was found. Tam treatment did not induce any change of M30 positivity in ER-positive tumors, whereas in ER-negative tumors, it produced a significant (P = 0.015) increase in the percentage of M30-positive cells in post-Tam versus pre-Tam biopsies. CONCLUSIONS: A short-term treatment with Tam at doses 4-8-fold higher than those in conventional schemes is associated with modifications of biological parameters associated with tumor cell proliferation, apoptosis, and neoangiogenesis in cervical cancer.

Chemoprevention of DMBA-induced mammary cancer in rats by dietary soy.

This study was designed to assess the potential chemopreventive effect of the administration of a standardized soy extract, SOYSELECT, on 7,12-Dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. Three groups, 24 females each, were used. Animals were fed either a phytoestrogen-free diet alone (control) or the same diet supplemented with 0.35% or 0.7% of soy extract. Treatment started at weaning and continued to the end of the study (24 weeks after DMBA administration). At day 50 of age all animals received via oral gavage 80 mg/kg DMBA. Only tumors subsequently classified as adenocarcinomas were considered for data evaluation. In rats on the soy diet, mammary tumors took a longer period of time to develop as compared to control rats. However, at the end of the study, no relevant difference in tumor incidence and multiplicity was observed among the groups. The most significant changes were seen between control and soy-treated groups when tumor dimension and results from histopathologic examination were considered. The latter, in fact, showed a dose-dependent reduction in the percentage of poorly differentiated tumors in treated animals. This change was statistically significant in animals receiving 0.7% soy. In addition, assessment of estrogen and progesterone receptor (ERalpha, PR) levels, revealed a significant reduction in the percentage of ERalpha and PR positive tumors in animals receiving 0.7% dietary soy, when compared to controls. Interestingly, genistein and daidzein plasma levels determined at the end of the study were within the range of those detected in people consuming large amounts of soyfoods.

Expression of bcl-2, bax-xL, and bcl-xS in endometrial and cervical tissues.

We assessed the protein expression levels of bcl-2, bax, bcl-xL, and bcl-xS in a group of 51 endometrial cancers and 8 normal samples as well as in 59 cervical neoplasms and in 15 normal cervical tissues. Neoplastic endometria (median, 1.30 absorbance units [AU]; range, 0.13-7.26 AU) had slightly higher bcl-2 levels than did normal tissue (median, 0.83 AU; range, 0.29-1.90 AU; P < .068), whereas bcl-2 was lower in neoplastic (median, 3.59 AU; range, 0.13-19.86 AU) than in normal cervical samples (median, 8.45 AU; range, 2.09-15.04 AU; P < .010). Bcl-xL levels were higher in endometrial carcinoma (median, 1.23 AU; range, 0.03(4.29 AU) than in normal tissues (median, 0.56 AU; range, 0.46-1.48 AU; P < .048), whereas no significant difference was observed in cervical tissues. Bax levels did not show any variation in either system. The protein bcl-xS was marginally detectable in only a few samples. In endometrial carcinoma, bcl-2 and bcl-xL levels were correlated inversely (r = -0.27; P < .054), whereas in cervical cancer, they were correlated directly (r = +0.40; P < .002). The different expression patterns of bcl-2 family members in endometrial and cervical tissues confirm the hypothesis of a strictly tissue-specific regulation of these proteins.

Growth inhibitory effects and radiosensitization induced by fatty aromatic acids on human cervical cancer cells.

Evidences have been reported that phenylacetic (PA) and phenylbutyric (PB) fatty aromatic acids can exert tumor growth inhibition in vitro and in vivo. Moreover, clinical trials also showed some activity for these drugs to modulate the expression of genes implicated in tumor growth, metastasis, immunogenicity, and to potentiate the efficacy of cytotoxic agents. The aim of the study was to examine the effects of PA and PB on the growth as well as sensitization to cisplatin and radiation in human cervical cancer cells. The effects of PA and PB on the proliferative activity and apoptosis induction in cervical tumor tissue was investigated. Both PA and PB exhibited a time- and dose-dependent antiproliferative activity in SW756 and ME180 cell lines: after 72-h treatment, the IC50 (concentration able to inhibit 50% of cell growth) of PB was 1.9 +/- 0.2 mM and 1.5 +/- 0.2 mM in SW756 and ME180 cells, respectively, while the IC50 of PA was 13.0 +/- 1.7 mM and 10.0 +/- 1.2 mM in SW756 and ME180 cells, respectively. In tumor tissue biopsies obtained from patients affected by squamous cervical cancer, both drugs resulted in a marked reduction of the percentage of bromodeoxyuridine-labeled cells compared with untreated samples [19.0 +/- 1.63% in untreated tissues with respect to 1.30 +/- 0.54% and 4.20 +/- 2.50% of stained cells after treatment with PA (30 mM) (P < 0.0001) and PB (5 mM) (P < 0.0001), respectively]. Moreover, analysis of the staining with M30 monoclonal antibody revealed that PA (30 mM) and PB (5 mM) were able to produce a marked increase in the number of stained apoptotic nuclei with respect to untreated samples. Finally, PB and PA were shown to enhance the sensitivity of SW756 to radiation and to exert an additive effect when combined with cisplatin. A significant reduction of the processed form of p21ras and rhoB proteins in the membrane fraction of cells exposed to PA and PB was observed. When farnesol, which is able to circumvent the enzymatic step inhibited by PA and PB, was added to the medium only a partial reversal of the growth inhibition and potentiation of sensitivity to radiation induced by PA and PB were found. In conclusion, the growth inhibitory properties of fatty aromatic acids suggest that these molecules could represent the prototype of a new class of compounds with some therapeutic potential in cervical cancer.