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Mechanisms of intrinsic resistance of serous ovarian cancers to standard treatment with carboplatin and paclitaxel are poorly understood. Seventeen primary serous ovarian cancers classified as responders or nonresponders to standard treatment were screened with DigiWest protein array analysis for 279 analytes. Histone methyl transferase EZH2, an interaction partner of ataxia telangiectasia mutated (ATM), was found as one of the most significantly represented proteins in responsive tumors. Survival analysis of 616 patients confirmed a better outcome in patients with high EZH2 expression, but a worse outcome in patients with low EZH2 and high-ATM-expressing tumors compared with patients with low EZH2 and low-ATM-expressing tumors. A proximity ligation assay further confirmed an association between ATM and EZH2 in tumors of patients with an increased disease-free survival. Knockdown of EZH2 resulted in treatment-resistant cells, but suppression of both EZH2 and ATM, or ATM alone, had no effect. DigiWest protein analysis of EZH2-knockdown cells revealed a decrease in proteins involved in mitotic processes and checkpoint regulation, suggesting that deregulated ATM may induce treatment resistance. IMPLICATIONS: Ovarian cancer is a malignancy with high mortality rates, with to date, no successful molecular characterization strategies. Our study uncovers in a comprehensive approach the involvement of checkpoint regulation via ATM and EZH2, potentially providing a new therapeutic perspective for further investigations., (©2019 American Association for Cancer Research.)

Background/aim: Borderline ovarian tumors (BOT) are malignant epithelial ovarian tumors with a very low incidence, therefore lacking sufficient clinical experience in diagnostics and treatment. This study characterized the histology, clinical features, diagnostics and therapy of BOT including patients treated at the Department of Gynecology and Obstetrics of the University Medicine Greifswald., Materials and Methods: In this retrospective, single-center study, patients with BOT treated between 1990 and 2010 were analyzed according to their histological and clinical reports., Results: A total of 54 patients were enrolled. The median age was 54.6 (range=23-83) years. Distribution of histological subtypes was: serous in 31 patients (57.4 %) and mucinous in 23 patients (42.6%). All patients underwent surgery. Eight patients (14.8%) were treated according to actual therapy recommendations during the initial surgery. Eight patients (14.8%) received adjuvant chemotherapy contrary to treatment recommendations. In the case of 36 patients (66.7%), a frozen section was taken intraoperatively, which matched the definitive histological result in 88.9%. During average follow-up of 70.3 months (range=0-231 months), two patients (3.7%) developed tumor recurrence after 9 and 29 months, respectively, two patients (3.7%) died of causes other than BOT., Conclusion: Our study critically demonstrated that until a few years ago, BOTs were not usually treated according to international therapy recommendations. Nevertheless, the rate of tumor recurrence was very low., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Immunotherapies, particularly checkpoint inhibitors, have set off a revolution in cancer therapy by releasing the power of the immune system. However, only little is known about the antigens that are essentially presented on cancer cells, capable of exposing them to immune cells. Large-scale HLA ligandome analysis has enabled us to exhaustively characterize the immunopeptidomic landscape of epithelial ovarian cancers (EOCs). Additional comparative profiling with the immunopeptidome of a variety of benign sources has unveiled a multitude of ovarian cancer antigens (MUC16, MSLN, LGALS1, IDO1, KLK10) to be presented by HLA class I and class II molecules exclusively on ovarian cancer cells. Most strikingly, ligands derived from mucin 16 and mesothelin, a molecular axis of prognostic importance in EOC, are prominent in a majority of patients. Differential gene-expression analysis has allowed us to confirm the relevance of these targets for EOC and further provided important insights into the relationship between gene transcript levels and HLA ligand presentation., Competing Interests: Conflict of interest statement: H.-G.R. is a shareholder of Immatics Biotechnologies, Tübingen, and CureVac GmbH, Tübingen. H.S. and D.J.K. are employees of Immatics Biotechnologies GmbH. The authors declare that Immatics did not provide neither financial nor scientific support in any direct relation to this manuscript or the underlying studies, and was not involved in data collection, analysis, or decision to publish., (Copyright © 2017 the Author(s). Published by PNAS.)

Many women with ovarian endometrioid carcinoma present with concurrent endometrial carcinoma. Organ-confined and low-grade synchronous endometrial and ovarian tumors (SEOs) clinically behave as independent primary tumors rather than a single advanced-stage carcinoma. We used 18 SEOs to investigate the ancestral relationship between the endometrial and ovarian components. Based on both targeted and exome sequencing, 17 of 18 patient cases of simultaneous cancer of the endometrium and ovary from our series showed evidence of a clonal relationship, ie, primary tumor and metastasis. Eleven patient cases fulfilled clinicopathological criteria that would lead to classification as independent endometrial and ovarian primary carcinomas, including being of FIGO stage T1a/1A, with organ-restricted growth and without surface involvement; 10 of 11 of these cases showed evidence of clonality. Our observations suggest that the disseminating cells amongst SEOs are restricted to physically accessible and microenvironment-compatible sites yet remain indolent, without the capacity for further dissemination., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To delineate the relevance of pelvic and para-aortic node involvement in optimally cytoreduced (residual tumour <1 cm) stage IIIC ovarian cancer patients., Methods: Ninety-five consecutive optimally cytoreduced (R ≤ 1 cm) patients with primary stage IIIc ovarian cancer underwent stage-related surgery and got adjuvant platinum-based chemotherapy. Median follow-up: 53.5 months. All patients got systematic lymphadenectomy. On average, 24.7 pelvic and para-aortic lymph nodes were removed per patient (range 1-60 nodes). Patients were stratified into three groups to evaluate node involvement (ratio: affected to resected nodes): (1) (=0); (2) (>0-≤ 0.5) >0 and ≤ 50 % of affected nodes; (3) (>0.5-≤ 1) >50 % of affected nodes. Clinical parameters were retrospectively evaluated. Kaplan-Meier survival curve was used to evaluate the prognostic value., Results: Most often serous histology, histologic grade 3 and a node ratio >0-≤ 0.5 (61.1 %) were detected. Complete cytoreduction (R = 0 mm) has significant best prognostic impact compared to R > 0 mm-1 cm (OS: p = 0.047, PFS: p = 0.00). Node involvement was associated with serous histology and grade 3. Increasing node ratio leads to significant decreased OS (p = 0.019) and significant best OS was associated with node ratio >0-≤0.5., Conclusions: The goal is optimal cytoreduction in advanced ovarian cancer. More extensive lymphadenectomy seems to play an important role in providing an accurate staging, and the node ratio might give prognostic information. Current prospective studies like the LION study (AGO-Ovar) had to investigate if these data have therapeutic implications and may be considered in future staging.

Aim: In locally advanced ovarian cancer with bowel involvement appropriate surgical treatment is still controversial. Objective was to delineate factors to select those most likely to benefit from radical surgery in patients with locally advanced ovarian cancer., Methods: Therefore, we retrospectively evaluated 207 consecutive patients with primary stage IIB-IV ovarian cancer who underwent primary surgery between 2000 and 2007. Every patient received stage-related surgery and adjuvant platinum-based chemotherapy. Median follow-up was 53.5 months. Data collected included stage, histology, extent of cytoreduction and type of bowel resection. Univariate survival analyses were performed to investigate variables associated with outcome., Results: Optimal cytoreduction (OCR) (R ≤ 1 cm) was achieved in 76.8%. Most patients presented histologic grade 2/3 (96.6%), serous ovarian cancers (84.1%) and lymph node involvement (52.2%). Complete cytoreduction (R = 0 mm) has significant best prognostic impact in FIGO IIB-IV (p = .026). Regarding bowel involvement, bowel resection was performed in 82 patients (39.6%). In this subgroup of patients complete cytoreduction led to significant better overall survival than R > 0 mm-1 cm, even in FIGO IIIC-IV patients (p = .027); this fact is independent of bowel resection. Noticeably, for survival bowel resection achieving residual tumor mass below 1 cm was also one main prognostic factor and even recurrence rate was associated with residual tumor mass., Conclusion: Our findings suggest that the major prognostic factor in patients with advanced ovarian cancer needing colorectal resection is completeness of cytoreduction. Therefore, in advanced ovarian cancer patients, multivisceral surgery is indicated to achieve OCR (R ≤ 1 cm) with or without bowel resection with best prognostic impact.

Aim: To delineate the relevance of pelvic and para-aortic node involvement in early-stage ovarian cancer., Patients and Methods: Data on 75 consecutive patients with primary stage T1 and 2 ovarian cancer treated at the Department of Gynecology, University Tuebingen, Germany were retrospectively analyzed. All patients underwent stage-related surgery with pelvic and para-aortic lymphadenectomy and adjuvant platinum-based chemotherapy (except pT1aG1). Median follow up was 53.5 months. Clinico-pathological parameters and the distribution pattern of node metastases were evaluated. Statistical analyses were performed using PASW., Results: Lymph node metastases were detectable in T1 and T2 in 6 (8%) of 75 patients. Three patients (4%) had lymph node metastases in the pelvic nodes only, 2 patients (2.7%) in the para-aortic nodes only; 1 patient (1.3%) both in the pelvic and para-aortic nodes. On multivariate analysis, histological grade 1/ 2 and 3 tumors, serous and endometrioid histology were independent predictors for node metastases, respectively. The risk of relapse was significantly higher with detection of node metastases (p=0.004)., Conclusion: A systematic lymphadenectomy in early-stage ovarian cancer leads to an upstaging in a few patients after detection of node metastases even in pelvic or para-aortic nodes, especially in patients with grade 3 tumours and serous cancers. Pelvic and para-aortic lymphadenectomy may detect node involvement in early-stage ovarian cancer and might be helpful in correct staging., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: This investigation aims to assess morbidity, mortality and postoperative outcomes of cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) in recurrent epithelial ovarian cancer (REOC) with peritoneal metastases (PM)., Methods: Consecutive patients with radiographic evidence of REOC with PM were scheduled for CRS and HIPEC at the Comprehensive Cancer Center, University Hospital Tübingen, Germany. Clinical data were retrospectively analyzed., Results: In total, 90 patients were analyzed. Complete cytoreduction and HIPEC could be performed in 69 % of patients. When categorizing patients with respect to the completeness of cytoreduction (CC-0/1 vs CC-2/3), there was no difference considering baseline demographic characteristics. Cumulative morbidity was 42 %. Morbidity rates did not statistically differ between CC-0/1 patients with HIPEC and CC-2/3 patients without HIPEC. No surgery-related and 90-day postoperative mortality was observed. In CC-0/1 patients, median overall survival was 35 months as opposed to 14 months in CC-2/3 patients. There was no difference in survival with respect to the peritoneal carcinomatosis index (PCI) as long as complete cytoreduction could be achieved., Conclusions: CRS and HIPEC can be performed with acceptable morbidity and low mortality in specialized centres. Our data do not suggest that HIPEC necessarily increases the risk of postoperative adverse events.

The SRY-related HMG-box family of transcription factors member SOX2 regulates stemness and pluripotency in embryonic stem cells and plays important roles during early embryogenesis. More recently, SOX2 expression was documented in several tumor types including ovarian carcinoma, suggesting an involvement of SOX2 in regulation of cancer stem cells (CSC). Intriguingly, however, studies exploring the predictive value of SOX2 protein expression with respect to histopathologic and clinical parameters report contradictory results in individual tumors, indicating that SOX2 may play tumor-specific roles. In this report, we analyze the functional relevance of SOX2 expression in human ovarian carcinoma. We report that in human serous ovarian carcinoma (SOC) cells, SOX2 expression increases the expression of CSC markers, the potential to form tumor spheres, and the in vivo tumor-initiating capacity, while leaving cellular proliferation unaltered. Moreover, SOX2-expressing cells display enhanced apoptosis resistance in response to conventional chemotherapies and TRAIL. Hence, our data show that SOX2 associates with stem cell state in ovarian carcinoma and induction of SOX2 imposes CSC properties on SOC cells. We propose the existence of SOX2-expressing ovarian CSCs as a mechanism of tumor aggressiveness and therapy resistance in human SOC.

Objective: To preclinical assess the feasibility of combining oncolytic measles vaccine virus (MeV) with suicide gene therapy for ovarian cancer treatment., Methods: We genetically engineered a recombinant MeV armed with a yeast-derived bifunctional suicide gene that encodes for cytosine deaminase and uracil phosphoribosyltransferase (MeV-SCD). From this suicide gene, a chimeric protein is produced that converts the non-toxic prodrug 5-fluorocytosine (5-FC) into highly cytotoxic 5-fluorouracil (5-FU) and directly into 5-fluorouridine monophosphate (5-FUMP) thereby bypassing an important mechanism of chemoresistance to 5-FU., Results: MeV-SCD was demonstrated to infect, replicate in and effectively lyse not only human ovarian cancer cell lines, but also primary tumor cells (albeit at lower efficiencies) that were derived from malignant ascites of ovarian cancer patients. Addition of the prodrug 5-FC significantly enhanced cell killing. Importantly, precision-cut tumor slices of human ovarian cancer patient specimens were efficiently infected with MeV-SCD. The prodrug-converting enzyme SCD was expressed by all infected tumor slices, thereby ensuring provision of the suicide gene arming function in patient-derived materials., Conclusions: With respect to safety and therapeutic impact, arming of oncolytic measles vaccine virus warrants further clinical investigation for ovarian cancer treatment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The transcription factor SOX2 has been extensively studied for its role in embryonic stem cell self-renewal and pluripotency. More recent data suggest oncogenic functions of SOX2 and demonstrate expression in several carcinoma types, predominantly of squamous cell origin. The gene SOX2 is located at chromosome 3q26, a region that is frequently amplified in ovarian high-grade serous carcinoma. In this study, we correlate SOX2 protein expression and gene-amplification status in ovarian carcinomas with histopathologic criteria and disease outcome. A total of 215 cases of ovarian carcinomas (154 serous, 39 endometrioid, 11 clear cell, 5 mucinous, and 6 transitional cell carcinomas) were analyzed by immunohistochemistry in a tissue microarray for nuclear expression of SOX2. A total of 60.5% of all carcinomas showed SOX2 expression with no significant difference between the major histologic types. Interestingly, SOX2 expression was predominantly a feature of high-grade tumors (G1: 36.4%, G2: 55.6%, G3: 64.0%, P=0.040). In 21.2% of these cases, fluorescence in situ hybridization analysis detected low-level SOX2 gene amplification which was not significantly associated with SOX2 protein expression. However, survival analysis of Stage II to IV high-grade serous carcinomas revealed a favorable effect of SOX2 expression (median disease-free survival 27 vs. 21 mo, P=0.041; median overall survival 39 vs. 25 mo, P=0.062). Further studies are needed to explore whether expression of SOX2 has a specific role in prognosis and therapy response.

Objective: Detection of disseminated tumor cells (DTCs) in the bone marrow (BM) of patients with breast cancer is associated with poor outcomes. Recent studies demonstrated that DTCs may serve as a prognostic factor in ovarian cancer. The aim of this 3-center study was to evaluate the impact of BM status on survival in a large cohort of patients with ovarian cancer., Materials and Methods: Four hundred ninety-five patients with primary ovarian cancer were included in this 3-center prospective study. Bone marrow aspirates were collected intraoperatively from the iliac crest. Disseminated tumor cells were identified by antibody staining and by cytomorphology. Clinical outcome was correlated with the presence of DTCs., Results: Disseminated tumor cells were detected in 27% of all BM aspirates. The number of cytokeratin-positive cells ranged from 1 to 42 per 2 × 10⁶ mononuclear cells. Disseminated tumor cell status did correlate with histologic subtype but not with any of the other established clinicopathologic factors. The overall survival was significantly shorter among DTC-positive patients compared to DTC-negative patients (51 months; 95% confidence interval, 37-65 months vs 33 months; 95% confidence interval, 23-43 months; P = 0.023). In the multivariate analysis, BM status, International Federation of Gynecology and Obstetrics stage, nodal status, resection status, and age were independent predictors of reduced overall survival, whereas only BM status, International Federation of Gynecology and Obstetrics stage, and resection status independently predicted progression-free survival., Conclusions: Tumor cell dissemination into the BM is a common phenomenon in ovarian cancer. Disseminated tumor cell detection has the potential to become an important biomarker for prognostication and disease monitoring in patients with ovarian cancer.

Purpose: Prognostic impact of nodal status or lymphadenectomy in advanced ovarian cancer is still unclear. Known best prognostic impact in advanced ovarian cancer has the residual tumor mass. The aim of this retrospective study is to examine the importance of nodal status in correlation with residual tumor mass., Methods: One hundred and fifty-seven consecutive patients with primary stage III ovarian cancer underwent surgery between 01/2000 and 06/2007 at the Department of gynecology and obstetrics, University Hospital, Tübingen, Germany. All patients got stage-related surgery and platin-based chemotherapy. Median follow-up time was 53.5 months, and all patients were included in the study., Results: Resection status and nodal status are significant prognostic factors in our study (P < 0.001). In FIGO III, patients without residual tumor (R0) had significant best OS and PFS independent to node status (N0/N+; P = 0.002) compared to patients with residual tumor. In contrast, node status had significant positive impact on PFS in patients without residual tumor and node negativity. With the increase in residual tumor, the influence of lymphnode metastases on prognosis is decreasing., Conclusion: Main intention of primary surgery is R0 resection with best prognosis in advanced stages. A systematic lymphadenectomy in cases with R0 resection or residual tumor <1 cm seems to be reasonable with positive impact on prognosis. Node status has impact on prognosis in patients with negative node after R0 resection with best PFS in FIGO III. Further prospective studies had to show whether systematic lymphadenectomy in suboptimally tumor-reduced patients can improve prognosis.

Purpose: Peritoneal recurrence of ovarian cancer is frequent after primary surgery and chemotherapy and has poor long-term survival. De novo cytoreductive surgery is crucial with the potential to improve prognosis, especially when combined with hyperthermic intraperitoneal chemotherapy (HIPEC)., Methods: The sampled data of 40 consecutive patients were retrospectively analyzed. Thirty-one patients were treated with cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy., Results: No patient was lost in the perioperative period, and the combined procedure was performed with acceptable morbidity. Colon-preserving cytoreductive surgery was associated with reduced morbidity., Conclusions: Patients suffering from peritoneal recurrence of ovarian cancer should be considered for radical reoperation with HIPEC in a center with expertise in multimodal therapeutic options. Organ-preserving cytoreductive surgery allows complete cytoreduction with the goal of decreasing morbidity.

Introduction: The presence of disseminated tumor cells (DTC) in the bone marrow (BM) of breast cancer patients is associated with poor prognosis. Several studies demonstrated that tumor cell dissemination may occur in gynecologic cancer and affect clinical outcome. The aim of our study was to evaluate the incidence of DTC and to assess their prognostic significance in patients with gynecologic malignancies., Methods: Bone marrow aspirates from 377 patients with primary ovarian (112), endometrial (141), cervical (102), and vulvar cancer (22) undergoing surgery at the Department of Gynecology and Obstetrics, University Hospital, Tuebingen, Germany between November 2001 and November 2007, were included into the study. Disseminated tumor cells were identified by immunocytochemistry using the pancytokeratin antibody A45B/B3 and by cytomorphology., Results: Disseminated tumor cells were detected in 19% of BM aspirates from patients with gynecological malignancies. Incidences of DTC in ovarian, endometrial, cervical, and vulvar cancer were 25%, 16%, 19%, and 5%, respectively. For patients with ovarian and endometrial cancer, no correlation with established clinicopathological factors was observed. In case of cervical cancer, BM positivity was correlated with International Federation of Gynecology and Obstetrics stage, tumor size, and nodal involvement. Bone marrow positivity of ovarian cancer patients was correlated with significantly shorter disease-free survival (P = 0.035). For other tumor entities, no association between BM status and clinical outcome could be observed., Conclusions: We conclude that up to 25% of patients with loco-regionally restricted gynecologic malignancies present with DTC at the time of diagnosis. For ovarian cancer patients, BM status affected clinical outcome.

Background: Most patients with primary epithelial ovarian cancer (PEOC) treated with carboplatin/paclitaxel will relapse between one to two years. Our purpose was to define the optimal calculation method for the adenosine triphosphate-tumor chemosensitivity assay (ATP-TCA) applied to PEOC treated with platinum-containing chemotherapy and to analyze its predictive relevance., Materials and Methods: ATP-TCA results from 80 PEOC specimens were analyzed applying three different methods: 50% inhibition concentration, sensitivity index (IndexSUM), and area under curve by testing multiple cut-off levels. Correlation between in vitro results and clinical outcome was performed for 61 (76%) patients by univariative and multivariative analysis. Tumor recurrence 6 months after chemotherapy was classified as platinum-resistance., Results: The IndexSUM set at > 250 had the highest test sensitivity, specificity, positive and negative predictive value of 90%, 43%, 62% and 81%, respectively. Patients whose tumors were shown to be resistant by ATP-TCA had a higher risk for recurrence (RR) compared to those who tested as sensitive (p < 0.003, RR = 3.3, 95% CI = 1.2-9.4). This result was confirmed after adjustment for FIGO stage by logistic regression (p < 0.004, Odds ratio = 8.3, 95% CI = 1.9-35.5). In multivariate analysis ATP-TCA and the FIGO-stage were independent predictive factors of early recurrence., Conclusion: ATP-TCA results in combination with the use of IndexSUM > 250 are best able to predict platinum resistance.

Purpose of Investigation: The role of progestogens in the genesis of ovarian cancer remains unclear although a rather protective behaviour has been suggested. Epidemiological studies indicate a possible increase in the risk for combined estrogen/progestin as compared to estrogen alone. It is ambigious whether a difference exists within the various progestogens. Apart from sex steroids, growth factors play a crucial role in the genesis of ovarian cancer, although as yet little investigated. In the present study we have explored the effect of progesterone (P), medroxyprogesterone acetate (MPA) and norethisterone (NET) on the proliferation of human ovarian cancer cells alone and in the presence of growth factors., Methods: For the experiments human ovarian cancer cells (OVCAR-3) were used. The progestogens were tested at the concentrations of 0.01 to 10 microM. The growth factor mixture consisted of EGF, FGF and IGF-I, each at a concentration of 10 pM. The incubation time was three or seven days. Proliferation rate was measured by an ATP-assay., Results: After three days' incubation the growth factors induced an increase in the proliferation rate of about 50%. Progesterone alone did not show any significant change as compared to the control values, whereas NET and MPA elicited a significant increase at 1 and 10 microM and at 1 microM, respectively. In the presence of growth factors none of the progestogens was able to inhibit the proliferative stimulation. After seven days' incubation the growth factors still showed an increase of about 50%. MPA alone had an inhibitory effect at 10 microM, for NET and P no effects were observed. Again in the presence of growth factors no progestogen was able to show an inhibitory effect., Conclusion: Our results indicate that progestogens do not have a protective role on the growth of pre-existing ovarian cancer cells, at least in the presence of growth factors. Further investigations are worthwile to evaluate possible differences between the effect of the various progestogens.

Purpose of Investigation: The etiology of ovarian cancer appears to be associated with a long-term influence of estrogens. However, evidence is accumulating that certain estradiol metabolites may play a decisive role in the carcinogenesis of estrogen-dependent diseases. In the present study we examined the effect of estradiol metabolites on the proliferation and apoptosis of human ovarian cancer cells in comparison to the effect of their parent substance., Methods: The ovarian cancer cell line OVCAR-3 was used for the experiments. 17Beta-estradiol (E2), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) and 16alpha-hydroxyestrone (16-OHE1) were incubated for seven days in the concentration range of 0.01 nM to 10 nM. Proliferation and apoptosis were measured by commercially available assay kits., Results: E2 enhanced proliferation rate and concomitantly reduced apoptotic rate of the ovarian cancer cells at physiological concentrations. 2-OHE2 had no significant effect, whereas 4-OHE2 elicited similar effects on proliferation and apoptotic rate as E2. The greatest proliferative and antiapoptotic effect was observed for 16-OHE1, the values being significantly different to the effects of E2., Conclusion: The pattern of endogenous estradiol metabolism may play a role in defining ovarian cancer risk. This may be of importance in certain predisposed women who are treated with hormone therapy in postmenopause.

Purpose: The absence of tumor-associated antigens (TAA) which might elicit an immune response is one reason for the disappointing results of therapeutical vaccines in cancer patients. Moreover, impaired expression of MHC class-I and components involved in antigen processing, such as TAP-1, -2, LMP-2, -7, and MECL-1, may lead to tumor escape from immune recognition. Expression profiling of TAA is one approach towards the design of well-defined and individualized anti-tumor vaccines., Methods: Quantitative polymerase chain reaction (qRT-PCR) is the method of choice to characterize immunologically relevant properties of individual tumors. However, the application of qRT-PCR as a surrogate parameter for the expression of TAAs depends upon the assumption that the level of an mRNA species correlates with the cellular level of the protein it encodes. Therefore, we additionally analyzed TAA expression by immunofluorescence and T cell recognition., Results: In the present study we were unable to confirm that impaired TAP-1 or -2 (transporter associated with antigen processing) expression characterized at the mRNA level is an appropriate surrogate parameter for down-regulated MHC class-I expression in breast cancer. In addition, we analyzed the expression pattern of TAAs in breast and ovarian cancer cell lines. Besides the well-known over-expression of HER-2/neu, CEA, and MUC-1, multiple antigens of the MAGE-family were frequently co-expressed. We investigated whether detection of TAAs by qRT-PCR correlates with monoclonal antibody staining, and which method could predict T cell recognition. We demonstrated a correlation between tumor cell lysis by HLA-A*0201-restricted, MUC-1-specific CTL and threshold levels of MUC-1-specific mRNA., Conclusion: MUC-1 is an example that TAA profiling by RT-PCR and flow cytometry can fail to correlate with each other and are of limited value in the prediction of T cell recognition.

Anti-idiotypic (Id) monoclonal antibodies can serve as surrogate for tumor-associated antigens in vaccination strategies. The murine anti-Id monoclonal antibody ACA125 that mimics the CA125 carbohydrate antigen expressed on ovarian cancer cells induces an anti-anti-Id antibody (Ab3) response that is associated with prolonged survival of ovarian cancer patients. To increase the Ab3 antibody response, we evaluated two strategies in a mouse model: (a) coinjection of human interleukin (IL)-6 together with the fusion protein chACA125, which consists of the anti-Id ACA125 single-chain Fv antibody joined to the human IgG1 CH2/CH3 domain; and (b) injection of the fusion protein chACA125-IL-6, which consists of the ACA125 single-chain Fv fused to human IL-6 via the IgG1 CH2/CH3 domain. Vaccination of mice with the chACA125-IL-6 fusion protein resulted in higher titers of anti-CA125 (Ab3) antibodies compared with application of the chACA125 antibody with or without systemic coadministration of IL-6. Application of the chACA125-IL-6 fusion protein did not elicit detectable antihuman IL-6 antibody titers, whereas coinjection of human IL-6 did. Taken together, these data suggest that the chACA125-IL-6 fusion protein directly stimulates ACA125-specific B cells via the IL-6 domain, whereas coinjection of IL-6 leads to an overall immune stimulation. Antigen-IL-6 fusion proteins will improve vaccination regimens and anticancer immunotherapeutic strategies by increasing the antigen-specific humoral immune response.

This phase II study evaluated the activity and toxicity of gemcitabine plus cisplatin as first-line treatment of patients with advanced ovarian cancer. Chemonaive patients >/=60-year-old with FIGO stage IIIC or IV epithelial ovarian carcinoma were enrolled. Patients received cisplatin 75 mg /m2 on day 1 and gemcitabine 1250 mg /m2 on day 1 (before cisplatin) and day 8 of a 21-day cycle. Of 44 female patients (median age, 70 years), 72.7% had stage IIIC disease and 67.4% had a Karnofsky performance status >/=80. Of the 37 response-evaluable patients (35 with measurable lesion[s] >/=2 cm), there were seven (18.9%) pathologic complete responses, two (5.4%) pathologic partial responses, two (5.4%) clinical complete responses, and 12 (32.4%) clinical partial responses, for an overall response rate of 62.2% (95% CI, 44.8%-77.5%), and a pathologic response rate of 24.3% (95% CI, 11.8%-41.2%). Median survival was 27.7 months (95% CI, 14.3-40.8 months). Grade 3/4 neutropenia and thrombocytopenia occurred in 59.5% and 30.2% of patients, respectively, with neutropenic fever in one patient. Grade 3 nausea /vomiting and alopecia occurred in 25.6% and 9.5% of patients, respectively. We conclude that gemcitabine plus cisplatin is active and feasible as first-line treatment of advanced epithelial ovarian cancer in patients >/=60 years. Further clinical trials adding gemcitabine to current standard, first-line treatment seem warranted in younger as well as older patients.

Radical debulking surgery allows for optimal cytoreduction in less than 50% of patients with advanced ovarian cancer. In spite of highly efficient chemotherapeutic regimens, the prognosis of patients with residual tumor masses larger than 1 cm in diameter following staging laparotomy is very poor. This observation led to the initiation of numerous trials evaluating the feasibility and efficiency of the use of primary chemotherapy followed by interval laparotomy in women with advanced ovarian cancer. The available data is presented and discussed in this review.

The aim of the present study was to assess whether the induction of specific immune responses by vaccination with the murine monoclonal anti-idiotypic antibody ACA125, which imitates the tumor-associated antigen CA125, has a positive influence on the survival of patients with recurrent ovarian carcinoma. Forty-two patients with platinum-pretreated recurrences were included in a clinical Phase I/II trial of consolidation in third-line therapy. Patients initially received four immunizations with 2 mg of alum-precipitated anti-idiotype ACA125 every 2 weeks and then monthly applications. No serious allergic reactions could be detected within a maximal control period of 56 months. Hyperimmune sera of 27 of 42 patients (64.2%) showed increased concentrations of human antimouse antibodies. Specific anti-anti-idiotypic antibodies as a marker for induced immunity were detected in 28 of 42 patients (66.7%). The survival of the whole ACA125-treated collective of patients after a mean of 12.6 antibody applications was 14.9 +/- 12.9 months. The survival of patients with a positive immune response was 19.9 +/- 13.1 months in contrast with 5.3 +/- 4.3 months in those patients without detectable anti-CA125 immunity (P < 0.0001). According to these results, vaccination with a suitable anti-idiotypic antibody offers an effective way to induce specific immunity against a primarily nonimmunogenic tumor antigen such as CA125 and is associated with a positive impact on the survival of patients with recurrent ovarian cancer with few side effects, which warrants a Phase III trial for ovarian cancer patients after primary therapy.

One possible reason for the poor immunogenicity of tumors is the induction of peripheral tolerance by tumor cells that fail to deliver costimulatory signals. Furthermore, T cells stimulated with wild-type tumor cells often fail to secrete cytokines. The present study has been undertaken to identify cytokines that cooperate with CD80 in T-cell activation in vitro toward human breast and ovarian carcinoma cell lines. Tumor cell-mediated T-lymphocyte activation was analyzed directly in allogeneic mixed lymphocyte/tumor cell cultures as proliferation and effector functions were assessed in cytotoxic T-cell assays. Interleukin-7 (IL-7) amplified the proliferative response toward CD80-transfected breast and ovarian carcinomas and stimulated predominantly CD4+ T lymphocytes. IL-12 represses the proliferative response of naive T cells but cooperates with CD80-mediated activation during secondary stimulations. In long-term T-cell cultures, IL-12 synergizes with CD80 expression to stimulate cytolytic CD8+ T-cell lines, which recognize a breast carcinoma line in a human histocompatibility leukocyte antigen-restricted manner. These studies illustrate that costimulation is necessary for tumor cells to function as alloantigen-presenting cells. Furthermore, when added after the priming of T cells with CD80-transfected tumor cells, IL-12 could be helpful in propagating sufficient T-cell numbers to be used in adoptive transfers during cellular immunotherapy.

Anti-idiotypic antibodies, which imitate a tumor-associated antigen by their variable region, offer an elegant method for the induction of a specific immune response, when used as a surrogate antigen for immunization. We generated anti-idiotypic antibodies imitating 2 different tumor-associated antigens. I. CA125 for ovarian carcinomas and II. 14C5, a tumor-associated cell substrate adhesion molecule on breast cancer cells, whereas the first approach could be introduced in a first clinical trial and the second was evaluated in an immunocompetent animal model. For the induction of an immune response against CA125, 18 patients with advanced ovarian cancer (n = 6) or heavily pretreated recurrences (n = 12) were immunized with the anti-idiotypic antibody MAb ACA125. Patients were treated with 2 mg anti-idiotype antibody every two weeks for 4 injections i.m. and then monthly. 12 of 18 patients demonstrated an anti-anti-idiotypic (Ab3) response, which was to a lower extent also directed against CA125 and 9 of 18 patients developed a CA125 specific cellular immune response by their peripheral blood lymphocytes. Based on this data a follow-up clinical trial in advanced ovarian cancer patients with minimal residual disease in an adjuvant approach after primary therapy was started to evaluate the effect of the immune response on the progression free survival. For immunotherapy of breast cancer, we generated a murine monoclonal anti-idiotypic antibody (MAb ACA14C5), which imitates a cell substrate adhesion molecule on breast cancer cells. The anti-idiotype was introduced in an immunocompetent animal to prove his capability on induction of an immune and tumor response. The results showed a highly significant difference in the tumor growth of the ACA14C5 treated group in contrast to the controls starting the immunization on day 6 after tumor cell application with 10 of 12 animals being cured from their tumor burden. Prophylactic immunization against the invasion antigen of breast cancer by anti-idiotypic antibodies showed protection against increasing tumor burden. However, in the situation of established tumors only minor responses could be detected. Vaccination with anti-idiotypic antibodies comprises an effective method for induction of a specific immune response against non-immunogenic tumor-associated antigens and should be therefore considered in immunological approaches to tumor therapy, where the primary structure and sequence of the antigen, e.g. CA125, is up to now not available.

Women suffering from advanced stage ovarian or mammary carcinoma frequently develop malignant ascites or pleural effusions consisting of tumor cells and tumor-associated lymphocytes (TALs). Locoregional immunotherapy with bispecific antibodies (bsAbs), which retarget T cells to tumor cells and induce their lysis, has been applied as an adjuvant treatment in the late stage of the disease. Until now, most of these therapies use peripheral blood mononuclear cells (PBMCs) as effector cells that have been stimulated and expanded ex vivo and loaded with bsAb before reinjection. Here we investigated whether TALs derived from malignant ascites or pleural effusions can be used as bsAb-guided effector cells without prior in vitro stimulation. For this we established a bsAb, HEA125xOKT3, which recognizes the epithelial antigen Egp34 on carcinoma cells and the CD3 molecule on T cells. BsAb HEA125xOKT3 induced lysis of various Egp34-expressing carcinoma lines by stimulated PBMCs. Optimal cytotoxicity was achieved at a bsAb concentration of 1 microg/ml. In three ovarian and two mammary carcinoma patients, we demonstrated efficient lytic activity of lymphocytes, isolated from malignant ascites or pleural effusion. Without prior stimulation, they lysed autologous tumor cells in the presence of bsAb HEA125xOKT3, indicating that they are already activated. Along this line, a subset of CD4+ and CD8+ unstimulated TALs expressed the early activation marker CD69. They are, however, negative for CD95, and only a small subpopulation of CD4+ TALs expresses CD25. OKT3/interleukin 2 stimulation of TALs increased the expression of activation markers on the CD4+ and CD8+ T-cell compartment. The activation markers CD69, CD25, CD95, and DR molecules are up-regulated on both T-cell types. However, lysis of autologous tumor cells by stimulated TALs is not significantly enhanced compared with unstimulated TALs. Our results may offer a novel and promising concept of adjuvant immunotherapy for ovarian and mammary carcinoma patients. Preactivation and expansion of PBMCs can be circumvented by exploring the cytotoxic capacity of unstimulated TALs in the presence of bsAbs in a locoregional therapeutic approach.

Apoptosis and cell proliferation play important roles in the cellular response to chemotherapy, and may have prognostic value. The percentage of apoptotic cells [apoptotic index (AI)] was evaluated in ovarian carcinomas of 25 patients by the terminal desoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) technique. All patients were surgically treated and received postoperatively chemotherapy consisting of cytoxan and cisplatin or carboplatin. As a marker of proliferation the mRNA expression of histone H3 in tumor tissue was determined. In addition, tumor vascularity was assessed by immunohistochemistry and factor VIII. Patients with high AI had significantly shorter survival times (p=0. 0001) or recurrence-free intervals (p=0.004) than patients with low AI. No significant relationship was found between AI and histone H3 mRNA expression, however, an inverse correlation of AI with microvessel density was detected. Tumors with high AI had significantly lower microvessel count than tumors with low AI (p=0. 002). The obtained data suggest that AI can be predictive of treatment outcome in ovarian cancer.

To establish the requirements for real-time transfer of an ultrasound examination via telecommunication network the following tests were performed: The ultrasound data were transferred from the video out of an ultrasound system to a basis terminal of the German Telekom. Simultaneously, an external video camera filmed the positioning and movements of the ultrasound transducer, and the verbal comments were recorded. These informations were transmitted to Karlsruhe and London, where they were rerouted to the examination room in Heidelberg. Here the informations were received on a Telecom reception unit/terminal and compared directly with the initial signal. The quality was sufficient if the moving ultrasound images and the camera image of the transducer as well as the oral comment were transmitted over 2 parallel ISDN lines. The delay to a real-time transmission of the examination process is only in the range of milliseconds. If only one ISDN line is used, the image quality is unsatisfactory, three parallel lines do not bring significant improvement of image quality. Telemedicine seems a new possibility to bring the knowledge of specialized centers to the practicing gynaecologists thus avoiding unnecessary referrals. Still unanswered, however, are the problem of liability, data protection and costs.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that may also function as an autocrine growth regulator. Thirty-one ovarian carcinomas were investigated for mRNA expression of VEGF and of a proliferation-dependent gene (histone H3) using slot-blot analysis. Tumor vascularity was assessed by immunohistochemistry and factor VIII. All tumors were demonstrated to express VEGF and histone H3, though to various degrees. There was a good correlation between VEGF mRNA values and histone H3 mRNA values (r = 0.71, p < 0.05). No correlation was found between tumor cell proliferation and tumor vascularity. There was no significant difference in relapse-free interval or overall survival between tumors with low and high VEGF expression. The close correlation of VEGF expression with tumor cell proliferation in this study raises the possibility of autocrine stimulation of ovarian carcinoma.

Whereas 65-70% of ovarian malignancies are of the epithelial type, the occurrence rate of stromal tumors is of approx. 7% and that of germ cell tumors of approx. 15%. Stromal tumors are mostly of the granulosa cell type, whereas germ cell tumors occur mainly as dysgerminoma (occurrence 0.9-2%), endodermal sinus tumors, or teratoma. Organ preservation is discussed in relation to the characteristics of these special tumor types. Granulosa cell tumors, representing 70% of the tumors of the gonadal stroma, occur unilaterally in approx. 97% of cases. 10-year-survival in stage I is over 90%. In stages II and III a complete remission after chemotherapy (acc. to the PVB, VAC, or BEP protocol) may be achieved in approx. 60% of cases. Due to these characteristics organ preservation seems feasible. Since dysgerminoma represent the most common malignant germ cell tumor in children, adolescents and pregnant women (up to 17% of all dysgerminoma are diagnosed during pregnancy), the wish for organ preservation is the more understandable. However, bilaterality, occurring in 20% of cases, has to be considered. Especially in large tumors lymphatic metastases also have to be taken into account. In cases of endodermal sinus tumors and teratoma, overall survival, mainly in patients with advanced disease, depends on the response to an aggressive chemotherapy. Preconditions for organ preservation are the patients' urgent childbearing desire, their information concerning the 5-7% risk of recurrence, an adequate oncologic postoperative care and optimally, after delivery, removal of the contralateral ovary and re-staging. The operative procedure requires removal of the corresponding adnexa, wedge dissection of the contralateral ovary, omentectomy, and depending on the histological tumor type a pelvic, possibly paraaortal lymph node dissection. No generally accepted standards are available for organ preserving surgery of stromal tumors, especially of the granulosa cell type. Prognosis is essentially influenced by a possible rupture being present, tumor size, cellular atypia, and the mitotic index. If one takes into consideration the possibility of lymph node metastases, at least a pelvic lymph node dissection should be recommended. In cases of metastases additionally a chemotherapy (VAC protocol) is indicated. Among germ cell tumors, dysgerminoma and non-dysgerminoma are treated differently. Non-dysgerminoma are endodermal sinus tumors, teratoma, embryonal carcinoma and mixed forms. For both groups operative management may aim at tumor reduction and in principle organ preservation. Whereas for dysgerminoma an adjuvant radiation therapy is feasible, in cases of non-dysgerminoma the response to a chemotherapy is the only factor influencing prognosis. Chemotherapy as adjuvant treatment is not indicated for pure dysgerminoma stage Ia, and pure solid teratoma stage Ia Gl. For all other dysgerminoma adjuvant chemotherapy, VAC protocol, and chemotherapy according the the BEP protocol for non-dysgerminoma is recommended. In cases of metastatic spread, in both groups an aggressive chemotherapy (BEP protocol) is most commonly performed.

Ovarian tumors during pregnancy are a rare event. In most cases the tumors are detected accidentially during routine examination, ultrasound or a caesarean section at term. The incidence of malignant ovarian tumors is about 1:10,000 to 1:40,000 pregnancies. Histologic subtypes and prognosis do not differ from tumors not associated with pregnancy, it seems however, that there are more lesions of borderline malignancy and of low grade. Therapy depends mostly on the age of gestation and tumor stage. Conservative surgery is recommended only in stage IA disease. Radical surgery and if necessary adjuvant therapy is recommended during the first trimester. In the third trimester a caesarean section can be followed by radical surgery, provided that there is a close cooperation between gynecologists and pediatricians. In the second trimester this regimen is possible only as an exception which includes a critical maternal risk-benefit assessment.

Endoscopic ovarian surgery is currently spreading tremendously, but also rather uncritically. The technical possibilities both of organ preserving and ablative endoscopic surgery are controversial, so that structuring of the indication for an endoscopic procedure as well as optimization of the endoscopic removal of ovarian tumor or adnexae is of utmost relevance. Therefore, a study was performed at the Department of Obstetrics and Gynaecology of the Heidelberg University with the following aims: Risk evaluation of operating into an ovarian malignancy at endoscopy for "presumably" benign cystic ovarian tumors in n = 100 cases in Heidelberg and literature survey Analysis of problems and complications during clinical application of laparoscopic lap sacs for removal of cystic adnexal tumors or adnexae (n = 50) Experimental examination of the risk of an endoscopic lap sacs to rupture during a procedure The risk of endoscopically operating into an ovarian cancer lies between 0.4 and 3% according to literature data. Despite maximal preoperative selection, mainly by ultrasound examination, in our group of 100 patients, in three women without preoperative signs of malignancy but with discreet intracystic structures in the ultrasound, an endoscopic adnexectomy with complete removal in a lap sac was performed, and though immediate section for microscopic examination was negative, final histology revealed one ovarian cancer la and 2 borderline tumors, same stage. In the time period analyzed, three further patients were referred to our center for secondary, delayed (median 3 months) staging after endoscopic procedures for presumed benign lesions. Clinical application of lap sacs proved the necessity of an intensive training. In 3 patients an intraperitoneal rupture of the sac occurred. Typical problems were volume discrepancies between sac respectively abdominal incision and tissue to be removed (28% of cases). The risk of rupture of the various lap sacs examined differed significantly (p < 0.05). Due to the complex pathological nature of cystic adnexal tumors, a 100% selection for the endoscopic approach is not feasible. Therefore, an endoscopic procedure should only be performed after optimal preoperative diagnosis and, in case of the slightest doubt, only if intraoperative microscopic examination is available, and the possibility to perform an immediate staging laparotomy. Sufficient information of the patient is relevant. Laparoscopic removal of tumor or adnexae should be performed in a lap sac. However, there is no absolute certainty of preventing spillage even with the lap sac, since not all endoscopic sacs available are of a sufficient quality.

Preoperative ultrasound as well as intraoperative laparoscopic diagnosis have both their limits, a fact that might be significant for laparoscopic surgical management. In particular, since in this surgical domain, where almost everything has become technically feasible, the operator must decide what is to the benefit of the patient. A possible solution could be laparoscopic ultrasound, i.e. ultrasound diagnosis per laparoscopy at the very site of the lesion. The advantages are evident. Due to the closeness to the organ to be examined, the frequency of the scan head can be extremely high, resulting in better resolution. Also, structures could be visualized by ultrasound which e.g., due to adhesions cannot be seen with the laparoscope. Moreover, ultrasound offers a view into the structures which can be seen only from the exterior with the laparoscope. To perform these examinations we used a specially designed scanhead (Toshiba): Instead of the optical system a crystal array was inserted into a conventional gastroscope. The result was a high resolution 7.5 MHz linear array at the distal end of a freely movable gastroscope. Colour doppler sonography is feasible with this scanhead. We examined 19 patients, 16 of them with ovarian tumours, and attempted visualization of the uterine myometrium/endometrium as well as of the liver. In six cases of ovarian tumours in which only cystic structures were found by preoperative transvaginal sonography, laparoscopic ultrasound additionally revealed solid inner structures. In 5 cases direct laparoscopic view of the ovarian lesions was impossible due to severe adhesions. They were, however, easily detected by intraoperative ultrasound.(ABSTRACT TRUNCATED AT 250 WORDS)