Tumoral programmed cell death 1 (PD1) expression in endometrial carcinoma is a prognostic marker for patient outcome.

OBJECTIVE: Immune checkpoint inhibitors have recently demonstrated benefit in patients with advanced and recurrent endometrial carcinoma. This retrospective study investigated immune checkpoint molecules in endometrial carcinoma as they pertain to the molecular subtypes, clinical outcomes, and predictive value. METHODS: Tumoral RNA expression of genes controlling the immune checkpoint, programmed cell death 1 (PD1, encoded by PDCD1), its ligand (PDL1, encoded by CD274), and interferon gamma (IFNG) was determined in 239 endometrial carcinoma tissues by quantitative polymerase chain reaction (qPCR) and compared with endometrial tissue from 25 controls. A total of 81 endometrial carcinoma tissues were analyzed using the ProMiSe molecular classification, and patient trajectories were analyzed for the entire cohort. Findings were validated in an independent cohort from The Cancer Genome Atlas (TCGA; n=548). RESULTS: PD1, PDL1, and IFNG expression was significantly higher in endometrial carcinoma when compared with non-malignant control tissue with a mean expression of 0.12, 0.05, and 0.05 in control tissue and 0.44, 0.31, and 0.35 in endometrial carcinoma, respectively. POLE-mutated and mismatch repair-deficient (MMRd) (immunologically hot) tumors showed the highest expression of PD1 and IFNG. Increased expression of PD1, PDL1, and IFNG was associated with improved recurrence-free (HR 0.32, p<0.001; HR 0.30, p<0.001; HR 0.47, p=0.012, respectively), disease-specific (HR 0.38, p<0.001; HR 0.29, p<0.001; HR 0.45, p=0.017, respectively), and overall survival (HR 0.56, p=0.003; HR 0.38, p<0.001; HR 0.58, p=0.006, respectively). Cox regression confirmed the prognostic significance of PD1 for recurrence-free survival (HR 0.39, p=0.009) and PDL1 for overall survival (HR 0.55, p=0.037). The prognostic value of tumoral PD1 on recurrence-free survival, disease-specific survival, and overall survival was confirmed in the TCGA cohort. CONCLUSIONS: Tumoral gene expression controlling the PD1 immune checkpoint, particularly expressed in "hot tumors", predicted recurrence-free, disease-specific, and overall survival in patients with endometrial carcinoma in two independent cohorts. Evaluation of these genes could be used to stratify patients who qualify for immune checkpoint inhibitors, which warrants prospective clinical trials.

An angiogenic tumor phenotype predicts poor prognosis in ovarian cancer.

OBJECTIVE: Epithelial ovarian cancer (OC) is the deadliest gynecological malignancy worldwide. Blocking angiogenesis with bevacizumab, an antibody targeting vascular endothelial growth factor (VEGF), shows efficacy in different lines of OC therapy. This study investigates the clinical impact of tumoral expression of angiogenesis-related genes and their association with bevacizumab response in OC in retrospective analysis of three independent cohorts. METHODS: mRNA expression of seven angiogenic genes (VEGF, VEGFR2, PDGFA, PDGFB, PDGFRA, PDGFRB, KIT) was quantified in an inception OC cohort (n = 195) and a transcriptional tumor angiogenesis score from 0 to 3 was established and linked to progression-free survival (PFS) and overall survival (OS). This score was corroborated in an independent publicly available cohort from The Cancer Genome Atlas (TCGA, n = 582) and prediction of therapeutic efficacy of bevacizumab by the angiogenesis score was analyzed in the Gene Expression Omnibus (GEO) dataset GSE140082 (n = 380) from the ICON7-trial. RESULTS: The tumor angiogenesis score prognosticated PFS and OS in patients with OC from the inception cohort (p < 0.001, respectively). Tumoral PDGFA expression (PFS: HR 2.46, p = 0.005; OS: HR 2.26, p = 0.011) and a high tumoral transcriptional angiogenesis score (PFS: HR 1.41, p = 0.018) were identified as independent predictors of clinical outcome. The transcriptional angiogenesis score exhibited a significant though smaller effect size on PFS in the TCGA cohort. However, in the ICON7-trial, the angiogenesis score was not associated with benefit of bevacizumab treatment. CONCLUSIONS: Our study indicates that tumoral expression of angiogenic genes is unfavorable in OC. The established score could be used to identify patients who respond to targeted angiogenic therapies, a concept that warrants prospective controlled clinical trials.

NADPH oxidase 4 expression in the normal endometrium and in endometrial cancer.

The aim of this study was to explore the role of NOX4 in the biology of the normal endometrium and endometrial cancer. NOX4 plays a key role in other adenocarcinomas and has been implicated in the pathogenesis of diabetes and obesity, which are important risk factors for endometrial cancer. NOX4 expression was assessed in 239 endometrial cancer and 25 normal endometrium samples by quantitative real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry. DNA methylation of the NOX4 promoter was determined by means of MethyLight PCR. Data were correlated with clinicopathological parameters and analyzed in the context of diabetes and body mass index. In the normal endometrium, NOX4 microRNA expression was significantly higher in the secretory transformed compared with proliferative endometrium ( p = 0.008). In endometrial cancer specimens, NOX4 expression did not differ between diabetic and non-diabetic patients, but was the highest in patients with a body mass index ≤ 26 ( p = 0.037). The lowest NOX4 expression was found in carcinosarcomas ( p = 0.007). High NOX4 expression predicted poorer clinical outcome with regard to overall survival, especially in non-diabetic patients and those with a body mass index > 20. Independent prognostic significance of NOX4 transcripts was retained in type I endometrial cancer and was the most meaningful in patients with a body mass index > 20. No prognostic impact was shown for NOX4 promoter methylation in endometrial cancer. For the first time, we demonstrate that NOX4 plays a considerable role in the cycle-dependent changes in the normal endometrium and in the biology of endometrial cancer.

Evaluation of folate receptor 1 (FOLR1) mRNA expression, its specific promoter methylation and global DNA hypomethylation in type I and type II ovarian cancers.

BACKGROUND: In this retrospective study we evaluated the respective correlations and clinical relevance of FOLR1 mRNA expression, FOLR1 promoter specific methylation and global DNA hypomethylation in type I and type II ovarian cancer. METHODS: Two hundred fifty four ovarian cancers, 13 borderline tumours and 60 samples of healthy fallopian epithelium and normal ovarian epithelium were retrospectively analysed for FOLR1 expression with RT-PCR. FOLR1 DNA promoter methylation and global DNA hypomethylation (measured by means of LINE1 DNA hypomethylation) were evaluated with MethyLight technique. RESULTS: No correlation between FOLR1 mRNA expression and its specific promoter DNA methylation was found neither in type I nor in type II cancers, however, high FOLR1 mRNA expression was found to be correlated with global DNA hypomethylation in type II cancers (p = 0.033). Strong FOLR1 mRNA expression was revealed for Grades 2-3, FIGO stages III-IV, residual disease > 0, and serous histotype. High FOLR1 expression was found to predict increased platinum sensitivity in type I cancers (odds ratio = 3.288; 1.256-10.75; p = 0.020). One-year survival analysis showed in type I cancers an independent better outcome for strong expression of FOLR1 in FIGO stage III and IV. For the entire follow up period no significant independent outcome for FOLR1 expression was revealed. In type I cancers LINE 1 DNA hypomethylation was found to exhibit a worse PFS and OS which were confirmed to be independent in multivariate COX regression model for both PFS (p = 0.026) and OS (p = 0.012). CONCLUSION: No correlations were found between FOLR1 expression and its specific promoter methylation, however, high FOLR1 mRNA expression was associated with DNA hypomethylation in type II cancers. FOLR1 mRNA expression did not prove to predict clinical outcome in type II cancers, although strong FOLR1 expression generally denotes ovarian cancers with highly aggressive phenotype. In type I cancers, however, strong FOLR1 expression has been found to be a reliable indicator of improved platinum responsiveness reflecting a transient better one-year follow up outcome in highly FOLR1 expressing type I cancers. An independent prognostic role of global DNA hypomethylation was demonstrated in type I tumours.

Expression and promotor hypermethylation of miR-34a in the various histological subtypes of ovarian cancer.

BACKGROUND: An increasing body of evidence shows that miR-34 family has tumor suppressive properties mediating apoptosis, cell cycle arrest and senescence. In ovarian cancer, miR34 family members were found to be under expressed. Particularly miR-34a has been revealed to be a direct transcriptional target of p53 which is frequently mutated in epithelial ovarian carcinomas especially in high grade serous cancer. Moreover, methylation of miR-34a CpG Islands was found to down-regulate miR-34a expression. The aim of this study was to investigate the clinical relevance of mir34a as well as its promoter methylation in a subset of 133 ovarian cancers with a special focus on the p53 mutation status, the dualistic type I and type II ovarian cancer model and the different histotypes. METHODS: One hundred thirty-three epithelial ovarian cancers and 8 samples of healthy ovarian surface epithelium were retrospectively analysed for miR-34a expression with quantitative real-time reverse transcription PCR (qRT-PCR). Gene-specific DNA methylation was evaluated with MethyLight technique. RESULTS: Significantly lower miR-34a expression was found in ovarian cancers than in healthy ovarian epithelium (p = 0.002). The expression of miR-34a was found lower in type II than in type I cancers (p = 0.037), in p53 mutated as compared to p53 wild type cancers (p = 0.003) and in high grade compared to in low grade cancers (p = 0.028). In multivariate COX regression model low expressing miR-34a cancers exhibited a reduced PFS (p = 0.039) and OS (p = 0.018). In serous cancers low miR-34a levels showed a worse OS confirmed also in multivariate analysis (p = 0.022). miR-34a promoter methylation was found higher in type II cancers than in type I (p = 0.006). mir34a expression and promoter methylation showed an inverse correlation in cancer samples (p = 0.05). CONCLUSION: We demonstrated a clinical independent role of miR-34a in epithelial ovarian cancers. Moreover, we corroborated the correlation between miR-34a expression and its promoter methylation in a large set of ovarian cancers. The inverse association between miR-34a expression and grading, p53 mutation status and dualistic tumor type classification, together with its prognostic relevance may underline the tumor-suppressive character of miR-34a in ovarian cancer.

Prophylactic long-acting granulocyte-colony stimulating factors (G-CSF) in gynecologic malignancies: an oncologic expert statement.

We reviewed the status of the use of the prophylactic long-acting granulocyte colony-stimulating factors (G-CSFs) pegfilgrastim and lipegfilgrastim in gynecologic malignancies. Long-acting G-CSFs should not be used in weekly regimens. Filgrastim is not indicated in patients with febrile and/or severe neutropenia after administration of long-acting G-CSF in the same cycle. One study has shown a moderate effect on febrile neutropenia of ciprofloxacin when co-administered with pegfilgrastim. There is broad evidence from meta-analyses that pegfilgrastim effectively reduces severe neutropenia. In parallel, its adverse effects have been studied extensively. All-cause mortality was significantly reduced by pegfilgrastim. The glycopegylated long-acting G-CSF, lipegfilgrastim has demonstrated antineutropenic efficacy similar to that of pegfilgrastimin in one breast cancer study. In another pivitol non-small cell lung cancer study, impaired survival was observed in the lipegfilgrastim group during the first 30 days of study. The European Medicines Agency claimed more profound safety data to be provided for lipegfilgrastim by 2017.

Interactions of human peritoneal mesothelial cells with serous ovarian cancer cell spheroids--evidence for a mechanical and paracrine barrier function of the peritoneal mesothelium.

BACKGROUND: Ovarian carcinoma spreads by implantation of tumor cells onto the peritoneal mesothelium. We established a 3-dimensional coculture model to simulate the interactions of ovarian carcinoma cell aggregates with human peritoneal mesothelial cells (HPMC). METHODS: Multicellular tumor spheroids (MCTS) of the human ovarian cancer cell line SK-OV-3 were directly inoculated onto either confluent HPMC monolayers or their submesothelial matrix or were cocultured with mesothelium without direct cellular contact. RESULTS AND DISCUSSIONS: Inoculation of MCTS onto submesothelial matrix resulted in rapid attachment (within 30 minutes) of the tumor cell aggregates followed by rapid dissemination (within 12 hours) and growth of tumor cells. Intact mesothelium increased the time required for MCTS attachment (up to 180 minutes) and led to almost complete inhibition of tumor cell dissemination and to 47% tumor growth suppression. Bromodeoxyuridine incorporation into tumor cell nuclei was almost completely abolished in cocultured MCTS. Growth also was inhibited in MCTS treated with supernatants of HPMC. Analysis of coculture supernatants revealed that HPMC-derived transforming growth factor ß (TGF-ß) was almost completely bound by MCTS. Addition of a function-blocking anti-TGF-ß antibody (30 µg/mL) to the cocultures abrogated the growth inhibitory effect of the mesothelium by 50%. CONCLUSIONS: The present model provides a dynamic system to study the complex interactions of ovarian carcinoma cells with HPMC over extended periods and suggests that the mesothelium constitutes a mechanical and partly TGF-ß-mediated paracrine barrier to the progression of ovarian cancer.

Endometrial Cancer. Guideline of the DGGG, DKG and DKH (S3-Level, AWMF Registry Number 032/034-OL, September 2022). Part 1 with Recommendations on the Epidemiology, Screening, Diagnosis and Hereditary Factors of Endometrial Cancer, Geriatric Assessment and Supply Structures.

Summary The S3-guideline on endometrial cancer, first published in April 2018, was reviewed in its entirety between April 2020 and January 2022 and updated. The review was carried out at the request of German Cancer Aid as part of the Oncology Guidelines Program and the lead coordinators were the German Society for Gynecology and Obstetrics (DGGG), the Gynecology Oncology Working Group (AGO) of the German Cancer Society (DKG) and the German Cancer Aid (DKH). The guideline update was based on a systematic search and assessment of the literature published between 2016 and 2020. All statements, recommendations and background texts were reviewed and either confirmed or amended. New statements and recommendations were included where necessary. Aim The use of evidence-based risk-adapted therapies to treat women with endometrial cancer of low risk prevents unnecessarily radical surgery and avoids non-beneficial adjuvant radiation therapy and/or chemotherapy. For women with endometrial cancer and a high risk of recurrence, the guideline defines the optimum level of radical surgery and indicates whether chemotherapy and/or adjuvant radiation therapy is necessary. This should improve the survival rates and quality of life of these patients. The S3-guideline on endometrial cancer and the quality indicators based on the guideline aim to provide the basis for the work of certified gynecological cancer centers. Methods The guideline was first compiled in 2018 in accordance with the requirements for S3-level guidelines and was updated in 2022. The update included an adaptation of the source guidelines identified using the German Instrument for Methodological Guideline Appraisal (DELBI). The update also used evidence reviews which were created based on selected literature obtained from systematic searches in selected literature databases using the PICO process. The Clinical Guidelines Service Group was tasked with carrying out a systematic search and assessment of the literature. Their results were used by interdisciplinary working groups as a basis for developing suggestions for recommendations and statements which were then modified during structured online consensus conferences and/or additionally amended online using the DELPHI process to achieve a consensus. Recommendations Part 1 of this short version of the guideline provides recommendations on epidemiology, screening, diagnosis, and hereditary factors. The epidemiology of endometrial cancer and the risk factors for developing endometrial cancer are presented. The options for screening and the methods used to diagnose endometrial cancer are outlined. Recommendations are given for the prevention, diagnosis, and therapy of hereditary forms of endometrial cancer. The use of geriatric assessment is considered and existing structures of care are presented.

Causal analyses with target trial emulation for real-world evidence removed large self-inflicted biases: systematic bias assessment of ovarian cancer treatment effectiveness.

BACKGROUND AND OBJECTIVES: Drawing causal conclusions from real-world data (RWD) poses methodological challenges and risk of bias. We aimed to systematically assess the type and impact of potential biases that may occur when analyzing RWD using the case of progressive ovarian cancer. METHODS: We retrospectively compared overall survival with and without second-line chemotherapy (LOT2) using electronic medical records. Potential biases were determined using directed acyclic graphs. We followed a stepwise analytic approach ranging from crude analysis and multivariable-adjusted Cox model up to a full causal analysis using a marginal structural Cox model with replicates emulating a reference randomized controlled trial (RCT). To assess biases, we compared effect estimates (hazard ratios [HRs]) of each approach to the HR of the reference trial. RESULTS: The reference trial showed an HR for second line vs. delayed therapy of 1.01 (95% confidence interval [95% CI]: 0.82-1.25). The corresponding HRs from the RWD analysis ranged from 0.51 for simple baseline adjustments to 1.41 (95% CI: 1.22-1.64) accounting for immortal time bias with time-varying covariates. Causal trial emulation yielded an HR of 1.12 (95% CI: 0.96-1.28). CONCLUSION: Our study, using ovarian cancer as an example, shows the importance of a thorough causal design and analysis if one is expecting RWD to emulate clinical trial results.

Clinical Impact of RANK Signalling in Ovarian Cancer.

Ovarian cancer (OC) is a gynaecological malignancy with poor clinical outcome and limited treatment options. The receptor activator of nuclear factor-κB (RANK) pathway, activated by RANK ligand (RANKL), critically controls bone metabolism, tumourigenesis and tumour immune responses. Denosumab, a monocloncal RANKL antibody, exerts tumour-suppressive effects in mice and humans. Here, we investigated the relevance of RANK signalling in OC. RANK, RANKL and OPG expression in 192 epithelial OC tissues was compared to expression in 35 non-malignant control tissues and related to clinico-pathological characteristics. Findings were validated in a cohort of 563 OC patients from The Cancer Genome Atlas (TCGA). The expression of RANK, RANKL and OPG was studied in four OC cell lines and the impact of RANK ligation or blockade on OC cell proliferation was determined. RANK, RANKL and OPG were expressed in epithelial and stromal cells in OC. RANKL expression was elevated in OC tissue, particularly in BRCA1/2 mutated tumours. High RANKL expression independently predicted reduced progression-free (PFS, p = 0.017) and overall survival (OS, p = 0.007), which could be validated in the TCGA cohort (PFS, p = 0.022; OS, p = 0.046, respectively). Expression of RANK and OPG in OC cells was induced by inflammatory cytokines IL-1ß and TNFα. Neither recombinant RANK ligation nor denosumab treatment affected OC cell proliferation. Our study independently links RANKL expression with poor clinical outcome in two unrelated OC cohorts. These findings implicate RANK signalling in the immunopathogenesis of OC and warrant clinical trials with denosumab in OC.

Part I of GANNET53: A European Multicenter Phase I/II Trial of the Hsp90 Inhibitor Ganetespib Combined With Weekly Paclitaxel in Women With High-Grade, Platinum-Resistant Epithelial Ovarian Cancer-A Study of the GANNET53 Consortium.

Background: Stabilized mutant p53 protein (mutp53) is a novel target in epithelial ovarian cancer. Due to aberrant conformation, mutp53 proteins depend on folding support by the Hsp90 chaperone. Hsp90 blockade induces degradation of mutp53, resulting in tumor cell cytotoxicity and increased sensitivity to chemotherapeutics. Preclinical synergy of the Hsp90 inhibitor ganetespib combined with paclitaxel provided the rationale for testing the combination in platinum-resistant ovarian cancer (PROC) patients in the GANNET53 trial (NCT02012192). Methods: Eligible patients had high-grade PROC with ≤ 4 prior lines of chemotherapy. Weekly paclitaxel (80 mg/m2) and increasing doses of ganetespib (100, 150 mg/m2) were given i.v. on days 1, 8, 15 in a 28 days cycle until disease progression or unacceptable toxicity. Endpoints were safety and determination of phase II dose. Dose limiting toxicity (DLT) was defined as grade 4 toxicity (with exceptions) occurring in cycles 1&2. Results: Ten patients (median age 59 years; range 43-70) were enrolled. No DLT occurred in cohort 1 (4 patients treated with paclitaxel + ganetespib 100 mg/m2), nor in cohorts 2 and 3 (6 patients treated with paclitaxel + ganetespib 150 mg/m2). The most common adverse event (AE) related to ganetespib was transient grade 1/2 diarrhea (n = 6). Related grade 1/2 AEs in >2 patients included QTc prolongation (n = 4), nausea (n = 3), anemia (n = 3), headache (n = 3), fatigue (n = 3), and dyspnoea (n = 3). Most frequently related grade 3/4 AEs were diarrhea (n = 3) and neutropenia (n = 2). There was 1 death on study due to hemorrhage from a duodenal ulcer. Three patients discontinued study treatment due to serious AEs (digestive hemorrhage n = 1, cardiac failure n = 1, abdominal pain and vomiting n = 1), 6 due to progressive disease, one due to investigator and patient decision. Two patients achieved a partial response (ORR 20%) and 4 patients a stable disease (disease control rate of 60%). Median PFS was 2.9 months (1.6 months in cohort 1 at 100 mg/m2 ganetespib, 5.1 months in cohorts 2+3 at 150 mg/m2 ganetespib). Conclusions: The combination of ganetespib 150 mg/m2 with paclitaxel 80 mg/m2 once weekly for 3 out of 4 weeks was generally well-tolerated with no DLTs, and therefore chosen for the randomized phase II trial.

Activated p38-MAPK and gemcitabine sensitivity in recurrent ovarian cancer.

BACKGROUND: Our study aimed to investigate if p38-MAPK determined in primary ovarian cancer can serve as a predictive marker for sensitivity to gemcitabine treatment in recurrent disease. MATERIALS AND METHODS: Activated (phosphorylated) p38-MAPK was immunohistochemically assessed in paraffin-embedded tumors obtained at primary debulking surgery from 45 patients treated with gemcitabine for platinum-resistant recurrence. The value of activated p38-MAPK in predicting sensitivity to gemcitabine treatment was statistically evaluated. RESULTS: Activated p38-MAPK was demonstrated in all healthy ovaries and all ovarian carcinomas examined. In controls, the median histological score (H-score) for activated p38-MAPK staining was 200, while in ovarian cancer the median H-score was 100. Activity of p38-MAPK in ovarian cancer tissue was not associated with overall response or survival after gemcitabine chemotherapy. CONCLUSION: P38-MAPK activity, determined by immunohistochemistry in ovarian cancer specimens obtained at primary diagnosis, cannot serve as a predictive marker for sensitivity to gemcitabine treatment in platinum-resistant disease.

L1CAM in the Early Enteric and Urogenital System.

L1 cell adhesion molecule (L1CAM) is a transmembrane molecule belonging to the L1 protein family. It has shown to be a key player in axonal guidance in the course of neuronal development. Furthermore, L1CAM is also crucial for the establishment of the enteric and urogenital organs and is aberrantly expressed in cancer originating in these organs. Carcinogenesis and embryogenesis follow a lot of similar molecular pathways, but unfortunately, comprehensive data on L1CAM expression and localization in human developing organs are lacking so far. In the present study we, therefore, examined the spatiotemporal distribution of L1CAM in the early human fetal period (weeks 8-12 of gestation) by means of immunohistochemistry and in situ hybridization (ISH). In the epithelia of the gastrointestinal organs, L1CAM localization cannot be observed in the examined stages most likely due to their advanced polarization and differentiation. Despite these results, our ISH data indicate weak L1CAM expression, but only in few epithelial cells. The genital tracts, however, are distinctly L1CAM positive throughout the entire fetal period. We, therefore, conclude that in embryogenesis L1CAM is crucial for further differentiation of epithelia.

Preserved coupling of oxidative phosphorylation but decreased mitochondrial respiratory capacity in IL-1beta-treated human peritoneal mesothelial cells.

The peritoneal mesothelium acts as a regulator of serosal responses to injury, infection, and neoplastic diseases. After inflammation of the serosal surfaces, proinflammatory cytokines induce an "activated" mesothelial cell phenotype, the mitochondrial aspect of which has not previously been studied. After incubation of cultured human peritoneal mesothelial cells with interleukin (IL)-1beta for 48 h, respiratory activity of suspended cells was analyzed by high-resolution respirometry. Citrate synthase (CS) and lactate dehydrogenase (LDH) activities were determined by spectrophotometry. Treatment with IL-1beta resulted in a significant decline of respiratory capacity (p < 0.05). Respiratory control ratios (i.e., uncoupled respiration at optimum carbonyl cyanide p-trifluoromethoxyphenylhydrazone concentration divided by oligomycin inhibited respiration measured in unpermeabilized cells) remained as high as 11, indicating well-coupled mitochondria and functional integrity of the inner mitochondrial membrane. Whereas respiratory capacities of the cells declined in proportion with decreased CS activity (p < 0.05), LDH activity increased (p < 0.05). Taken together, these results indicate that IL-1beta exposure of peritoneal mesothelial cells does not lead to irreversible defects or inhibition of specific components of the respiratory chain, but is associated with a decrease of mitochondrial content of the cells that is correlated with an increase in LDH (and thus glycolytic) capacity.

Evaluating L1CAM expression in human endometrial cancer using qRT-PCR.

BACKGROUND: Management of endometrial carcinoma (EC) still needs improvement of risk assessment. Recently, L1CAM immunohistochemical (IHC) evaluation showed a unique value to predict the outcome of early EC. However IHC results are often conflicting for lack of inter-laboratory standardisation. METHODS: Here, as a proof of concept and to increase reproducibility we assayed eighty-two EC and 26 normal endometrium samples for L1CAM expression (L1CAMEXP) via qRT-PCR. The IHC evaluation was performed in 50 cancer samples. Moreover, we aimed to substantiate the in-vitro findings of L1CAM regulation through its promoter methylation (L1CAMMET), miR-34a expression and miR-34a promoter methylation. DNA methylation was assessed with MethyLight PCR technique. RESULTS: High overall concordant results between IHC and RT-PCR evaluations were found. L1CAMEXP was detected in 11% of cancer specimens. These positive cancers exhibited a worse DFS (p=0.032) and OS (p=0.016) in a multivariate COX-regression model. L1CAMEXP predicted distant failure (p=0.007) and L1CAMMET predicted risk-reduction of lymph-node involvement (p=0.005). Inverse correlations between L1CAMEXP and L1CAMMET (p=0.004) and between L1CAMEXP and miR-34a expression (p=0.002) were found. CONCLUSIONS: In conclusion qRT-PCR analysis is a reliable approach to evaluate L1CAM status in EC and L1CAMEXP was highly predictive for distant failure and poor outcome, confirming the large IHC-based studies. Interestingly, L1CAMMET was able to assess the risk of pelvic lymph-node involvement. Especially the latter finding has to be confirmed in larger prospective series.

Disruption of the integrity of human peritoneal mesothelium by interleukin-1beta and tumor necrosis factor-alpha.

Inflammatory and neoplastic disease processes of the abdominal cavity are frequently associated with disruption of the integrity of the peritoneal mesothelium. In the present study, we analyzed the effects of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on the morphology and expression of adhesion molecules of human peritoneal mesothelial cells (HPMC). Treatment of HPMC with IL-1beta and TNF-alpha resulted in a time- and dose-dependent alteration of the normal cobblestone morphology of the mesothelium with loss of polarization, cellular retraction and exposure of the submesothelial matrix. The effect was already observable after 6 h of treatment and was most pronounced at a dose of 10 ng/ml of IL-1beta or TNF-alpha. These morphological alterations were associated with a significant rearrangement of the expression of mesothelial adhesion molecules as detected by flow cytometry. IL-1beta and TNF-alpha both led to a loss of the expression of the hemidesmosomal integrin subunits alpha6 ( P<0.01 and P<0.001) and beta4 ( P<0.01) and an increased expression of the integrin subunit alpha5 ( P<0.001 and P<0.01). IL-1beta furthermore upregulated the expression of the integrin subunits alpha1, alpha2 and the adhesion molecule CD44 while the latter was downregulated by TNF-alpha. Our data indicate that IL-1beta and TNF-alpha may significantly affect disease processes of the abdominal cavity by their potential to disrupt the mesothelial basal cell-matrix adhesion and, thus, the integrity of the peritoneal mesothelial cell lining.

Austrian Arbeitsgemeinschaft für Gynäkologische Onkologie (AGO) guideline for prophylaxis with granulocyte colony-stimulating factors (G-CSF) in gynecologic malignancies, including breast cancer.

The current knowledge and recommendations on the clinical use of granulocyte colony-stimulating factors (G-CSF) in gynecologic cancers including breast cancer, along with the clinical experience of the members of the working group of the Austrian Arbeitsgemeinschaft für Gynäkologische Onkologie (AGO), have been summarized. G-CSF is either administered as primary or secondary prophylaxis of febrile neutropenia. The term "primary prophylaxis" denotes the prophylactic use of G-CSF as early as during the first cycle of a new chemotherapeutic regimen. Secondary prophylaxis, on the other hand, defines the use of G-CSF after development of grade 4 neutropenia or febrile neutropenia in a preceding cycle of a particular chemotherapeutic regimen. When chemotherapy regimens are associated with a > 20 % risk of febrile neutropenia such as TAC (docetaxel-doxorubicin-cyclophosphamide), primary prophylaxis with G-CSF is indicated. When chemotherapy regimens are associated with a 10-20 % risk of febrile neutropenia, the decision for primary prophylaxis with G-CSF is based upon patient-related risk factors such as age > 65 years, previous cytotoxic treatment(s) and/or radiation therapy, preexisting tumor-related neutropenia or bone marrow involvement, preexisting neutropenia, infections/open sores, reduced Karnofsky performance status/WHO performance status and reduced nutritional status, advanced malignant disease, history of prior febrile neutropenia, impaired kidney function, and hepatic failure particularly with hyperbilirubinaemia. The patient's individual overall febrile neutropenia risk should be assessed prior to each chemotherapy cycle.

Epithelial growth factor receptor status in primary and recurrent ovarian cancer.

Success of epidermal growth factor receptor (EGFR) targeting agents in different cancer types is related to EGFR gene mutations and/or copy number gains. We investigated the EGFR gene status and protein expression by DNA mutational analysis, fluorescence in situ hybridization (FISH), and immunohistochemistry in tumor tissues from 80 patients with primary and corresponding recurrent ovarian serous carcinomas. The patients were classified into six groups with ascending EGFR gene copy numbers. EGFR amplification and high polysomy (FISH+) was present in a significant fraction of the primary (20%) and recurrent (22%) ovarian carcinomas. On mutational analysis, only one tumor with a silent EGFR mutation was observed, and this was the only carcinoma with high-level amplification. EGFR protein immunoexpression was seen in 28% of primary and 33% of recurrent carcinomas and correlated to amplification in the primary tumors (P = 0.003). In recurrent carcinoma, moderate and strong EGFR expression was associated with amplification (P = 0.034). These molecular events potentially have impact on the responsiveness to EGFR targeting agents in ovarian cancer.

Ovarian carcinoma cells and IL-1beta-activated human peritoneal mesothelial cells are possible sources of vascular endothelial growth factor in inflammatory and malignant peritoneal effusions.

OBJECTIVE: Inflammatory or malignant peritoneal diseases are associated with high levels of ascitic vascular endothelial growth factor (VEGF). We compared the VEGF secretion by human peritoneal mesothelial cells (HPMC) and ovarian carcinoma (OVCA) cells and its regulation by pro-inflammatory cytokines. MATERIALS AND METHODS: VEGF secretion in cultured HPMC, established human OVCA cell lines, and inflammatory or OVCA-associated ascites was determined by enzyme linked immunosorbent assay. RESULTS: HPMC constitutively produced VEGF at median levels of 43 +/- 7 pg/10(5) cells. Treatment of HPMC with 1 ng/ml IL-1beta (567 +/- 213 pg/10(5) cells) or TNF-alpha (89 +/- 1 pg/10(5) cells) resulted in a 13-fold (P < 0.01) or 2-fold (P < 0.05) elevation of the VEGF secretion. In OVCA, the constitutive VEGF expression was 8-fold higher than VEGF levels in HPMC (364 +/- 185 pg/10(5) cells; P < 0.001). VEGF secretion in OVCA cells was also increased by IL-1beta (514 +/- 105 pg/10(5) cells; P < 0.01) or TNF-alpha (458 +/- 168 pg/10(5) cells; P < 0.01) reaching similar levels as in IL-1beta-activated HPMC. Median VEGF levels in malignant ascites (2761 +/- 1549 pg/ml) were 11-fold higher compared with levels in inflammatory fluids (244 +/- 170 pg/ml; P < 0.01). VEGF levels in both inflammatory- and OVCA-associated fluids correlated with ascitic IL-1beta levels (P < 0.05). CONCLUSION: We identified ovarian cancer cells and/or IL-1beta-activated peritoneal mesothelial cells as important sources of ascitic VEGF. The present data indicate that IL-1beta-triggered VEGF production by neoplastic and normal cells is a common pathomechanism for ascites formation in both inflammatory and malignant conditions.