Dominique Könsgen

Mass Spectrometry–Based Metabolic Profiling Reveals Different Metabolite Patterns in Invasive Ovarian Carcinomas and Ovarian Borderline Tumors

Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes. We have used a metabolite profiling approach to test the hypothesis that quantitative signatures of primary metabolites can be used to characterize molecular changes in ovarian tumor tissues. Sixty-six invasive ovarian carcinomas and nine borderline tumors of the ovary were analyzed by gas chromatography/time-of-flight mass spectrometry (GC-TOF MS) using a novel contamination-free injector system. After automated mass spectral deconvolution, 291 metabolites were detected, of which 114 (39.1%) were annotated as known compounds. By t test statistics with P < 0.01, 51 metabolites were significantly different between borderline tumors and carcinomas, with a false discovery rate of 7.8%, estimated with repeated permutation analysis. Principal component analysis (PCA) revealed four principal components that were significantly different between both groups, with the highest significance found for the second component (P = 0.00000009). PCA as well as additional supervised predictive models allowed a separation of 88% of the borderline tumors from the carcinomas. Our study shows for the first time that large-scale metabolic profiling using GC-TOF MS is suitable for analysis of fresh frozen human tumor samples, and that there is a consistent and significant change in primary metabolism of ovarian tumors, which can be detected using multivariate statistical approaches. We conclude that metabolomics is a promising high-throughput, automated approach in addition to functional genomics and proteomics for analyses of molecular changes in malignant tumors.

Expression of estrogen receptor-related receptors, a subfamily of orphan nuclear receptors, as new tumor biomarkers in ovarian cancer cells

A subfamily of orphan receptors, estrogen receptor-related receptors (ERRs), has been demonstrated to modulate the transcription of some estrogen responsive genes via variant estrogen response elements (EREs). This study was conducted to determine whether human ERRα, ERRβ, and ERRγ might be involved in the tumorigenesis of ovarian cancer. RT-PCR was performed to analyze the expression of hERRα, hERRβ, hERRβ-2, and hERRγ mRNA in five ovarian cancer cell lines as well as 33 samples of ovarian cancer and 12 samples of normal ovary. Serum CA-125 levels were also analyzed in all samples by ELISA. Progression-free survival and overall survival of patients with different expression of ERRs were analyzed by the Kaplan–Meier method. To analyze the subcellular localization of ERRα, a green fluorescent protein (GFP)-reporter plasmid of hERRα was constructed and transfected into the ovarian cancer cell line OVCAR-3. Expression of hERRα-GFP fusion protein was observed in the nucleus of OVCAR-3 ovarian cancer cell lines. We observed increased expression of hERRα mRNA (P=0.020) and hERRγ mRNA (P=0.045) in ovarian cancers compared to normal ovaries. In contrast, hERRβ was only observed in 9.1% of ovarian cancers. We found a positive correlation between the serum CA-125 levels and hERRα expression (P=0.012), but not hERRβ and hERRγ expression. Survival analysis showed that the hERRα-positive group has a reduced overall survival (P=0.015), and the ERRγ-positive group has a longer progression-free survival (P=0.020). In multivariate analysis, expression of hERRα was an independent prognostic factor for poor survival (relative risk, 3.032; 95% CI, 1.27–6.06). Based on our results, ERRs may play an important role in ovarian cancer. hERRα may represent a biomarker of poor prognosis, and hERRγ may be a new therapeutic target in ovarian cancer.

A prognostic gene expression index in ovarian cancer - validation across different independent data sets.

Ovarian carcinoma has the highest mortality rate among gynaecological malignancies. In this project, we investigated the hypothesis that molecular markers are able to predict outcome of ovarian cancer independently of classical clinical predictors, and that these molecular markers can be validated using independent data sets. We applied a semi‐supervised method for prediction of patient survival. Microarrays from a cohort of 80 ovarian carcinomas (TOC cohort) were used for the development of a predictive model, which was then evaluated in an entirely independent cohort of 118 carcinomas (Duke cohort). A 300‐gene ovarian prognostic index (OPI) was generated and validated in a leave‐one‐out approach in the TOC cohort (Kaplan‐Meier analysis, p = 0.0087). In a second validation step, the prognostic power of the OPI was confirmed in an independent data set (Duke cohort, p = 0.0063). In multivariate analysis, the OPI was independent of the post‐operative residual tumour, the main clinico‐pathological prognostic parameter with an adjusted hazard ratio of 6.4 (TOC cohort, CI 1.8–23.5, p = 0.0049) and 1.9 (Duke cohort, CI 1.2–3.0, p = 0.0068). We constructed a combined score of molecular data (OPI) and clinical parameters (residual tumour), which was able to define patient groups with highly significant differences in survival. The integrated analysis of gene expression data as well as residual tumour can be used for optimized assessment of the prognosis of platinum‐taxol‐treated ovarian cancer. As traditional treatment options are limited, this analysis may be able to optimize clinical management and to identify those patients who would be candidates for new therapeutic strategies. Copyright

Microheterogeneity of transthyretin in serum and ascitic fluid of ovarian cancer patients.

BackgroundTransthyretin (TTR), a traditional biomarker for nutritional and inflammatory status exists in different molecular variants of yet unknown importance. A truncated form of TTR has recently been described to be part of a set of biomarkers for the diagnosis of ovarian cancer. The main aim of the study was therefore to characterize differences in microheterogeneity between ascitic fluid and plasma of women affected with ovarian cancer and to evaluate the tumor site as the possible source of TTR.MethodsSubjects were 48 women with primary invasive epithelial ovarian cancer or recurrent ovarian carcinoma. The control group consisted of 20 postmenopausal women. TTR and retinol-binding protein (RBP) levels were measured by enzyme-linked immunoassay (ELISA) and C-reactive protein (CRP) levels by a high-sensitivity latex particle turbidimetric assay. The molecular heterogeneity of TTR was analysed using immunoprecipitation and matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Presence of TTR in tumor tissue was determined with indirect peroxidase immunostaining.ResultsTTR and RBP (μg/ml) levels in serum were 148.5 ± 96.7 and 22.5 ± 14.8 in affected women compared to 363.3 ± 105.5 and 55.8 ± 9.3 in healthy postmenopausal women (p < 0.01). In ascitic fluid, levels were 1.02 ± 0.24 and 4.63 ± 1.57 μg/ml, respectively. The mean levels of TTR and RBP in serum showed a tendency to decrease with the severity of the disease and were lower in affected women whose CRP levels were > 40 mg/ml (p = 0.08 for TTR; p < 0.05 for RBP). No differences in TTR microheterogeneity were observed between TTR isolated from serum of affected and healthy women or from ascitic fluid. TTR occurred rather consistently in four variants. Mass signals were at 13758 ± 7, 13876 ± 13 (greatest intensity), 13924 ± 21 and 14062 ± 24 Da, representing native, S-cysteinylated, S-cysteinglycinylated and glutathionylated TTR, respectively. Serum of healthy and affected women as well as ascitic fluid contained the truncated fragment of TTR (12828 ± 11 Da). No immunoreactive TTR was observed in the tumor sites.ConclusionThe severity of the cancer associated catabolism as well as the inflammation status affect serum TTR and RBP levels. Neither TTR nor its truncated form originates from tumor tissue and its occurrence in ascites may well reflect the filtration from blood into ascitic fluid.

An Integrated Clinical-Genomics Approach Identifies a Candidate Multi-Analyte Blood Test for Serous Ovarian Carcinoma

Purpose: Cancer of the ovary confers the worst prognosis among women with gynecologic malignancies, underscoring the need to develop new biomarkers for detection of early disease, particularly those that can be readily monitored in the blood. Experimental Design: We developed an algorithm to identify secreted proteins encoded among ∼22,500 genes on commercial oligonucleotide arrays and applied it to gene expression profiles of 67 stage I to IV serous papillary carcinomas and 9 crudely enriched normal ovarian tissues, to identify putative diagnostic markers. ELISAs were used to validate increased levels of secreted proteins in patient sera encoded by genes with differentially high expression. Results: We identified 275 genes predicted to encode secreted proteins with increased/decreased expression in ovarian cancers (<0.5- or >2-fold, P < 0.001). The serum levels of four of these proteins (matrix metalloproteinase-7, osteopontin, secretory leukoprotease inhibitor, and kallikrein 10) were significantly elevated in a series of 67 independent patients with serous ovarian carcinomas compared with 67 healthy controls (P < 0.001, Wilcoxon rank sum test). Optimized support vector machine classifiers with as few as two of these markers (osteopontin or kallikrein 10/matrix metalloproteinase-7) in combination with CA-125 yielded sensitivity and specificity values ranging from 96% to 98.7% and 99.7% to 100%, respectively, with the ability to discern early-stage disease from normal, healthy controls. Conclusions: Our data suggest that this assay combination warrants further investigation as a multi-analyte diagnostic test for serous ovarian adenocarcinoma.

Topoisomerase IIα mRNA and protein expression in ovarian carcinoma: Correlation with clinicopathological factors and prognosis

Topoisomerase IIα (Top IIα) is a nuclear enzyme that plays a central role in DNA metabolism, and is a molecular target for a variety of chemotherapeutic agents. Top IIα has recently gained attention as a biomarker for therapy response and patient survival. In this study, we attempted to assess the feasibility of measuring Top IIα gene expression in RNA, isolated from archival formalin-fixed paraffin-embedded tissue specimens, which are used routinely in pathology laboratories. We have employed a new technique on the basis of magnetic particles’ separation and purification of nucleic acids, and evaluated both protein and mRNA expressions from the same routinely processed tissue blocks. We investigated the expression of Top IIα mRNA and protein by real-time reverse transcription polymerase chain reaction and immunohistochemistry, in a cohort of 133 primary ovarian carcinomas, and evaluated the association between Top IIα expression and clincopathological variables as well as patient outcome. Elevated Top IIα mRNA expression was observed in high-grade tumors (P=0.003) and advanced stage disease (P=0.011). In univariate Kaplan–Meier analysis, patients with higher expression of Top IIα nuclear protein had a significantly decreased overall survival (P=0.045). Interestingly, we detected cytoplasmic protein expression of Top IIα in a subset of samples. Cytoplasmic expression of Top IIα was associated with the expression of chromosomal region maintenance/exportin 1 (CRM1)—a nuclear export protein (P=0.008). Our study suggests that Top IIα overexpression is involved in the progression of ovarian cancer in a subset of the patients. Our results encourage the further evaluation of the prognostic and predictive values of Top IIα expression in ovarian carcinoma, which might help to assess the patients’ risk profile, and the planning of an individualized therapy.

Expression of multidrug resistance-associated protein 1 in invasive ovarian carcinoma: implication for prognosis

Aims:  Multidrug resistance is a major impediment in chemotherapeutic treatment of ovarian carcinoma patients. The aim of this study was to investigate the expression of multidrug resistance‐associated protein 1 (MRP1) and to assess the possible associations with clinicopathological variables and patient outcome in primary ovarian carcinoma.

Decreased IL-1 RA concentration in ascites is associated with a significant improvement in overall survival in ovarian cancer

BACKGROUND Cytokines play a major role in promoting the growth and metastatic spread of cancer cells. Interleukin-1 alpha and beta (IL-1) and IL-1 RA are known to be critically involved in carcinogenesis and in various solid tumors. There are limited data on expression of IL-1 alpha, beta and RA in serum and ascites in patients with advanced ovarian cancer. Objectives of this study were to investigate the level of IL-1 alpha, IL-1 beta and IL-1 RA in serum and ascites from patients with ovarian cancer and their impact on the prognosis. METHODS Fifty-three women with ovarian cancer (OC) (33 patients with primary OC and 20 with relapsed OC) and 50 women with benign gynaecological diseases as a control group (CG) were enrolled onto this prospective study. IL-1 alpha, beta and RA levels were analyzed in serum and ascites by ELISA technique. RESULTS The median age was 55 years (range 19-80) in the ovarian cancer group and 40 years (range 15-89) in the controls. The distribution of histological type of ovarian cancer was as follows: serous-papillary 43 (81.1%), 4 (7.5%) mucinous, 3 (5.7%) endometroid and 3 (5.7%) clear cell carcinoma. The concentrations of IL-1 beta and RA in ascites or peritoneal fluid were significantly increased in patients with OC in comparison to the CG, for both cytokines (p<0.0001); also the concentration of IL-1 RA in serum was increased in OC (p=0.003) vs. CG. An increased level of IL-1 beta in ascites correlated significantly with a poorer histopathological grading (p=0.038). IL-1 RA concentration in ascites was correlated with advanced FIGO stage (p=0.049) and the IL-1 RA serum level with ascites volume (< or =500 ml vs. >500 ml) (p=0.046). Patients with IL-1 RA level in ascites lower than the cut off value of 695.6 pg/ml showed a significant better progression-free median survival (24.6 vs. 12.8 months, p=0.008) and postoperative median overall survival (34.6 vs. 17 months, p=0.01) in comparison to patients with an IL-1 RA level in ascites higher than the cut off level. Additionally, a higher expression of IL-1 beta in serum (p=0.004) and ascites (p=0.05) reduced significantly the progression-free survival. In the multivariate analysis, expression of IL-1 RA in ascites was an independent prognostic factor for good progression-free and postoperative overall survival (HR, 0.39 95% CI, 0.18-0.83, p=0.01, HR, 0.36 95% CI, 0.16-0.8, p=0.01). CONCLUSIONS IL-1 RA levels in ascites lower than the cut off value of 695.6 pg/ml are associated with a significant improvement in postoperative and progression-free survival. IL-1 RA shows a prognostic relevance in ovarian cancer.

The cellular ratio of immune tolerance (immunoCRIT) is a definite marker for aggressiveness of solid tumors and may explain tumor dissemination patterns

The adaptive immune system is involved in tumor establishment and aggressiveness. Tumors of the ovaries, an immune-privileged organ, spread via transceolomic routes and rarely to distant organs. This is contrary to tumors of non-immune privileged organs, which often disseminate hematogenously to distant organs. Epigenetics-based immune cell quantification allows direct comparison of the immune status in benign and malignant tissues and in blood. Here, we introduce the “cellular ratio of immune tolerance” (immunoCRIT) as defined by the ratio of regulatory T cells to total T lymphocytes. The immunoCRIT was analyzed on 273 benign tissue samples of colorectal, bronchial, renal and ovarian origin as well as in 808 samples from primary colorectal, bronchial, mammary and ovarian cancers. ImmunoCRIT is strongly increased in all cancerous tissues and gradually augmented strictly dependent on tumor aggressiveness. In peripheral blood of ovarian cancer patients, immunoCRIT incrementally increases from primary diagnosis to disease recurrence, at which distant metastases frequently occur. We postulate that non-pathological immunoCRIT values observed in peripheral blood of immune privileged ovarian tumor patients are sufficient to prevent hematogenous spread at primary diagnosis. Contrarily, non-immune privileged tumors establish high immunoCRIT in an immunological environment equivalent to the bloodstream and thus spread hematogenously to distant organs. In summary, our data suggest that the immunoCRIT is a powerful marker for tumor aggressiveness and disease dissemination.