Alexander Mustea

Quantitative DNA Methylation Analysis of FOXP3 as a New Method for Counting Regulatory T Cells in Peripheral Blood and Solid Tissue

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.

Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes

Exosomes are membrane vesicles that are released from many different cell types. Tumor derived-exosomes play a role in immune suppression. We hypothesized that in ovarian carcinoma patients exosomes initially produced at the local abdominal site may become systemic. We examined paired samples of ascites and blood from ovarian carcinoma patients for the presence of exosomes. We also studied the requirements for exosomal uptake by immune cells, the role of phosphatidyl-serine (PS) as uptake signal and the effect of exosome application on tumor growth. We used exosomes from ovarian carcinoma cell lines, malignant ascites and sera from ovarian carcinoma patients isolated by ultracentrifugation. PS-displayed by exosomes was detected by Anexin-V-FITC staining of latex beads adsorbed exosomes. For uptake experiments, labeled exosomes were exposed to cells in the presence or absence of cold Annexin-V as competitor. Uptake was examined by fluorescent microscopy and cytofluorographic analysis. Effects of exosomes on tumor growth were studied using SKOV3ip ovarian carcinoma cells in CD1 nu/nu mice. We found that malignant ascites-derived exosomes cargo tumor progression related proteins such as L1CAM, CD24, ADAM10, and EMMPRIN. We observed that exosomes become systemic via the blood stream. Uptake of ovarian carcinoma exosomes by NK cells was found to require PS at the exosomal surface but the presence of PS was not sufficient. Application of malignant ascites-derived exosomes to tumor bearing mice resulted in augmented tumor growth. Exosomes from the serum of tumor patients could be isolated from only one ml of blood and this analysis could serve for diagnostic purposes. We propose that tumor-derived exosomes could play a role in tumor progression.

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.

Epigenetic quantification of tumor-infiltrating T-lymphocytes

The immune system plays a pivotal role in tumor establishment. However, the role of T-lymphocytes within the tumor microenvironment as major cellular component of the adaptive effector immune response and their counterpart, regulatory T-cells (Treg), responsible for suppressive immune modulation, is not completely understood. This is partly due to the lack of reliable technical solutions for specific cell quantification in solid tissues. Previous reports indicated that epigenetic marks of immune cells, such as the Treg specifically demethylated region (TSDR) within the FOXP3 gene, may be exploited as robust analytical tool for Treg-quantification. Here, we expand the concept of epigenetic immunophenotyping to overall T-lymphocytes (oTL). This tool allows cell quantification with at least equivalent precision to FACS and is adoptable for analysis of blood and solid tissues. Based on this method, we analyse the frequency of Treg, oTL and their ratio in independent cohorts of healthy and tumorous ovarian, colorectal and bronchial tissues with 616 partly donor-matched samples. We find a shift of the median ratio of Treg-to-oTL from 3-8% in healthy tissue to 18-25% in all tumor entities. Epigenetically determined oTL frequencies correlate with the outcome of colorectal and ovarian cancers. Together, our data show that the composition of immune cells in tumor microenvironments can be quantitatively assessed by epigenetic measurements. This composition is disturbed in solid tumors, indicating a fundamental mechanism of tumor immune evasion. Epigenetic quantification of T-lymphocytes serves as independent clinical parameter for outcome prognosis.

KRAS mutation analysis in ovarian samples using a high sensitivity biochip assay

BackgroundMutations in the KRAS gene are one of the most frequent genetic abnormalities in ovarian carcinoma. They are of renewed interest as new epidermal growth factor receptor (EGFR)-targeted therapies are being investigated for use in ovarian carcinoma. As KRAS mutations are associated with poor response and resistance to EGFR-targeting drugs, this study was conducted to obtain more information on the spectrum of KRAS mutations in ovarian carcinoma.MethodsThe presence of KRAS mutations in codon 12 and 13 was analyzed in frozen and formalin-fixed paraffin-embedded (FFPE) tissue with a low density biochip platform. 381 malignant (29 borderline malignancy, 270 primary carcinomas, and 82 recurrent carcinomas) and 22 benign tissue samples from a total of 394 patients were examined. KRAS mutational status of each sample was correlated with dignity, FIGO stage, grade, histology, and survival.ResultsKRAS mutations were found in 60 (15%) samples with 58 samples deriving from malignant tissue and 2 samples deriving from benign tissue. In 55 (92%) samples codon 12 was found to be mutated. Frozen and FFPE samples concurred with respect to KRAS mutation status.ConclusionKRAS mutation is a common event in ovarian cancer primarily in carcinomas of lower grade, lower FIGO stage, and mucinous histotype. The KRAS mutational status is no prognostic factor for patients treated with standard therapy. However, in line with experience from colorectal cancer and non-small-cell-lung cancer (NSCLC), it may be important for prediction of response to EGFR-targeted therapies.

Adjuvant Therapy in Lymph Node–Positive Vulvar Cancer: The AGO-CaRE-1 Study

Background: Women with node-positive vulvar cancer have a high risk for disease recurrence. Indication criteria for adjuvant radiotherapy are controversial. This study was designed to further understand the role of adjuvant therapy in node-positive disease. Methods: Patients with primary squamous-cell vulvar cancer treated at 29 gynecologic cancer centers in Germany from 1998 through 2008 were included in this retrospective exploratory multicenter cohort study. Of 1618 documented patients, 1249 had surgical groin staging and known lymph node status and were further analyzed. All statistical tests were two-sided. Results: Four hundred forty-seven of 1249 patients (35.8%) had lymph node metastases (N+). The majority of N+ patients had one (172 [38.5%]) or two (102 [22.8%]) positive nodes. The three-year progression-free survival (PFS) rate of N+ patients was 35.2%, and the overall survival (OS) rate 56.2% compared with 75.2% and 90.2% in node-negative patients (N-). Two hundred forty-four (54.6%) N+ patients had adjuvant therapy, of which 183 (40.9%) had radiotherapy directed at the groins (+/-other fields). Three-year PFS and OS rates in these patients were better compared with N+ patients without adjuvant treatment (PFS: 39.6% vs 25.9%, hazard ratio [HR] = 0.67, 95% confidence interval [CI[= 0.51 to 0.88, P = .004; OS: 57.7% vs 51.4%, HR = 0.79, 95% CI = 0.56 to 1.11, P = .17). This effect was statistically significant in multivariable analysis adjusted for age, Eastern Cooperative Oncology Group, Union internationale contre le cancer stage, grade, invasion depth, and number of positive nodes (PFS: HR = 0.58, 95% CI = 0.43 to 0.78, P < .001; OS: HR = 0.63, 95% CI = 0.43 to 0.91, P = .01). Conclusion: This large multicenter study in vulvar cancer observed that adjuvant radiotherapy was associated with improved prognosis in node-positive patients and will hopefully help to overcome concerns regarding adjuvant treatment. However, outcome after adjuvant radiotherapy remains poor compared with node-negative patients. Adjuvant chemoradiation could be a possible strategy to improve therapy because it is superior to radiotherapy alone in other squamous cell carcinomas.

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.

Epigenetic silencing of argininosuccinate synthetase confers resistance to platinum-induced cell death but collateral sensitivity to arginine auxotrophy in ovarian cancer

Evidence indicates that acquired resistance of cancers to chemotherapeutic agents can occur via epigenetic mechanisms. Down‐regulation of expression of argininosuccinate synthetase (ASS1), the rate‐limiting enzyme in the biosynthesis of arginine, has been associated with the development of platinum resistance in ovarian cancer treated with platinum‐based chemotherapy. The aim of the present study was to analyse epigenetic regulation of ASS1 in ovarian cancer tissue taken at diagnosis and relapse and determine its significance as a predictor of clinical outcome in patients treated with platinum‐based chemotherapy. In addition, expression and epigenetic regulation of ASS1 were analysed in human ovarian cancer cell lines, and ASS1 expression correlated with the ability of the lines to grow in media containing cisplatin, carboplatin or taxol or in arginine‐depleted media. Our results show that aberrant methylation in the ASS1 promoter correlated with transcriptional silencing in ovarian cancer cell lines. ASS1 silencing conferred selective resistance to platinum‐based drugs and conferred arginine auxotrophy and sensitivity to arginine deprivation. In ovarian cancer, ASS1 methylation at diagnosis was associated with significantly reduced overall survival (p = 0.01) and relapse‐free survival (p = 0.01). In patients who relapse, ASS1 methylation was significantly more frequent at relapse (p = 0.008). These data establish epigenetic inactivation of ASS1 as a determinant of response to platinum chemotherapy and imply that transcriptional silencing of ASS1 contributes to treatment failure and clinical relapse in ovarian cancer. The collateral sensitivity of cells lacking endogenous ASS1 to arginine depletion suggests novel therapeutic strategies for the management of relapsed ovarian cancer.

Polo-like Kinase Plk2 Is an Epigenetic Determinant of Chemosensitivity and Clinical Outcomes in Ovarian Cancer

Resistance to platinum- and taxane-based chemotherapy remains a major clinical impediment to effective management of epithelial ovarian cancer (EOC). To gain insights into resistance mechanisms, we compared gene and confirmed expression patterns of novel EOC cell lines selected for paclitaxel and carboplatin resistance. Here, we report that resistance can be conferred by downregulation of the Polo-like kinase Plk2. Mechanistic investigations revealed that downregulation occurred at the level of transcription via associated DNA methylation of the CpG island in the Plk2 gene promoter in cell lines, primary tumors, and patient sera. Inhibitory RNA (RNAi)-mediated knockdown and ectopic overexpression established a critical functional role for Plk2 in determining apoptotic sensitivity to paclitaxel and carboplatin. In drug-resistant human EOC cell lines, Plk2 promoter methylation varied with the degree of drug resistance and transcriptional silencing of the promoter. RNAi-dependent knockdown of Plk2 abrogated G(2)-M cell-cycle blockade by paclitaxel, conferring resistance to both paclitaxel and platinum. Conversely, ectopic expression of Plk2 restored sensitivity to G(2)-M cell-cycle blockade and cytotoxicity triggered by paclitaxel. In clinical cases, DNA methylation of the Plk2 CpG island in tumor tissue was associated with a higher risk of relapse in patients treated postoperatively with carboplatin and paclitaxel (P = 0.003). This trend was also reflected in the analysis of matched serum samples. Taken together, our results implicate Plk2 as a clinically important determinant of chemosensitivity, in support of the candidacy of Plk2 as a theranostic marker to inform EOC management.

Monitoring of IL-10 in the serum of patients with advanced ovarian cancer: Results from a prospective pilot-study

OBJECTIVES The fact that ovarian cancer remains confined to the peritoneal cavity even in advanced stages has allowed us to surmise that local immunosuppressive factors could be involved in the tumor biology of ovarian cancer. In this context, IL-10 can be one of the key factors. By studying the kinetics of IL-10 concentrations prior to and after surgery, this study attempts to reveal once more the ability of tumor micro-environment to produce IL-10. Some studies indicate that IL-10 concentration correlates with the tumor burden and can thus predict the surgical outcome. Data concerning this aim from patients with ovarian cancer do not seem to exist. METHODS In this prospective study, serum blood was collected from 27 patients, one day prior to surgery as well as 24h, 4 and 8 days after surgery. The concentration of IL-10 was determined using ELISA. RESULTS While IL-10 levels rise within the first day post-OP, they are found to be reduced significantly when measured at later time points. IL-10 levels also correlate statistically significantly with the tumor grade, with lower IL-10 levels observed in well-differentiated and higher IL-10 levels in undifferentiated or only poorly differentiated tumors. CONCLUSION IL-10 expression levels appear to be a good surrogate marker for tumor grading. If validated, this may in future contribute to the understanding of the biology stage cancers.