Identification and Characterization of a Novel Indoleamine 2,3-Dioxygenase 1 Protein Degrader for Glioblastoma.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunosuppressive enzyme that inhibits the antitumor immune response through both tryptophan metabolism and non-enzymatic functions. To date, most IDO1-targeted approaches have focused on inhibiting tryptophan metabolism. However, this class of drugs has failed to improve the overall survival of patients with cancer. Here, we developed and characterized proteolysis targeting chimeras (PROTACs) that degrade the IDO1 protein. IDO1-PROTACs were tested for their effects on IDO1 enzyme and non-enzyme activities. After screening a library of IDO1-PROTAC derivatives, a compound was identified that potently degraded the IDO1 protein through cereblon-mediated proteasomal degradation. The IDO1-PROTAC: (i) inhibited IDO1 enzyme activity and IDO1-mediated NF-κB phosphorylation in cultured human glioblastoma (GBM) cells, (ii) degraded the IDO1 protein within intracranial brain tumors in vivo, and (iii) mediated a survival benefit in mice with well-established brain tumors. This study identified and characterized a new IDO1 protein degrader with therapeutic potential for patients with glioblastoma.

Translational randomized phase II trial of cabozantinib in combination with nivolumab in advanced, recurrent, or metastatic endometrial cancer.

BACKGROUND: Combining immunotherapy and antiangiogenic agents is a promising treatment strategy in endometrial cancer. To date, no biomarkers for response have been identified and data on post-immunotherapy progression are lacking. We explored the combination of a checkpoint inhibitor (nivolumab) and an antiangiogenic agent (cabozantinib) in immunotherapy-naïve endometrial cancer and in patients whose disease progressed on previous immunotherapy with baseline biopsy for immune profiling. PATIENTS AND METHODS: In this phase II trial (ClinicalTrials.gov NCT03367741, registered December 11, 2017), women with recurrent endometrial cancer were randomized 2:1 to nivolumab with cabozantinib (Arm A) or nivolumab alone (Arm B). The primary endpoint was Response Evaluation Criteria in Solid Tumors-defined progression-free survival (PFS). Patients with carcinosarcoma or prior immune checkpoint inhibitor received combination treatment (Arm C). Baseline biopsy and serial peripheral blood mononuclear cell (PBMC) samples were analyzed and associations between patient outcome and immune data from cytometry by time of flight (CyTOF) and PBMCs were explored. RESULTS: Median PFS was 5.3 (90% CI 3.5 to 9.2) months in Arm A (n=36) and 1.9 (90% CI 1.6 to 3.4) months in Arm B (n=18) (HR=0.59, 90% CI 0.35 to 0.98; log-rank p=0.09, meeting the prespecified statistical significance criteria). The most common treatment-related adverse events in Arm A were diarrhea (50%) and elevated liver enzymes (aspartate aminotransferase 47%, alanine aminotransferase 42%). In-depth baseline CyTOF analysis across treatment arms (n=40) identified 35 immune-cell subsets. Among immunotherapy-pretreated patients in Arm C, non-progressors had significantly higher proportions of activated tissue-resident (CD103+CD69+) ɣδ T cells than progressors (adjusted p=0.009). CONCLUSIONS: Adding cabozantinib to nivolumab significantly improved outcomes in heavily pretreated endometrial cancer. A subgroup of immunotherapy-pretreated patients identified by baseline immune profile and potentially benefiting from combination with antiangiogenics requires further investigation.

Phase I, Pharmacogenomic, Drug Interaction Study of Sorafenib and Bevacizumab in Combination with Paclitaxel in Patients with Advanced Refractory Solid Tumors.

VEGF blockade does not uniformly result in clinical benefit. We evaluated safety, dose-limiting toxicities (DLT), recommended phase II dose (RP2D), antitumor efficacy, and exploratory biomarkers including pharmacogenomics and pharmacokinetics with sorafenib, bevacizumab, and paclitaxel in patients with refractory cancers. The study had a "3 + 3" design, using paclitaxel 80 mg/m2 every week for 3 weeks, in every 4 week cycles, bevacizumab 5 mg/kg every 2 weeks, and sorafenib 200 or 400 mg twice a day, 5 or 7 days/week (5/7, 7/7). The MTD cohort was expanded. Twenty-seven patients enrolled in 3 cohorts: sorafenib 200 mg twice a day 5/7, 200 mg twice a day 7/7, and 400 mg twice a day 5/7. DLTs were grade 3 neutropenia >7 days (cohort 1, 1), grade 3 hypertension (cohort 2, 1), grade 3 hand-foot skin reaction (HFSR; cohort 3, 2). MTD was sorafenib 200 mg twice a day 7/7. Six DLTs occurred in cohort 2 expansion: grade 3 HFSR (2), grade 2 HFSR with sorafenib delay >7 days (2), grade 4 cerebrovascular accident (1), grade 3 neutropenia >7 days (1). RP2D was sorafenib 200 mg twice a day 5/7. Most patients (62%) dose reduced sorafenib to 200 mg daily 5/7 after a median 3 (range, 2-17) cycles. Response rates were 48% overall (27) and 64% for ovarian cancers (14). VEGF-A-1154AA and -7TT recessive homozygous genotypes conferred worse overall survival versus alternative genotypes (7 vs. 22 months). Intermittent, low-dose sorafenib (200 mg twice a day 5/7) combined with bevacizumab and paclitaxel was tolerable and had high antitumor efficacy in patients with refractory cancer (NCT00572078).

CD73: an emerging checkpoint for cancer immunotherapy.

CD73 is a novel immune checkpoint associated with adenosine metabolism that promotes tumor progression by suppressing antitumor immune response and promoting angiogenesis. The inhibition of CD73, in combination with immune checkpoint blockade, targeted therapy or conventional therapy, improves antitumor effects in numerous preclinical mouse models of cancer. Emerging evidence suggests that the combination of anti-CD73 and immune checkpoint blockade has promising clinical activity in patients with advanced solid tumors. In this review, we will discuss the specific role of CD73 on both tumor cells and nontumor cells in regulating tumor immunity and tumorigenesis and provide an update on the current view of the antitumor activity of targeting CD73 by mAb or small molecule selective inhibitors in preclinical and clinical settings.

Niraparib monotherapy for late-line treatment of ovarian cancer (QUADRA): a multicentre, open-label, single-arm, phase 2 trial.

BACKGROUND: Late-line treatment options for patients with ovarian cancer are few, with the proportion of patients achieving an overall response typically less than 10%, and median overall survival after third-line therapy of 5-9 months. In this study (QUADRA), we investigated the activity of niraparib monotherapy as the fourth or later line of therapy. METHODS: QUADRA was a multicentre, open-label, single-arm, phase 2 study that evaluated the safety and activity of niraparib in adult patients (≥18 years) with relapsed, high-grade serous (grade 2 or 3) epithelial ovarian, fallopian tube, or primary peritoneal cancer who had been treated with three or more previous chemotherapy regimens. The study was done in the USA and Canada, and 56 sites screened patients (50 sites treated at least one patient). Patients received oral niraparib 300 mg once daily continuously, beginning on day 1 and every cycle (28 days) thereafter until disease progression. The primary objective was the proportion of patients achieving an investigator-assessed confirmed overall response in patients with homologous recombination deficiency (HRD)-positive tumours (including patients with BRCA and without BRCA mutations) sensitive to their last platinum-based therapy who had received three or four previous anticancer therapy regimens (primary efficacy population). Efficacy analyses were additionally done in all dosed patients with measurable disease at baseline. FINDINGS: Between April 1, 2015 and Nov 1, 2017, we screened 729 patients for eligibility and enrolled 463 patients, who were initiated on niraparib therapy. At the time of database lock (April 11, 2018), enrolment had closed and the study was ongoing, with 21 patients still on treatment. Patients had received a median of four (IQR 3-5) previous lines of therapy, and the median follow-up for overall survival was 12·2 months (IQR 3·7-22·1). 151 (33%) of 463 patients were resistant and 161 (35%) of 463 patients were refractory to the last administered platinum therapy. 13 (28%) of 47 patients in the primary efficacy population achieved an overall response according to RECIST (95% CI 15·6-42·6; one-sided p=0·00053). The most common drug-related grade 3 or worse treatment-emergent adverse events were anaemia (113 [24%] of 463 patients) and thrombocytopenia (95 [21%] of 463 patients). The most common treatment-emergent serious adverse events were small intestinal obstruction (34 [7%] of 463 patients), thrombocytopenia (34 [7%] of 463 patients), and vomiting (27 [6%] of 463 patients). One death due to gastric haemorrhage was considered treatment related. INTERPRETATION: We observed clinically relevant activity of niraparib among women with heavily pretreated ovarian cancer, especially in patients with HRD-positive platinum-sensitive disease, which includes not only patients with a BRCA mutation but also a population with BRCA wild-type disease. We identified no new safety signals. Our data support expansion of the treatment indication for poly(ADP-ribose) polymerase inhibitors to include patients with HRD-positive ovarian cancer beyond those with BRCA mutations. FUNDING: Tesaro.

Association of chemotherapy and radiotherapy sequence with overall survival in locoregionally advanced endometrial cancer.

OBJECTIVE: The optimal adjuvant management of women with FIGO Stage III-IVA endometrial cancer (EC) is unclear. While recent prospective data suggest that treatment with pelvic radiotherapy (RT) prior to chemotherapy (CT) is not associated with a survival benefit compared to CT alone, no prospective randomized trial has included a treatment arm in which CT is given before RT. METHODS: An observational cohort study was performed on women with FIGO Stage III-IVA Type 1 (grade 1-2, endometrioid) EC who underwent hysterectomy and received multi-agent CT and/or RT from 2004 to 2014 at Commission on Cancer-accredited hospitals. Multivariable parametric accelerated failure time models were performed to estimate the association of sequence of adjuvant CT and RT with overall survival (OS) using propensity score-adjusted matched cohorts. RESULTS: Of 5795 women identified, 1260 (21.7%) received RT only, 2465 (42.5%) received CT only, 593 (9.7%) received RT before CT, and 1506 (26.0%) received RT after CT. Women who received RT after CT experienced significantly longer 5-year OS than women who received RT before CT (5-year OS: 80.1% vs 73.3%; time-ratio (TR) = 1.37, 95% CI = 1.18-1.58, P < 0.001), CT only (68.9%; TR = 1.33, 95% CI = 1.19-1.48, P < 0.001), or RT only (64.5%, TR = 1.50, 95% CI = 1.32-1.70, P < 0.001). CONCLUSIONS: For women with advanced EC, treatment with multi-agent CT followed by RT is associated with longer OS compared with treatment with RT followed by CT or either treatment alone. These hypothesis-generating data support inclusion in future prospective trials of regimens in which multi-agent CT starts prior to RT.

siRNA Delivery Using Dithiocarbamate-Anchored Oligonucleotides on Gold Nanorods.

We present a robust method for loading small interfering RNA (siRNA) duplexes onto the surfaces of gold nanorods (GNRs) at high density, using near-infrared laser irradiation to trigger their intracellular release with subsequent knockdown activity. Citrate-stabilized GNRs were first coated with oleylsulfobetaine, a zwitterionic amphiphile with low cytotoxicity, which produced stable dispersions at high ionic strength. Amine-modified siRNA duplexes were converted into dithiocarbamate (DTC) ligands and adsorbed onto GNR surfaces in a single incubation step at 0.5 M NaCl, simplifying the charge screening process. The DTC anchors were effective at minimizing premature siRNA desorption and release, a common but often overlooked problem in the use of gold nanoparticles as oligonucleotide carriers. The activity of GNR-siRNA complexes was evaluated systematically against an eGFP-producing ovarian cancer cell line (SKOV-3) using folate receptor-mediated uptake. Efficient knockdown was achieved by using a femtosecond-pulsed laser source to release DTC-anchored siRNA, with essentially no contributions from spontaneous (dark) RNA desorption. GNRs coated with thiol-anchored siRNA duplexes were less effective and also permitted low levels of knockdown activity without photothermal activation. Optimized siRNA delivery conditions were applied toward the targeted knockdown of transglutaminase 2, whose expression is associated with the progression of recurrent ovarian cancer, with a reduction in activity of >80% achieved after a single pulsed laser treatment.

Concurrent Detection of Cellular and Molecular Cancer Markers Using an Immunomagnetic Flow System.

We report a detection system for simultaneous measurement of cellular and molecular markers of cancer. Magnetic beads conjugated with antibodies against a specific antigen are used to capture both free molecules and whole cells overexpressing the antigen. The target-bound beads then flow through a microfluidic chamber where they are drawn to a glass surface by an external magnetic field. The cells and molecules captured on the surface are quantitatively analyzed using fluorescent microscopy. The system was characterized by detecting free folate receptor (FR) and an FR+ cancer cell line (KB) in culture media. The system detected as low as 10 pM of FR and captured 87% of the spiked KB cells at a volumetric throughput of 3 mL/min. We further demonstrated the detection of 100 KB cells and 200 pM FR spiked into healthy human blood to simulate detection of rare cells and protein biomarkers present in a cancer patient's blood sample. The FR concentration was measured to be 244 pM (including the intrinsic FR present in the blood), and the total number of KB cells in the sample was estimated to be 98. The potential of this approach in clinical diagnostics was also demonstrated by detecting both FR+ cells and free FR in an ascites sample obtained from an ovarian cancer patient. Because of the system's capability to detect multiple targets at the same time, its high throughput, and its overall simplicity, we expect it to be highly useful in a wide range of research settings.

Circulating tumor cell detection using a parallel flow micro-aperture chip system.

We report on-chip isolation and detection of circulating tumor cells (CTCs) from blood samples using a system that integrates a microchip with immunomagnetics, high-throughput fluidics and size-based filtration. CTCs in a sample are targeted via their surface antigens using magnetic beads functionalized with antibodies. The mixture is then run through a fluidic chamber that contains a micro-fabricated chip with arrays of 8 µm diameter apertures. The fluid runs parallel to the microchip while a magnetic field is generated underneath to draw the beads and cells bound to them toward the chip surface for detection of CTCs that are larger than the apertures and clear out free beads and other smaller particles bound to them. The parallel flow configuration allows high volumetric flow rates, which reduces nonspecific binding to the chip surface and enables multiple circulations of the sample fluid through the system in a short period of time. In this study we first present models of the magnetic and fluidic forces in the system using a finite element method. We then verify the simulation results experimentally to determine an optimal flow rate. Next, we characterize the system by detecting cancer cell lines spiked into healthy human blood and show that on average 89% of the spiked MCF-7 breast cancer cells were detected. We finally demonstrate detection of CTCs in 49 out of 50 blood samples obtained from non-small cell lung cancer (NSCLC) patients and pancreatic cancer (PANC) patients. The number of CTCs detected ranges from 2 to 122 per 8 mL s of blood. We also demonstrate a statistically significant difference between the CTC counts of NSCLC patients who have received therapy and those who have not.

Endoglin for targeted cancer treatment.

Endoglin is a homodimeric cell membrane glycoprotein receptor for transforming growth factor ß and bone morphogenetic proteins. Endoglin is essential for angiogenesis, being densely expressed on proliferating endothelial cells and upregulated during hypoxia. Its expression is implicated in development of resistance to vascular endothelial growth factor (VEGF) inhibition. TRC105 is an antibody that binds endoglin and prevents endothelial cell activation. Targeting endoglin and the VEGF pathway concurrently improves treatment in vitro and appears to reverse resistance to bevacizumab in some refractory cancer patients. Randomized trials are under way to assess the clinical benefit of adding TRC105 therapy to bevacizumab therapy. Further trials are under way to assess the activity of TRC105 with small-molecule inhibitors of the VEGF pathway in renal cell carcinoma, hepatocellular carcinoma, and soft tissue sarcoma. Stratification of soft tissue sarcomas based on endoglin expression levels is proposed to identify patients most likely to benefit from TRC105 treatment. The development of a TRC105 antibody-drug conjugate is also described.

N-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients.

Glycosylated proteins play important roles in a broad spectrum of biochemical and biological processes, and prior reports have suggested that changes in protein glycosylation occur during cancer initiation and progression. Ovarian cancer (OC) is a fatal malignancy, most commonly diagnosed after the development of metastases. Therefore, early detection of OC is key to improving survival. To this end, specific changes of the serum glycome have been proposed as possible biomarkers for different types of cancers. In this study, we extend this concept to OC. To characterize differences in total N-glycan levels, serum samples provided by 20 healthy control women were compared to those acquired from patients diagnosed with late-stage recurrent OC who were enrolled in an experimental treatment trial prior to receiving therapy (N=19) and one month later, prior to the second treatment cycle (N=11). Additionally, analyses of the N-glycans associated with IgG and characterization of the relative abundance levels of core vs outer-arm fucosylation were also performed. The N-linked glycomic profiles revealed increased abundances of tri- and tetra-branched structures with varying degrees of sialylation and fucosylation and an apparent decrease in the levels of "bisecting" glycans in OC samples compared to controls. Increased levels of a-galactosylation structures were observed on N-linked glycans derived from IgG, which were independent of the presence of fucose residues. Elevated levels of outer-arm fucosylation were also identified in the OC samples. These results allowed the control samples to be distinguished from the baseline ovarian cancer patients prior to receiving the experimental treatment. In some cases, the pre-treatment samples could be distinguished from the post-experimental treatment samples, as many of those patients showed a further progression of the disease.

A phase 1 and pharmacodynamic study of decitabine in combination with carboplatin in patients with recurrent, platinum-resistant, epithelial ovarian cancer.

BACKGROUND: Aberrant DNA methylation is a hallmark of cancer, and DNA methyltransferase inhibitors have demonstrated clinical efficacy in hematologic malignancies. On the basis of preclinical studies indicating that hypomethylating agents can reverse platinum resistance in ovarian cancer cells, the authors conducted a phase 1 trial of low-dose decitabine combined with carboplatin in patients with recurrent, platinum-resistant ovarian cancer. METHODS: Decitabine was administered intravenously daily for 5 days, before carboplatin (area under the curve, 5) on Day 8 of a 28-day cycle. By using a standard 3 + 3 dose escalation, decitabine was tested at 2 dose levels: 10 mg/m(2) (7 patients) or 20 mg/m(2) (3 patients). Peripheral blood mononuclear cells (PBMCs) and plasma collected on Days 1 (pretreatment), 5, 8, and 15 were used to assess global (LINE-1 repetitive element) and gene-specific DNA methylation. RESULTS: Dose-limiting toxicity (DLT) at the 20-mg/m(2) dose was grade 4 neutropenia (2 patients), and no DLTs were observed at 10 mg/m(2). The most common toxicities were nausea, allergic reactions, neutropenia, fatigue, anorexia, vomiting, and abdominal pain, the majority being grades 1-2. One complete response was observed, and 3 additional patients had stable disease for >/=6 months. LINE-1 hypomethylation on Days 8 and 15 was detected in DNA from PBMCs. Of 5 ovarian cancer-associated methylated genes, HOXA11 and BRCA1 were demethylated in plasma on Days 8 and 15. CONCLUSIONS: Repetitive low-dose decitabine is tolerated when combined with carboplatin in ovarian cancer patients, and demonstrates biological (ie, DNA-hypomethylating) activity, justifying further testing for clinical efficacy. Cancer 2010. (c) 2010 American Cancer Society.

Role of epigenomics in ovarian and endometrial cancers.

Ovarian cancer is the most lethal gynecologic malignancy and while constituting only 3% of all female cancers, it causes 14,600 deaths in the USA annually. Endometrial cancer, the most diagnosed and second-most fatal gynecologic cancer, afflicts over 40,000 US women annually, causing an estimated 7780 deaths in 2009. In both advanced ovarian and endometrial carcinomas, the majority of initially therapy-responsive tumors eventually evolve to a fully drug-resistant phenotype. In addition to genetic mutations, epigenetic anomalies are frequent in both gynecologic malignancies, including aberrant DNA methylation, atypical histone modifications and dysregulated expression of distinct microRNAs, resulting in altered gene-expression patterns favoring cell survival. In this article, we summarize the most recent hypotheses regarding the role of epigenetics in ovarian and endometrial cancers, including a possible role in tumor 'stemness' and also evaluate the possible therapeutic benefits of reversal of these oncogenic chromatin aberrations.

Epigenetic therapies for chemoresensitization of epithelial ovarian cancer.

Epigenetic drugs have been shown to enhance gene expression and drug sensitivity in ovarian cancer cell lines and animal models. Based on promising preclinical studies, DNA methylation inhibitors in combination with existing chemotherapeutic agents have the potential for overcoming acquired drug resistance, laying the foundation for this specific class of epigenetic drug in ovarian cancer clinical trials. The recent completion of phase I trials of decitabine has yielded important information on dosing schedules and biological endpoints for evaluating patient responses. In addition, epigenetic drug effects on pharmacodyamic targets are beginning to emerge, and predictive epigenetic biomarkers and next generation epigenome therapeutics are being developed for application in clinical settings for ovarian cancer patients.

Minireview: epigenetic changes in ovarian cancer.

Epigenetic aberrations, including DNA methylation, histone modifications, and micro-RNA dysregulation, are now well established in the development and progression of ovarian cancer, and their gradual accumulation is associated with advancing disease stage and grade. Epigenetic aberrations are relatively stable, associated with distinct disease subtypes, and present in circulating serum, representing promising diagnostic, prognostic, and pharmacodynamic biomarkers. In contrast to DNA mutations and deletions, aberrant gene-repressive epigenetic modifications are potentially reversible by epigenetic therapies, including inhibitors of DNA methylation or histone-modifying enzymes. Although epigenetic monotherapies have not shown activity against solid tumors, including ovarian cancer, preclinical studies suggest they will be effective when used in combination with one another or with conventional chemotherapeutics, and combinatorial epigenetic therapy regiments are being examined in cancer clinical trials. A greater understanding of the role of epigenetics in ovarian neoplasia will provide for improved interventions against this devastating malignancy.

A phase I study of lapatinib in combination with carboplatin in women with platinum sensitive recurrent ovarian carcinoma.

OBJECTIVES: To determine the maximum tolerated dose (MTD), spectrum of toxicities, clinical activity, and pharmacokinetics of carboplatin given in combination with lapatinib in women with a first recurrence of platinum sensitive epithelial ovarian carcinoma. METHODS: Patients with measurable, platinum sensitive recurrent epithelial ovarian carcinoma were eligible. Cohorts of 3-6 patients were to receive up to 6 cycles of intravenous carboplatin AUC of 6 every 21 days in combination with escalating dosages of oral lapatinib (starting at a dose of 750 mg daily). Toxicity was assessed using NCI CTC for Adverse Events. Clinical response was monitored using RECIST criteria. Pharmacokinetic (PK) analysis was performed for the second cohort of patients. RESULTS: Twelve patients were enrolled. No dose limiting toxicity was noted. Two of 6 patients in the first cohort had unanticipated excessive delays in treatment due to non-dose limiting G3 neutropenia. Therefore, the study was modified to reduce the carboplatin dose in the second cohort. The median number of courses administered to the 11 evaluable patients in these two cohorts was 2.8 (range 1-6). Drug-related grade 3 or 4 toxicities included non-dose limiting G4 thrombocytopenia (n=1), and non-dose limiting G3 neutropenia (n=3). Of the 11 patients who received >or=1 course of therapy, 3 (27%) had a partial response, and 3 (27%) had stable disease. The pharmacokinetics of carboplatin were not significantly altered by concomitant administration of lapatinib. CONCLUSIONS: This regimen of lapatinib and carboplatin was associated with unacceptable non-dose limiting toxicities, excessive treatment delays and limited clinical responses.

Hypermethylation of 18S and 28S ribosomal DNAs predicts progression-free survival in patients with ovarian cancer.

PURPOSE: Repetitive ribosomal DNA (rDNA) genes are GC-rich clusters in the human genome. The aim of the study was to determine the methylation status of two rDNA subunits, the 18S and 28S genes, in ovarian tumors and to correlate methylation levels with clinicopathologic features in a cohort of ovarian cancer patients. EXPERIMENTAL DESIGN: 18S and 28S rDNA methylation was examined by quantitative methylation-specific PCR in 74 late-stage ovarian cancers, 9 histologically uninvolved, and 11 normal ovarian surface epithelial samples. In addition, methylation and gene expression levels of 18S and 28S rDNAs in two ovarian cancer cell lines were examined by reverse transcription-PCR before and after treatment with the demethylating drug 5'-aza-2'-deoxycytidine. RESULTS: The methylation level (amount of methylated rDNA/beta-actin) of 18S and 28S rDNAs was significantly higher (P < 0.05) in tumors than in normal ovarian surface epithelial samples. Methylation of 18S and 28S rDNA was highly correlated (R2= 0.842). Multivariate analysis by Cox regression found that rDNA hypermethylation [hazard ratio (HR), 0.25; P < 0.01], but not age (HR, 1.29; P = 0.291) and stage (HR, 1.09; P = 0.709), was independently associated with longer progression-free survival. In ovarian cancer cell lines, methylation levels of rDNA correlated with gene down-regulation and 5'-aza-2'-deoxycytidine treatment resulted in a moderate increase in 18S and 28S rDNA gene expressions. CONCLUSION: This is the first report of rDNA hypermethylation in ovarian tumors. Furthermore, rDNA methylation levels were higher in patients with long progression-free survival versus patients with short survival. Thus, rDNA methylation as a prognostic marker in ovarian cancer warrants further investigation.