Genes associated with bowel metastases in ovarian cancer.

OBJECTIVE: This study is designed to identify genes and pathways that could promote metastasis to the bowel in high-grade serous ovarian cancer (OC) and evaluate their associations with clinical outcomes. METHODS: We performed RNA sequencing of OC primary tumors (PTs) and their corresponding bowel metastases (n = 21 discovery set; n = 18 replication set). Differentially expressed genes (DEGs) were those expressed at least 2-fold higher in bowel metastases (BMets) than PTs in at least 30% of patients (P < .05) with no increased expression in paired benign bowel tissue and were validated with quantitative reverse transcription PCR. Using an independent OC cohort (n = 333), associations between DEGs in PTs and surgical and clinical outcomes were performed. Immunohistochemistry and mouse xenograft studies were performed to confirm the role of LRRC15 in promoting metastasis. RESULTS: Among 27 DEGs in the discovery set, 21 were confirmed in the replication set: SFRP2, Col11A1, LRRC15, ADAM12, ADAMTS12, MFAP5, LUM, PLPP4, FAP, POSTN, GRP, MMP11, MMP13, C1QTNF3, EPYC, DIO2, KCNA1, NETO1, NTM, MYH13, and PVALB. Higher expression of more than half of the genes in the PT was associated with an increased requirement for bowel resection at primary surgery and an inability to achieve complete cytoreduction. Increased expression of LRRC15 in BMets was confirmed by immunohistochemistry and knockdown of LRRC15 significantly inhibited tumor progression in mice. CONCLUSIONS: We identified 21 genes that are overexpressed in bowel metastases among patients with OC. Our findings will help select potential molecular targets for the prevention and treatment of malignant bowel obstruction in OC.

An integrative approach to assess X-chromosome inactivation using allele-specific expression with applications to epithelial ovarian cancer.

X-chromosome inactivation (XCI) epigenetically silences transcription of an X chromosome in females; patterns of XCI are thought to be aberrant in women's cancers, but are understudied due to statistical challenges. We develop a two-stage statistical framework to assess skewed XCI and evaluate gene-level patterns of XCI for an individual sample by integration of RNA sequence, copy number alteration, and genotype data. Our method relies on allele-specific expression (ASE) to directly measure XCI and does not rely on male samples or paired normal tissue for comparison. We model ASE using a two-component mixture of beta distributions, allowing estimation for a given sample of the degree of skewness (based on a composite likelihood ratio test) and the posterior probability that a given gene escapes XCI (using a Bayesian beta-binomial mixture model). To illustrate the utility of our approach, we applied these methods to data from tumors of ovarian cancer patients. Among 99 patients, 45 tumors were informative for analysis and showed evidence of XCI skewed toward a particular parental chromosome. For 397 X-linked genes, we observed tumor XCI patterns largely consistent with previously identified consensus states based on multiple normal tissue types. However, 37 genes differed in XCI state between ovarian tumors and the consensus state; 17 genes aberrantly escaped XCI in ovarian tumors (including many oncogenes), whereas 20 genes were unexpectedly inactivated in ovarian tumors (including many tumor suppressor genes). These results provide evidence of the importance of XCI in ovarian cancer and demonstrate the utility of our two-stage analysis.

TP53 mutations, tetraploidy and homologous recombination repair defects in early stage high-grade serous ovarian cancer.

To determine early somatic changes in high-grade serous ovarian cancer (HGSOC), we performed whole genome sequencing on a rare collection of 16 low stage HGSOCs. The majority showed extensive structural alterations (one had an ultramutated profile), exhibited high levels of p53 immunoreactivity, and harboured a TP53 mutation, deletion or inactivation. BRCA1 and BRCA2 mutations were observed in two tumors, with nine showing evidence of a homologous recombination (HR) defect. Combined Analysis with The Cancer Genome Atlas (TCGA) indicated that low and late stage HGSOCs have similar mutation and copy number profiles. We also found evidence that deleterious TP53 mutations are the earliest events, followed by deletions or loss of heterozygosity (LOH) of chromosomes carrying TP53, BRCA1 or BRCA2. Inactivation of HR appears to be an early event, as 62.5% of tumours showed a LOH pattern suggestive of HR defects. Three tumours with the highest ploidy had little genome-wide LOH, yet one of these had a homozygous somatic frame-shift BRCA2 mutation, suggesting that some carcinomas begin as tetraploid then descend into diploidy accompanied by genome-wide LOH. Lastly, we found evidence that structural variants (SV) cluster in HGSOC, but are absent in one ultramutated tumor, providing insights into the pathogenesis of low stage HGSOC.

Expression signature distinguishing two tumour transcriptome classes associated with progression-free survival among rare histological types of epithelial ovarian cancer.

BACKGROUND: The mechanisms of recurrence have been under-studied in rare histologies of invasive epithelial ovarian cancer (EOC) (endometrioid, clear cell, mucinous, and low-grade serous). We hypothesised the existence of an expression signature predictive of outcome in the rarer histologies. METHODS: In split discovery and validation analysis of 131 Mayo Clinic EOC cases, we used clustering to determine clinically relevant transcriptome classes using microarray gene expression measurements. The signature was validated in 967 EOC tumours (91 rare histological subtypes) with recurrence information. RESULTS: We found two validated transcriptome classes associated with progression-free survival (PFS) in the Mayo Clinic EOC cases (P=8.24 × 10(-3)). This signature was further validated in the public expression data sets involving the rare EOC histologies, where these two classes were also predictive of PFS (P=1.43 × 10(-3)). In contrast, the signatures were not predictive of PFS in the high-grade serous EOC cases. Moreover, genes upregulated in Class-1 (with better outcome) were showed enrichment in steroid hormone biosynthesis (false discovery rate, FDR=0.005%) and WNT signalling pathway (FDR=1.46%); genes upregulated in Class-2 were enriched in cell cycle (FDR=0.86%) and toll-like receptor pathways (FDR=2.37%). CONCLUSIONS: These findings provide important biological insights into the rarer EOC histologies that may aid in the development of targeted treatment options for the rarer histologies.

The inflammatory microenvironment in epithelial ovarian cancer: a role for TLR4 and MyD88 and related proteins.

The tumor-associated inflammatory microenvironment may play a pivotal role in epithelial ovarian cancer (EOC) carcinogenesis and outcomes, but a detailed profile in patient-derived tumors is needed. Here, we investigated the expression of TLR4- and MyD88-associated markers in tumors from over 500 EOC patients using immunohistochemical staining. We demonstrate that high expression of TLR4 and MyD88 predicts poorer overall survival in patients with EOC; most likely, this is due to their association with serous histology and features of high tumor burden and aggressiveness, including stage, grade, and ascites at surgery. Combined TLR4 and MyD88 expression appears to serve as an independent risk factor for shortened survival time, even after covariate adjustment (both moderate HR 1.1 [95 % CI 0.7-1.8], both strong HR 2.1 [95 % CI 1.1-3.8], both weak as referent; p = 0.027). We reveal that in EOC tissues with elevated expression of both TLR4 and MyD88 and activated NF-κB signaling pathway, expression of hsp60, hsp70, beta 2 defensin, and HMGB1 are also enriched. In total, these results suggest that activation of TLR4/MyD88/NF-κB signaling by endogenous ligands may contribute to an inflammatory microenvironment that drives a more aggressive phenotype with poorer clinical outcome in EOC patients.

Molecular classification of high grade endometrioid and clear cell ovarian cancer using TCGA gene expression signatures.

BACKGROUND: It is unclear whether the transcriptional subtypes of high grade serous ovarian cancer (HGSOC) apply to high grade clear cell (HGCCOC) or high grade endometrioid ovarian cancer (HGEOC). We aim to delineate transcriptional profiles of HGCCOCs and HGEOCs. METHODS: We used Agilent microarrays to determine gene expression profiles of 276 well annotated ovarian cancers (OCs) including 37 HGCCOCs and 66 HGEOCs. We excluded low grade OCs as these are known to be distinct molecular entities. We applied the prespecified TCGA and CLOVAR gene signatures using consensus non-negative matrix factorization (NMF). RESULTS: We confirm the presence of four TCGA transcriptional subtypes and their significant prognostic relevance (p<0.001) across all three histological subtypes (HGSOC, HGCCOC and HGEOCs). However, we also demonstrate that 22/37 (59%) HGCCOCs and 30/67 (45%) HGEOCs form 2 additional separate clusters with distinct gene signatures. Importantly, of the HGCCOC and HGEOCs that clustered separately 62% and 65% were early stage (FIGO I/II), respectively. These finding were confirmed using the reduced CLOVAR gene set for classification where most early stage HGCCOCs and HGEOCs formed a distinct cluster of their own. When restricting the analysis to the four TCGA signatures (ssGSEA or NMF with CLOVAR genes) most early stage HGCCOCs and HGEOC were assigned to the differentiated subtype. CONCLUSIONS: Using transcriptional profiling the current study suggests that HGCCOCs and HGEOCs of advanced stage group together with HGSOCs. However, HGCCOCs and HGEOCs of early disease stages may have distinct transcriptional signatures similar to those seen in their low grade counterparts.

In vivo anti-tumor activity of the PARP inhibitor niraparib in homologous recombination deficient and proficient ovarian carcinoma.

OBJECTIVE: Poly(ADP-ribose) polymerase (PARP) inhibitors have yielded encouraging responses in high-grade serous ovarian carcinomas (HGSOCs), but the optimal treatment setting remains unknown. We assessed the effect of niraparib on HGSOC patient-derived xenograft (PDX) models as well as the relationship between certain markers of homologous recombination (HR) status, including BRCA1/2 mutations and formation of RAD51 foci after DNA damage, and response of these PDXs to niraparib in vivo. METHODS: Massively parallel sequencing was performed on HGSOCs to identify mutations contributing to HR deficiency. HR pathway integrity was assessed using fluorescence microscopy-based RAD51 focus formation assays. Effects of niraparib (MK-4827) on treatment-naïve PDX tumor growth as monotherapy, in combination with carboplatin/paclitaxel, and as maintenance therapy were assessed by transabdominal ultrasound. Niraparib responses were correlated with changes in levels of poly(ADP-ribose), PARP1, and repair proteins by western blotting. RESULTS: Five PDX models were evaluated in vivo. Tumor regressions were induced by single-agent niraparib in one of two PDX models with deleterious BRCA2 mutations and in a PDX with RAD51C promoter methylation. Diminished formation of RAD51 foci failed to predict response, but Artemis loss was associated with resistance. Niraparib generally failed to enhance responses to carboplatin/paclitaxel chemotherapy, but maintenance niraparib therapy delayed progression in a BRCA2-deficient PDX. CONCLUSIONS: Mutations in HR genes are neither necessary nor sufficient to predict response to niraparib. Assessment of repair status through multiple complementary assays is needed to guide PARP inhibitor therapy, design future clinical trials and identify ovarian cancer patients most likely to benefit from PARP inhibition.

Genome-wide investigation of regional blood-based DNA methylation adjusted for complete blood counts implicates BNC2 in ovarian cancer.

Due to its potential as a biomarker for early cancer detection, blood-based DNA methylation (DNAm) is of interest in cancer research. Specifically, highly predictive mechanisms for early detection of epithelial ovarian cancer (EOC) are desired, so previous studies have compared DNAm between EOC cases and controls. However, case-control studies are confounded by the distribution of white blood cell types through an immune response induced by the cancer. Rather than determining the distribution of the cell types manually or investigating isolated cell types, an alternative approach involves the use of complete blood count (CBC), which is routinely collected. In the analysis of an EOC case-control study of DNAm, we incorporate CBC measures to adjust for this confounding and compare DNAm between 242 EOC cases and 181 age-matched controls (assayed on the Illumina Infinium HumanMethylation27 or HumanMethylation450 Beadchips), at both the individual CpG and CpG island levels. We found that adjustment for leukocyte distribution using CBC measurements dramatically reduced confounding, with 62 single CpG sites found to be associated with EOC status after adjustment (P < 5E-8). Additionally, regional DNAm was assessed by applying principal components analysis to CpG islands. The top associated CpG island (P = 7E-6) was located in the promoter/transcription start site of the human basonuclin 2 gene (BNC2), a known susceptibility gene for EOC risk identified through GWAS. Follow-up studies are necessary to establish the role of BNC2 in blood-based DNA and EOC, including prospective studies to validate this region as a potential biomarker and predictor of EOC susceptibility.

Epigenome-wide ovarian cancer analysis identifies a methylation profile differentiating clear-cell histology with epigenetic silencing of the HERG K+ channel.

Ovarian cancer remains the leading cause of death in women with gynecologic malignancies, despite surgical advances and the development of more effective chemotherapeutics. As increasing evidence indicates that clear-cell ovarian cancer may have unique pathogenesis, further understanding of molecular features may enable us to begin to understand the underlying biology and histology-specific information for improved outcomes. To study epigenetics in clear-cell ovarian cancer, fresh frozen tumor DNA (n = 485) was assayed on Illumina Infinium HumanMethylation450 BeadChips. We identified a clear-cell ovarian cancer tumor methylation profile (n = 163) which we validated in two independent replication sets (set 1, n = 163; set 2, n = 159), highlighting 22 CpG loci associated with nine genes (VWA1, FOXP1, FGFRL1, LINC00340, KCNH2, ANK1, ATXN2, NDRG21 and SLC16A11). Nearly all of the differentially methylated CpGs showed a propensity toward hypermethylation among clear-cell cases. Several loci methylation inversely correlated with tumor gene expression, most notably KCNH2 (HERG, a potassium channel) (P = 9.5 × 10(-7)), indicating epigenetic silencing. In addition, a predicted methylation class mainly represented by the clear-cell cases (20 clear cell out of 23 cases) had improved survival time. Although these analyses included only 30 clear-cell carcinomas, results suggest that loss of expression of KCNH2 (HERG) by methylation could be a good prognostic marker, given that overexpression of the potassium (K(+)) channel Eag family members promotes increased proliferation and results in poor prognosis. Validation in a bigger cohort of clear-cell tumors of the ovary is warranted.

Regulatory T cells, inherited variation, and clinical outcome in epithelial ovarian cancer.

The immune system constitutes one of the host factors modifying outcomes in ovarian cancer. Regulatory T cells (Tregs) are believed to be a major factor in preventing the immune response from destroying ovarian cancers. Understanding mechanisms that regulate Tregs in the tumor microenvironment could lead to the identification of novel targets aimed at reducing their influence. In this study, we used immunofluorescence-based microscopy to enumerate Tregs, total CD4 T cells, and CD8(+) cytotoxic T cells in fresh frozen tumors from over 400 patients with ovarian cancer (>80 % high-grade serous). We sought to determine whether Tregs were associated with survival and genetic variation in 79 genes known to influence Treg induction, trafficking, or function. We used Cox regression, accounting for known prognostic factors, to estimate hazard ratios (HRs) associated with T cell counts and ratios. We found that the ratios of CD8 T cells and total CD4 T cells to Tregs were associated with improved overall survival (CD8/Treg HR 0.84, p = 0.0089; CD4/Treg HR 0.88, p = 0.046) and with genetic variation in IL-10 (p = 0.0073 and 0.01, respectively). In multivariate analyses, the associations between the ratios and overall survival remained similar (IL-10 and clinical covariate-adjusted CD8/Treg HR 0.85, p = 0.031; CD4/Treg HR 0.87, p = 0.093), suggesting that this association was not driven by variation in IL-10. Thus, integration of novel tumor phenotyping measures with extensive clinical and genetic information suggests that the ratio of T cells to Tregs may be prognostic of outcome in ovarian cancer, regardless of inherited genotype in genes related to Tregs.

Plasma immune analytes in patients with epithelial ovarian cancer.

OBJECTIVES: Inflammation is a common feature of epithelial ovarian cancer (EOC), and measurement of plasma markers of inflammation might identify candidate markers for use in screening or presurgical evaluation of patients with adnexal masses. METHODS: Plasma specimens from cohorts of 100 patients with advanced EOC (AJCC Stage III and IV), 50 patients with early stage EOC (Stage I and II), and 50 patients with benign surgical conditions were assayed for concentrations of multiple cytokines, toll-like receptor agonists, and vascular growth factors via ELISA and electrochemiluminescence. Immune proteins were then analyzed for association with EOC. Differences in plasma protein levels between benign, early, and advanced EOC patient groups were assessed with and without adjustment for plasma cancer antigen 125 (CA-125) levels. RESULTS: Out of 23 proteins tested, six-including interferon gamma (IFNγ), interleukin 6 (IL-6), IL-8, IL-10, tumor necrosis factor alpha (TNFα), and placental growth factor (PlGF)-were univariately associated with EOC (all p<0.005), and one-IL-6-was associated with early stage EOC (p<0.0001). Heat shock protein 90kDa beta member 1 (HSP90B1, gp96) was associated with EOC and early stage EOC with borderline statistical significance (p=0.039 and p=0.026, respectively). However, when adjusted for (CA-125), only HSP90B1 independently predicted EOC (p=0.008), as well as early stage EOC (p=0.014). CONCLUSIONS: Multiple plasma cytokines, including IFNγ, IL-6, IL-8, IL-10, TNFα, PlGF, and HSP90B1 are associated with EOC. Of these, HSP90B1 is associated with EOC independent from the biomarker CA-125.

Prior oral contraceptive use in ovarian cancer patients: assessing associations with overall and progression-free survival.

BACKGROUND: Prior studies have described a reduced risk of developing ovarian cancer with the use of oral contraceptives. In this context, we decided to examine if oral contraceptive use prior to a diagnosis of ovarian cancer is associated with better overall and progression-free survival. METHODS: This retrospective cohort study included ovarian cancer patients who were seen at the Mayo Clinic in Rochester, Minnesota from 2000 through 2013. Patients completed a risk factor questionnaire about previous oral contraceptive use, and clinical data were extracted from the electronic medical record. RESULTS: A total of 1398 ovarian cancer patients responded to questions on oral contraceptive use; 571 reported no prior use with all others having responded affirmatively to oral contraceptive use. Univariate analyses found that oral contraceptive use (for example, ever versus never) was associated with better overall survival (hazard ratio (HR) 0.73 (95 % confidence interval (CI): 0.62, 0.86); p = 0.0002) and better progression-free survival (HR 0.71 (95 % CI: 0.61, 0.83); p < 0.0001). In multivariate analyses, contraceptive use continued to yield a favorable, statistically significant association with progression-free survival, but such was not the case with overall survival. CONCLUSIONS: This study suggests that previous oral contraceptive use is associated with improved progression-free survival in patients diagnosed with ovarian cancer.

Assessment of published models and prognostic variables in epithelial ovarian cancer at Mayo Clinic.

OBJECTIVES: Epithelial ovarian cancer (EOC) is an aggressive disease in which first line therapy consists of a surgical staging/debulking procedure and platinum based chemotherapy. There is significant interest in clinically applicable, easy to use prognostic tools to estimate risk of recurrence and overall survival. In this study we used a large prospectively collected cohort of women with EOC to validate currently published models and assess prognostic variables. METHODS: Women with invasive ovarian, peritoneal, or fallopian tube cancer diagnosed between 2000 and 2011 and prospectively enrolled into the Mayo Clinic Ovarian Cancer registry were identified. Demographics and known prognostic markers as well as epidemiologic exposure variables were abstracted from the medical record and collected via questionnaire. Six previously published models of overall and recurrence-free survival were assessed for external validity. In addition, predictors of outcome were assessed in our dataset. RESULTS: Previously published models validated with a range of c-statistics (0.587-0.827), though application of models containing variables which are not part of routine practice were somewhat limited by missing data; utilization of all applicable models and comparison of results are suggested. Examination of prognostic variables identified only the presence of ascites and ASA score to be independent predictors of prognosis in our dataset, albeit with marginal gain in prognostic information, after accounting for stage and debulking. CONCLUSIONS: Existing prognostic models for newly diagnosed EOC showed acceptable calibration in our cohort for clinical application. However, modeling of prospective variables in our dataset reiterates that stage and debulking remains the most important predictors of prognosis in this setting.

The immune system in the pathogenesis of ovarian cancer.

Clinical outcomes in ovarian cancer are heterogeneous even when considering common features such as stage, response to therapy, and grade. This disparity in outcomes warrants further exploration into tumor and host characteristics. One compelling host characteristic is the immune response to ovarian cancer. While several studies have confirmed a prominent role for the immune system in modifying the clinical course of the disease, recent genetic and protein analyses also suggest a role in disease incidence. Recent studies also show that anti-tumor immunity is often negated by immune suppressive cells present in the tumor microenvironment. These suppressive immune cells also directly enhance the pathogenesis through the release of various cytokines and chemokines, which together form an integrated pathologic network. Thus, future research into immunotherapy targeting ovarian cancer will likely become increasingly focused on combination approaches that simultaneously augment immunity while preventing local immune suppression or by disrupting critical cytokine networks.

Hormone-receptor expression and ovarian cancer survival: an Ovarian Tumor Tissue Analysis consortium study.

BACKGROUND: Few biomarkers of ovarian cancer prognosis have been established, partly because subtype-specific associations might be obscured in studies combining all histopathological subtypes. We examined whether tumour expression of the progesterone receptor (PR) and oestrogen receptor (ER) was associated with subtype-specific survival. METHODS: 12 studies participating in the Ovarian Tumor Tissue Analysis consortium contributed tissue microarray sections and clinical data to our study. Participants included in our analysis had been diagnosed with invasive serous, mucinous, endometrioid, or clear-cell carcinomas of the ovary. For a patient to be eligible, tissue microarrays, clinical follow-up data, age at diagnosis, and tumour grade and stage had to be available. Clinical data were obtained from medical records, cancer registries, death certificates, pathology reports, and review of histological slides. PR and ER statuses were assessed by central immunohistochemistry analysis done by masked pathologists. PR and ER staining was defined as negative (<1% tumour cell nuclei), weak (1 to <50%), or strong (≥50%). Associations with disease-specific survival were assessed. FINDINGS: 2933 women with invasive epithelial ovarian cancer were included: 1742 with high-grade serous carcinoma, 110 with low-grade serous carcinoma, 207 with mucinous carcinoma, 484 with endometrioid carcinoma, and 390 with clear-cell carcinoma. PR expression was associated with improved disease-specific survival in endometrioid carcinoma (log-rank p<0·0001) and high-grade serous carcinoma (log-rank p=0·0006), and ER expression was associated with improved disease-specific survival in endometrioid carcinoma (log-rank p<0·0001). We recorded no significant associations for mucinous, clear-cell, or low-grade serous carcinoma. Positive hormone-receptor expression (weak or strong staining for PR or ER, or both) was associated with significantly improved disease-specific survival in endometrioid carcinoma compared with negative hormone-receptor expression, independent of study site, age, stage, and grade (hazard ratio 0·33, 95% CI 0·21-0·51; p<0·0001). Strong PR expression was independently associated with improved disease-specific survival in high-grade serous carcinoma (0·71, 0·55-0·91; p=0·0080), but weak PR expression was not (1·02, 0·89-1·18; p=0·74). INTERPRETATION: PR and ER are prognostic biomarkers for endometrioid and high-grade serous ovarian cancers. Clinical trials, stratified by subtype and biomarker status, are needed to establish whether hormone-receptor status predicts response to endocrine treatment, and whether it could guide personalised treatment for ovarian cancer. FUNDING: Carraresi Foundation and others.

Nutritional status, CT body composition measures and survival in ovarian cancer.

OBJECTIVE: Body composition measures (BCMs) are an important predictor of nutritional status in patients with cancer. Poor nutritional status is common in ovarian cancer (OC) and is a well-known variable that influences cancer treatment and outcome. We aim to establish the role of BCMs measured by computed tomography (CT) in predicting outcomes in patients with OC. METHODS: We retrospectively searched our institutional database for patients with stage IIIC/IV OC who underwent surgery as primary treatment at Mayo Clinic between 1996 and 2005 and had adequate presurgical CT images available. For each patient, 1 axial CT image at the level of the 3rd lumbar vertebra was evaluated. Adipose and lean tissues were discriminated using commercially available software. Cox models were fit to evaluate the relationship between patient factors and overall survival (OS). Associations were summarized using hazard ratios (HRs) and corresponding 95% CIs. RESULTS: A total of 82 patients were identified, with a median age of 68.4 years. OS at 1 and 5 years was 84.1% and 24.1%, respectively. Older age (P=.01), stage IV disease (P<.001), and subcutaneous and muscular fat<77.21cm(2) (P<.001) were independently associated with poor OS. Longer hospital stay was independently predicted by albumin≤3g/dL (P=.03), suboptimal surgery (P=.02), and subcutaneous and muscular fat<77.21cm(2) (P<.001). Surgical complications were independently predicted only by albumin≤3g/dL (P<.01). CONCLUSIONS: CT BCMs, as indicators of nutritional status, are independent predictors of longer hospital stay and poor OS in patients with OC.

Tumor-infiltrating programmed death receptor-1+ dendritic cells mediate immune suppression in ovarian cancer.

Within the ovarian cancer microenvironment, there are several mechanisms that suppress the actions of antitumor immune effectors. Delineating the complex immune microenvironment is an important goal toward developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell (DC)-associated B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, programmed death receptor-1 (PD-1), is also expressed on myeloid cells, complicating interpretations of how B7-H1 regulates DC function in the tumor. In this study, we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1(+) B7-H1(+) DCs have a classical DC phenotype (i.e., CD11c(+)CD11b(+)CD8(-)), but are immature, suppressive, and respond poorly to danger signals. Accumulation of PD-1(+)B7-H1(+) DCs in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell-associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DCs suppressed NF-κB activation, release of immune regulatory cytokines, and upregulation of costimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector Ag-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1(+)B7-H1(+) DCs in mediating immune suppression in ovarian cancer.

MHC class II epitope nesting modulates dendritic cell function and improves generation of antigen-specific CD4 helper T cells.

CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.

A factor found in the IgG fraction of serum of patients with paraneoplastic bilateral diffuse uveal melanocytic proliferation causes proliferation of cultured human melanocytes.

PURPOSE: To determine if there is a factor in the serum of patients with bilateral diffuse uveal melanocytic proliferation (BDUMP) that causes melanocytic proliferation. METHODS: Human melanocytes and melanoma cells were grown and exposed to serum or plasma of patients with BDUMP, other neoplastic conditions, or control media. Preliminary studies using serum were conducted in an unmasked fashion. In addition, IgG-depleted and IgG-enriched plasma was also tested in a similar fashion. Experiments using plasma were conducted triple masked. To show that the proliferation was melanocyte selective, human dermal fibroblasts, keratinocytes, and ovarian cancer cells were treated with plasma of the BDUMP cases or controls, and the effect of this exposure on their proliferation was quantified. RESULTS: At 72 hours, the serum of BDUMP patients caused statistically significant increased proliferation of normal human melanocytes. Further studies at 6 days demonstrated similar findings. In addition, melanocytes grown in BDUMP serum exhibited a disorganized morphology with foci of multilayered cells. Cultured melanoma cells also showed statistically significant increase in growth in serum from BDUMP patients compared with controls. Masked plasma studies further confirmed these findings and showed that the IgG fraction appeared to contain the melanocyte growth-stimulating factor. The human fibroblasts, keratinocytes, and ovarian cancer cells did not show an increase in growth with the BDUMP plasma treatment. CONCLUSION: Patients with BDUMP have a factor in the IgG fraction that selectively causes melanocyte proliferation. How it causes proliferation of human melanocytes and melanoma cells needs to be further elucidated.

Genomics of the NF-κB signaling pathway: hypothesized role in ovarian cancer.

OBJECTIVE: We sought to review evidence linking nuclear factor-kappa B (NF-κB) to ovarian cancer and to identify genetic variants involved in NF-κB signaling. METHODS: PubMed was reviewed to inform on ovarian cancer biology and NF-κB signaling and to identify key genes. Public linkage disequilibrium (LD) data were analyzed to identify informative inherited variants (tagSNPs) using ldSelect. RESULTS: We identified 319 key NF-κB genes including five NF-κB subunits, 167 activating genes, and 55 inhibiting genes. We found that the 1000 Genomes Project was the most informative LD source for most genes (92.8%), and we identified 13,027 LD bins (r (2) ≥ 0.9, minor allele frequency ≥ 0.05) and 1,018 putative-functional variants worthy of investigation. We also report that reliance on a commonly used genome-wide SNP array and genotype imputation with HapMap Phase II data provides data on only 74% of the common inherited NF-κB SNPs of interest. CONCLUSIONS: Compelling evidence suggests that NF-κB plays a critical role in ovarian cancer, yet inherited variation in these genes has not been thoroughly assessed in relation to disease risk or outcome. We present a collection of variants in key genes and suggest creation of a custom genotyping array as an optimal approach.