Targeted immune therapy of ovarian cancer.

Ovarian cancer has the highest mortality rate of the cancers unique to women. According to Siegel and colleagues, in 2012 there were an estimated 22,240 new cases of ovarian cancer and estimated 14,030 deaths [ 1 ]. Moreover, the majority (65%-75%) of women with ovarian cancer are diagnosed with advanced stage disease (III and IV), and only about 15%-20% of these women are free of disease recurrence at ten years [ 2 - 4 ]. Major subtypes of ovarian cancer include serous, endometrioid, clear cell and mucinous histologies, with heterogeneous characteristics [ 5 ]. Nevertheless, there are unique features about ovarian cancer that may shed light on its basic biology, hopefully leading to improvements in therapeutics and outcome. One of those unique features is the complex interaction of the immune system in both ovarian cancer initiation and progression which has been well documented over the last two decades and is the central topic of this review. This review covers a broad range of topics related to the immunobiology and immunotherapy of ovarian cancer.

Recent evaluations of associations between genetic variation and progression in ovarian cancer could also provide clues as to potential immune targets. Goode and colleagues evaluated germline variation in 26 inflammation-related genes in a population of 325 patients with ovarian cancer [ 153 ]. Of those 26 genes, SNPs in CCR3, CCL2, IL1B, IL18, and ALOX5 were related to survival in patients. Both arachidonate 5-lipoxigenase (ALOX5) which converts arachidonic acid into leukotrienes and IL-18 which promotes IFN-γ production in NK and TH1 cells which were previously associated with increased risk of developing ovarian cancer as described above. The chemokine receptor CCR3, the product of CCR3, binds and responds to a variety of chemokines, including CCL11, CCL26, CCL7, CCL13, and CCL5. The receptor is involved in chemotaxis in a wide variety of leukocytes such as CD4 T cells. CCR2 (monocyte chemotactic protein 1) is important for recruitment of monocytes (macrophages) to sites of inflammation. IL1B encodes IL1beta, a pro-inflammatory cytokine that also has multiple effects on both the immune system and directly on ovarian cancer cells [ 154 ]. In another prospective study of 147 advanced ovarian cancer patients, an association between two IL10 promoter polymorphisms and disease-free and overall survival overall survival was observed, possibly due to its relationship with optimal tumor debulking [ 155 ]. Lastly, in addition to cytokines, genes encoding other modulators of the immune system have been implicated in disease recurrence and progression. TLR4, normally expressed at high levels on immune cells, detects lipopolysaccharide from Gram-negative bacteria and is important in the activation of the innate immune system through NF-κB. In a clinical outcomes study of ovarian cancer patients, a putative microRNA binding site polymorphism in Toll-like receptor 4 ( TLR4) (rs7869402) was associated with inferior survival and response to treatment [ 40 ]. We recently analyzed expression quantitative trait loci (eQTL) and sequence-based tagging single nucleotide polymorphisms (tagSNPs) for 79 Treg associated genes in invasive ovarian cancer cases. We found that poorer survival was associated with minor alleles at SNPs in TNFRSF18 (OX40) in the mucinous subtype, in CD80 in the endometrioid subtype, CD25 in endometrioid subtype, and CTLA4 in the clear cell subtype [ 156 , 157 ]. As the products of these genes are known to affect induction, trafficking, or immunosuppressive function of immune cells, these results suggest the need for follow-up phenotypic studies and suggest that the role of the immune system may differ among the various subtypes of cancer.

The immune system is constituted by a heterogeneous population of both cellular and molecular effectors which function in an organized and integrated manner to eradicate disease and maintain the overall health of the host while minimizing off-target activity (e.g. autoimmunity). The immune system is typically divided into two different, but interacting, systems referred to as the innate and the adaptive mechanisms. The innate immune system is the first line of defense that consists of a group of cellular and humoral factors. The cellular component of innate immunity includes natural killer (NK) T cells, mast cells, eosinophils, basophils, macrophages, neutrophils and dendritic cells, while the humoral factors primarily include cytokines and complement. The major functions of the innate immune system include recruiting immune cells into sites of infection, activation of complement, identification of foreign substances, and preparation and activation of the adaptive immune systems. Innate responses are immediate, maximal, and antigen-independent. In the absence of stimulation, non-activated innate cells are well known to maintain tolerance and prevent inappropriate immune activation. The adaptive immune response involves both T and B lymphocytes. T lymphocytes produce cytokines or cytolytic molecules, while B cells produce antibodies. Primary adaptive immune responses usually develop after one week and are highly antigen-specific. Like the innate immune system, subsets of lymphocytes, e.g. regulatory T cells (Treg), are also known to regulate immunity and prevent autoimmunity. Lastly, another key feature of the adaptive immune response is generation of immune memory and the resulting rapid and robust responses to previously encountered antigens, a fundamental reason for the widespread clinical success of vaccines.

Adoptive T cell therapy involves ex vivo activation, expansion, and re-infusion of either natural or engineered antigen-specific T cells [ 237 - 240 ]. The approach has been used successfully for the treatment of melanoma and other cancer, and is envisioned to be useful for ovarian cancer [ 241 ]. From a technical and infrastructure standpoint, adoptive T cell therapy remains a challenge. A few clinical trials of adoptive T cell therapy in ovarian cancer have been reported. The first clinical trial, in patients with recurrent disease, by Kershaw examined the feasibility of infusion of T cells engineered to express a FRα-receptor antibody coupled to the signaling motif of the Fc receptor γ chain [ 242 ]. While the infusions were found to be safe, no clinical activity was observed, likely due to the observation that the infused T cells did not persist for long periods of time in large numbers [ 241 ] and potentially an inhibitory factor which developed in a half of the patients. In another trial patients were infused with non-modified MUC1-specific Th1 effector T cells [ 243 , 244 ]. MUC1-specific T cells were derived by repeated stimulation and expansion of leukapheresis-derived cells with the 20mer MUC1 peptide, GSTAPPAHGVTSAPATAPAP. Three doses ranging from 10 8 to 10 9 cells (largely CD4 T cells), were administered intraperitoneally into seven patients. Three patients were discontinued treatment due to local inflammation or obstruction at the intraperitoneal port; in the four remaining patients, treatment was shown to increase systemic MUC1 immune responses, which could have contributed to the long term survival seen in two of the patients. Similarly, in another study, 7 subjects with recurrent ovarian cancer underwent up to 4 cycles with in vitro generated CTLs that were specific for MUC1. The tumor marker CA-125 was non-statistically reduced after the first month of immunotherapy. One subject was free of disease since December 2000 at the time of publication [ 245 ].

Farletuzumab (MORAb-003) is a humanized monoclonal antibody, optimized from the original murine LK26 antibody, which has high affinity for FRα [ 206 ]. Preclinical in vivo and in vitro studies have shown that farletuzumab which binds specifically to ovarian cancer cells, has high efficacy in preventing tumor growth with little or no toxicity [ 207 , 208 ]. A trial by Konner reported that farletuzumab as a single therapy in platinum-resistant ovarian cancer patients was safe and well tolerated at all doses tested and that 35% of the patients treated with one cycle (four weekly doses) had stable disease and three of these patients received extended therapy showing 3% to 17% mean decrease in tumor size. Another trial analyzed farletuzumab as single-agent therapy or in combination with platinum and taxane in platinum-sensitive ovarian cancer patients with first recurrence of disease [ 209 , 210 ]. In that trial, the group treated with farletuzumab combination therapy showed a 7% complete response, 63% partial response and 89% of the patients achieved normal CA125. Moreover, four patients still remained in remission with median extension of the secondary remission being eleven months [ 211 ]. Despite these very positive results, the developing company, Eisai Inc., revealed in the first quarter of 2013 via a news release that a double blinded phase III in platinum-sensitive disease failed to achieve the endpoint of improved progression free survival but further analyses are ongoing to determine if subsets of patients experienced benefits. HER-2/neu antibodies, namely trastuzumab and pertuzumab, have been extensively studied as therapeutics for the small subset of patients with tumors that overexpress HER-2/neu. Trastuzumab (Herceptin) is a humanized monoclonal antibody that targets the HER-2/neu extracellular domain and inhibits HER-2/neu-positive tumor cell proliferation. This antibody is standard of care for patients with HER-2/neu + breast cancer [ 212 ]. Few clinical trials have been reported on the use of trastuzumab in ovarian cancer. One study done in 41 patients showed that trastuzumab given as single agent therapy demonstrated a low response rate (~7%) and a 2-month median progression-free interval [ 162 ]. A phase II clinical study looked at the efficacy and tolerance of trastuzumab when combined with paclitaxel and carboplatin in seven patients with taxane/carboplatin resistant ovarian cancer and HER-2/neu overexpression finding that combination treatment elicited a clinical response in three patients with a median PFS of 2.9 months and overall survival of 12.3 months [ 213 ]. Another trial done in 33 patients with mucinous ovarian carcinoma showed ~18% HER-2 expression, and of the three patients treated with chemotherapy and trastuzumab, one had complete effective response and another had partial response, suggesting that trastuzumab may be effective for this subset of individuals [ 214 ]. Pertuzumab (rhuMAb 2C4, Omnitarg) is a HER-2/neu targeted monoclonal antibody that binds to an epitope distinct from that of trastuzumab, inhibiting heterodimer formation with other HER-family receptors and halting the proliferation of ovarian cancer [ 215 , 216 ]. The first clinical study was done by Gordon who showed that as a monotherapy pertuzumab, in recurrent ovarian cancer, demonstrated a 4.3% partial response rate and stable disease in 6.8% for six months with a median PFS of 6.6 weeks in patients with recurrent ovarian carcinoma [ 215 ]. A study done in platinum-resistant patients showed that the combination therapy of pertuzumab with gemcitabine resulted in a response rate of 13.8% with 2.9 months PFS as compared with a 4.6% response rate and PFS of 1.5 months in the placebo group [ 217 ]. A randomized study of 152 platinum-sensitive patients evaluated the activity of pertuzumab and carboplatin-based therapy compared with carboplatin therapy alone [ 218 ]. In their preliminary report of their study, the investigators demonstrated that pertuzumab with carboplatin was well tolerated, had a 64% response rate and PFS of 34.1 weeks in comparison with a 52% response rate and PFS of 31.3 weeks in the single-agent carboplatin group, suggesting some activity [ 219 ]. However, in their final report, the team found no evidence that inclusion of pertuzumab into chemotherapy improves either progression-free survival or response to therapy rates [ 218 ]. Abagovomab is an anti-idiotype monoclonal antibody whose variable region mirrors CA-125. Abagovomab does not bind directly to CA-125, but it acts as a surrogate antigen able to generate antibody responses against CA-125 protein. In a Phase Ib/II study on 119 patients with advanced ovarian cancer who were resistant to standard therapies, treatment with abagovomab appeared to have led to improved survival time (23.4 months compared to 4.9 months) in those patients who responded to the vaccination with the generation of an immune response [ 220 , 221 ]. The vaccine had little side effects and two additional phase I trials were done to determine the best dose, schedule and route of administration, which paved the way to a Phase III double blinded clinical trial [ 220 ]. The phase III enrolled 888 women in first remission with a 2:1 ratio. By the final patient visit, the median anti-idiotypic antibody level was a robust 493 μg/ml. But, despite the strong immune response, neither recurrence free or overall survival was prolonged and further development of the drug for ovarian cancer was apparently terminated despite impressive early trial data [ 222 ]. This pathway is very reminiscent of that of oregovomab, a high affinity monoclonal antibody that binds directly to CA-125. Oregovomab has been tested in various phase II and III clinical trials as both frontline and maintenance therapy [ 223 - 225 ]. Unlike the antibodies described above, Catumaxomab is a trifunctional antibody with bi-specificity for EpCAM on tumor cells and CD3 on T cells. Trifunctional antibodies induce a multi-cell complex of tumor cells, T cells, and accessory immune cells (e.g. macrophages, DCs and NK cells) [ 226 ]. This drug has been approved in Europe since 2009 for the treatment of ascites associated with EpCAM-positive cancers. This drug has also been tested in the clinical setting as a new therapeutic strategy for ovarian cancer patients with symptomatic malignant ascites. A clinical study from Burges and colleagues showed good tolerance of patients with recurrent ascites due to ovarian cancer [ 227 ]. The trial showed a significant decrease in ascites production in all patients with a decrease in the number of EpCAM+ tumor cells from 540,000/10 6 total cells before treatment to 39/10 6 following the last infusion. Another trial in patients with malignant ascites due to epithelial cancers (129 ovarian and 129 non-ovarian) assessed the efficacy and safety of Catumaxomab and paracentesis compared with paracentesis alone [ 228 ]. This study showed Catumaxomab reduced the ascites volume and the time between paracentesis in both cancer groups but most significantly in the ovarian cancer group along with an overall survival of 110 days compared to 81 days in the control group. Baumann and colleagues recently published results of a phase IIa trial in which 45 patients with platinum-resistant (or refractory) disease were treated with one of two doses of drug given as monotherapy directly intraperitoneally [ 229 ]. With only one partial response and seven disease stabilizations, the activity was considered very modest. Despite the modest activity, there are clinical trials underway, at the time of preparation of this review, testing different strategies such as treatment with the antibody prior to initiation of chemotherapy at time of recurrence (either biochemical or RECIST). Siltuximab (CNTO328), a human-mouse chimeric IL-6 antibody, has been used in multiple clinical trials for a variety of cancers, including ovarian, renal, and prostate [ 134 , 230 - 233 ]. In a phase II trial of 18 ovarian cancer patients, one individual had a partial response and 7 had stable disease for a period of time [ 140 ]. In another early stage monotherapy trial which included 29 ovarian cancer patients, no objective responses were observed suggesting that ovarian cancer can easily evade the IL-6 blockade [ 234 ]. The use of monoclonal antibody-based checkpoint blockade for the treatment of ovarian cancer is also being developed as well targeting CTLA-4 or the PD-1/PD-L1 axis. The anti-CTLA-4 antibody widely being tested is ipilimumab. The first indication that this drug may be useful came from Hodi and colleagues who enrolled two ovarian cancer patients in an early trial and demonstrated reduction in CA-125 in one patient and stabilization in another [ 235 ]. At the time of writing of this review, no other results in ovarian have been published, however, ClinicalTrials.gov ( www.clinictrials.gov ) lists a phase II study testing the safety and efficacy of ipilimumab monotherapy following completion of chemotherapy in recurrent platinum sensitive ovarian cancer patients with residual disease in the second, third, or fourth line therapy. The safety and preliminary activity of anti-PD-L1 has recently been reported in 17 patients with ovarian cancer. One of 17 had a partial response and 3 patients had disease stabilization for at least 24 weeks [ 236 ].

In contrast to agents that directly increase or decrease specific immune cell subsets, another concept that has recently been introduced is the disruption of integrated cytokine networks. One integrated cytokine network for which there is considerable published support, termed the ‘TNF network’ – consisting of TNF, CXCL12, and IL-6 – was recently identified by Kulbe and colleagues in human ovarian cancer specimens ( Figure 3 ) [ 134 ]. TNFα expression and function in cancers including ovarian cancer has long been studied and its expression is highly correlated with increased expression CXCL12 and IL-6, and this results in the ability of TNFα to mediate a variety of effects [ 135 ]. The effect of TNFα on a tumor depends on both timing and dose. A single, high-dose exposure promotes tumor regression while chronic, low-dose expression of TNFα is tumor promoting [ 136 , 137 ]. In vitro studies of ovarian cancer cell lines using RNAi to decrease expression of TNFα did not result in any changes in proliferation or apoptosis. However, when the same cells were injected in vivo , a decrease in tumor burden, restricted distribution, and increased apoptosis was evident [ 135 ]. Kulbe and colleagues suggest that these differential effects of TNFα blockade in vivo are due to disruption of a multi-cytokine network constituted by TNFα, CXCL12 and IL-6 that results in reduced angiogenesis, reduced infiltration of regulatory cells and NOTCH signaling [ 134 ]. High level expression of IL-6 has been found in ovarian cancer [ 138 ]. IL-6 directly promotes cancer growth and progression through several mechanisms including proliferation, angiogenesis, and decreased sensitivity to apoptotic signals [ 139 , 140 ]. High levels of IL-6 in the serum of ovarian cancer patients have been associated with shorter survival [ 141 , 142 ]. Additionally, high levels of IL-6 are often detected in the malignant ascites of ovarian cancer patients [ 143 ]. IL-6 mediates its pathologic effects on cancers via several mechanisms, including activation of the growth promoting JAK/STAT, PI3K/Akt, and Ras/MEK/IRK pathways [ 141 , 142 , 144 , 145 ]. IL-6 is also thought to be important in the generation of thrombocytosis (elevated platelets) commonly observed in ovarian cancer patients and associated with shortened survival [ 146 ]. Thus, IL-6 has emerged as a logical candidate for targeted therapy [reviewed in [ 139 , 147 ]]. CXCR4 has been previously reported to be constitutively expressed in ovarian cancer and its ligand, CXCL12 is secreted by cells in the tumor microenvironment in addition to ovarian cancer cells [reviewed in [ 148 ]]. Ligation of CXCR4 has several effects, including stimulation of proliferation, vascular endothelial growth factor-mediated angiogenesis, and recruitment of immune cells [ 149 - 152 ]. In immunocompetent mouse model studies blockade of the CXCL12/CXCR4 signaling increased survival, increased tumor cell apoptosis, and reduces Treg infiltration [ 152 ].

Despite early clinical trials data that immunotherapy approaches may have efficacy, the approaches (mainly monoclonal antibody therapy) that reach phase III clinical trials overwhelmingly fail in ovarian cancer. This is due to at least two reasons. First, early trials fail to define companion predictors that may better inform the design of the intent-to-treat population. As such, trials are typically open to all patients significantly reducing power to detect an impact on the disease. Second and most importantly, treatments are almost done in patients with very advanced refractory disease, a disease state that is characterized by increased plasticity of the tumor cells allowing them to rapidly adapt to hostile microenvironments. One way to solve this problem is to detect ovarian cancer much earlier in the course of disease or develop methods to prevent disease altogether. As discussed, there is significant evidence to suggest that the immune system is involved in the initiation of ovarian cancer, potentially paving the way to preventive strategies such as anti-inflammatory drugs and vaccines. Alternatively, there is general agreement in the field that combination approaches could lead to synergism and perhaps greater efficacy in the setting of established disease. A number of strategies have emerged, some of which are being tested clinically. For example, the efficacy of trastuzumab therapy may be improved by combining with agents that boost T cell immunity. In one study, Zum Buschenfelde found that pretreatment of HER-2/neu + ovarian tumor cells with trastuzumab enhanced the cytolytic activity of HER-2/neu-specific T cells against the HER-2/neuoverexpressing tumors in vitro [ 251 ]. Although the mechanism is unclear, it is possible that trastuzumab promotes the internalization and degradation of HER-2/neu, resulting in increased presentation of HER-2/neu MHC class I epitopes which may lead to greater activation and expansion of HER-2/neu-specific T cells. A clinical trial of combination trastuzumab and HER-2/neu vaccination in breast cancer was recently reported by Disis and colleagues [ 252 ]. Twenty-two patients with HER-2/neu-overexpressing metastatic breast cancer receiving trastuzumab therapy were vaccinated with an HER-2/neu T cell peptide-based vaccine. The patients had either no evidence of disease or stable masses at the time of therapy. The combination was well tolerated. While many patients had pre-existing immunity to HER-2/neu while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Importantly, at a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached. Alternatively, the development of checkpoint blockade opens up a range of possibilities. For example, we recently reported on the development of a combination approach incorporating anti-PD-1 antibody and a multi-peptide vaccine [ 81 ]. The vaccine was unique and was developed to target the inherent plasticity in breast cancer aimed at generating T cells against both epithelial tumor cells as well tumor-initiating breast cancer stem cells. We found that combination therapy when given during rapid tumor growth was able to regress 75% of tumors and prolonged the vaccine-induced progression-free survival period. The combination resulting in regressing tumors were infiltrated with increased numbers of Tc1 and Tc2 CD8 T cells with a memory precursor phenotype. We also observed that combination therapy enhanced the antigen reactivity of tumor-infiltrating CD8 T cells as determined by increase in IFN-γ, TNF-α, IL-2, IL-5, and IL-4 production. Given the recent success of anti-PD-1 monotherapy, our results are encouraging for developing future combination therapies for the treatment of cancer patients. Alternatively, targeted therapeutics, such as small molecule inhibitors, could also work in concert with vaccines. An example is inhibitors of transforming growth factor-β (TGF-β), a growth factor with multiple roles in cancer [ 253 , 254 ]. TGF-β promotes tumor progression, invasion, and metastasis by inducing epithelial to mesenchymal transition (EMT), migration, and release of vascular endothelial growth factor [ 253 ]. TGF-β also directly inhibits cytotoxic actions of tumor-infiltrating CD8 T cells [ 255 ]. Thus, an agent that simultaneously blocks immunosuppression and tumor progression may be more effective by providing sufficient time for the immune system to expand and destroy residual tumor burden. Many combinatorial strategies are currently being tested and time will tell whether such mixing will be therapeutically effective.

Therapeutic vaccines are an attractive type of active immunotherapy that offers several advantages such as ease of manufacturing and non-autologous approach. There have been several types of vaccines developed for ovarian cancer such as protein, DNA, viral and peptide vaccines. Only a few recently developed novel vaccines with potential clinical activity are highlighted in this review. Le and colleagues recently reported a novel approach employing a Listeria monocytogenes (Lm) based vaccination. Lm is a highly pathogenic foodborne microorganism that causes serious central nervous system and gastrointestinal pathologies. The microorganism is unique in that it is an obligate intracellular bacterium that specifically infects myeloid cells such as DC and therefore could enhance antigen presentation. Le and colleagues recently used an attenuated Lm vector encoding mesothelin to vaccinate treatment refractory patients with various solid tumors including patients with ovarian cancer [ 246 ]. Patients were only given a single dose, the vaccine was well tolerated, and patients generated mesothelin immune responses in about 60% of immunized individuals. Importantly, although only a phase I trial, 37% of patients survived ≥ 15 months. These results are encouraging because the approach is not limited to specific HLA genotypes and could possibly overcome many of the problems, such as inefficient antigen presentation encountered with whole protein approaches. Peptide-based cancer vaccines are typically short-minimal fragments whose utility is limited to those patients with specific HLA molecules One way to overcome this problem, while avoiding issues related to deficient processing of whole recombinant protein is to use single or pooled synthetic long peptides [ 247 ]. This approach was used recently by Sabbatini and colleagues who developed a pool of NY-ESO-1 overlapping peptides [ 248 ]. Twenty patients with advanced ovarian cancer in second or third remission were given five challenges with vaccine in Montanide-ISA and poly-ICLC as vaccine adjuvants. Greater than 90% of patients developed immunity to the peptides and in some cases at very high levels, suggesting that this strategy is very efficient in immunizing against self antigens. It is less clear if the immune responses could easily recognize naturally processed whole protein. Nonetheless, some early indications of improved survival were observed in patients whose tumors demonstrated expression of NY-ESO-1. While most of the vaccines being tested in the setting of ovarian cancer target the generation of T cells, the fact that antibody immunity correlates with disease outcomes in patients makes antibody-inducing vaccines a logical option to explore. To that end, Kaumaya determined the activity of a HER-2/neu antibody-inducing chimeric peptide (aa 628-647, and aa 316-339) vaccine with a promiscuous T-cell epitope (aa 280-302) from the measles virus fusion protein (MVF) in patients with metastatic solid tumors including patients with advanced ovarian cancer [ 249 ]. This study demonstrated that this vaccine is moderately well tolerated (20% severe adverse events) and elicits IgG antibodies at all doses. Moreover, of six patients that showed clinical benefit, two were patients with ovarian cancer who had a high antibody response to the vaccine [ 249 ]. Overall, the few vaccine trials that have been conducted reveal that patients can mount immunity safely to tumor antigens paving the way for advanced clinical trials designed to test clinical efficacy. Optimism remains that vaccines may be able to, in subsets of patients, eradicate residual tumor to prevent recurrence or disease progression. Indeed, Hernando recently published a case report demonstrating vaccine potency in a single patient with metastatic ovarian cancer [ 250 ]. The patient had been previously treated twice with surgical debulking and platinum based therapy. At third recurrence, four months following the last therapy, the patient presented with an axillary lymph node metastasis and a para-aortic mass. At that time, a vaccination regimen with autologous dendritic cells engineered to express FRα was initiated. CT scans before and three months after the last treatment showed a marked, albeit partial, shrinkage in tumor. CA-125 levels which were rising just prior to vaccine, immediately declined to baseline following the first two vaccinations. A follow-up CT scan at sixteen months following the start of vaccination showed greater than 50% regression. Immune monitoring revealed that the patient developed strong FRα-specific T cell immunity to the vaccine. Remarkably, at eleven months following the last vaccine, CA-125 levels remained at baseline showing that vaccines can induce long-term remission.

Some studies suggest that chronic immune activation and inflammation is an important mediator of ovarian carcinogenesis [ 6 ]. Several theories exist regarding the causes of chronic inflammation. The incessant ovulation model postulates that multiple rounds of ovulation and the subsequent inflammatory response to the wound leads to an oncogenic event(s). Many epidemiologic studies have implicated that increased number of ovulations is a risk factor for ovarian cancer development [ 7 ]. For example, protection from ovarian cancer has been observed in association with increased parity [ 8 , 9 ], oral contraceptive use [ 10 , 11 ], breast feeding [ 12 , 13 ], older age at first menses (for premenopausal women) [ 14 ], all of which impact total lifetime number of ovulations. One of these studies, which has been further substantiated by more recent investigations, found protective effects of “anovulatory time” by combining information on both increased oral contraceptive use and parity as well as age at first and last menses [ 15 ] supporting the theory of incessant ovulation as a mechanism of oncogenesis. Recent studies support a role of macrophages in inducing DNA damage during repair of the wound left by ovulation. For example, one study found not only that superovulated mice had elevated macrophage counts in their oviducts, but that oxidative stress induced by hydrogen peroxide or macrophage-secreted mediators initiated DNA damage in immortalized baboon tubal epithelial cell lines [ 16 ]. Inflammation may also be due to chronic exposures to external triggers of immunity leading to persistent infiltration of immune cells that cause injury to surrounding epithelium, damage DNA through release of reactive oxygen species, or produce cytokines that promote proliferation. One proposed environmental exposure shown to induce inflammation in animal models is talcum powder [ 17 ]. Composed primarily of magnesium silicate, this moisture absorbing substance has been linked to ovarian cancer risk in a number of studies [ 6 , 18 - 20 ] although selection bias and confounding were strong concerns for the studies that found these associations [ 21 , 22 ]. The strong correlation between endometriosis and ovarian cancer also supports the chronic inflammation hypothesis. Endometriosis involves endometrial cells growing outside of the uterus and has been investigated extensively as a risk factor for ovarian cancer. Several epidemiologic studies have found an association between history of endometriosis and invasive epithelial ovarian cancer and most recently a pooled analysis of 13 case-control studies demonstrated that history of endometriosis increased the risk of clear cell, low grade serous, and endometrioid invasive ovarian cancers, but not mucinous or high grade serous ovarian cancers [ 23 ]. Exposure to pathogens might also cause chronic inflammation that would aid in tumor promotion and progression. Retrograde menstruation provides a potential mechanism by which pathogens could reach fallopian tube epithelial cells to establish infection [ 24 ]. While there is little evidence that specific pathogens contribute to most ovarian cancer cases, some studies have found a relationship between pelvic inflammatory disease and ovarian cancer risk. [ 6 , 25 , 26 ]. Consistent with the idea that chronic inflammation - whether mediated by ovulation induced damage, foreign agents, or both - is an underlying cause of ovarian cancer, non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with a decreased risk of ovarian cancer but results have been inconsistent [ 6 , 20 , 27 ]. Studies evaluating inherited single nucleotide polymorphisms (i.e. SNPs) support a role for chronic inflammation. SNPs in genes can affect expression and function of their protein products [ 28 ]. Apart from typical epidemiologic approaches, the ability to examine the impact of genetic variation in disease processes over the past decade represents a significant advance in understanding the role of the immune system in disease risk. While genome wide association studies to date have found significant associations with ovarian cancer and single nucleotide polymorphisms (SNPs) in genes that are not directly immune-related [ 29 - 31 ], several groups have conducted candidate gene studies that evaluated SNPs in immune-related genes, including NFKB1 [ 32 ], NOD2 [ 33 ], NFKBIA [ 34 ], CASP8 [ 35 , 36 ], MBL2 [ 37 ], PTGS2 [ 38 ], ALOX5 [ 39 ], IL1A [ 39 ], IL1R1 [ 40 ], TNFS10 [ 41 ] and IL18 [ 42 ]. While this is not an all-inclusive list of studies that have assessed immune related SNPs in relation to ovarian cancer risk, it does start to provide insight into contribution of immune genes in this disease. The suggestion that initiation of ovarian cancer is mediated by inflammation is supported by studies examining blood-based markers of inflammation including C-reactive protein (CRP). CRP levels in the blood rise in response to IL-6 released during local inflammatory processes, [ 43 ]. Its physiological role is to bind to phosphocholine on the surface of dead cells in order to activate the complement system. CRP protein levels in the blood are associated with a wide range of diseases, such as diabetes and atherosclerosis [ 44 ]. Toriola showed in the Finnish Maternity Cohort case-control study that there was an association between elevated pre-diagnostic CRP levels in the blood and ovarian cancer (OR=2.0, 95% CI: 1.1-3.4) [ 45 ]. In another analysis, which included participants from three pooled prospective nested cohort studies, Lundin and colleagues reported a similar finding in which high levels of CRP were associated with a 4.4 fold increased risk of developing ovarian cancer [ 46 ]. The increased availability of assays to determine cytokine levels has recently led to studies evaluating other circulating markers of inflammation. Using a nested approach with three prospective cohort studies, Clenenden and colleagues found that IL-6, as well as IL-2, IL-4, IL-12, and IL-13 levels were associated with increased risk of developing epithelial ovarian cancer [ 47 ]. Overall, the studies demonstrating that elevated CRP and IL-6 levels are associated with an elevated risk of ovarian cancer support a role for inflammation, likely subclinical, in initiating the disease. In summary, there is support of the notion that chronic inflammation in the reproductive tract is involved in ovarian cancer development. Unfortunately, to the best of our knowledge, no studies have been reported which directly compare in either a prospective cohort or case-control setting that smoldering subclinical inflammation drives the development of neoplasia of the ovary. With high-throughput genetic approaches having been developed in recent years, such as genome-wide SNP analysis and multiplexed cytokine analysis, it has become possible to interrogate mechanisms at the population level. The studies being largely association-based remain difficult to interpret at present, particularly the genetic studies; however, the identified targets provide a future pipeline of possible immunologic targets to prevent ovarian cancer onset ( Figure 1 ).

A completely different kind of inflammation follows tumor development [ 48 ]. Observations that date back several decades have established that there are natural immune responses to ovarian tumors and these immune responses have a profound impact on the clinical course of the disease. Although infiltration of immune effectors into ovarian cancers was observed as early as 1982 by Haskill and colleagues [ 49 ] it would not be until nearly two decades later that the prognostic significance of these cells was appreciated. In their seminal publication in 2003, Zhang and colleagues showed that T cell infiltration into ovarian tumors was associated with improved survival [ 50 ]. Among 74 patients with complete clinical responses after debulking and platinum-based therapy, the five-year survival rate was an extraordinary 73.9% among those patients with CD3 + T cells within their tumor compared to 11.9% among patients without infiltrating T cells [ 50 ]. This study also revealed that in tumors with high numbers of tumor-infiltrating T cells, the expression of monokines induced by IFN-γ, macrophage-derived chemokines, and secondary lymphoid-tissue chemokines were significantly increased as compared with tumors lacking T cells, which may represent novel targets for improving anti-tumor immunity [ 50 ]. CD8 cytotoxic T lymphocytes (CTL), thought to be the primary mediators of anti-tumor immune responses, recognize antigens displayed in the context of major histocompatibility complex (HLA) class I molecules expressed on ovarian cancer cells. To examine a role of CTL in ovarian cancer, Sato and colleagues studied 117 ovarian cancer cases and found improved survival in patients who had higher numbers of infiltrating CD8+ T cells compared to patients without infiltrating CD8 T cells (median survival 55 vs. 26 months) [ 51 ]. These findings were largely confirmed by Leffers [ 52 ]. Transcriptomic profiling of serous ovarian tumors with high and low CD8 T cell infiltration found two genes differentially expressed in cancers with high versus low CD8 T cell infiltration, namely, interferon regulatory factor 1 (upregulator of MHC class I) and C-X-C chemokine receptor 6 (enhances T cell proliferation) [ 53 , 54 ]. The role of CD4 helper T cells is less clear due to shared expression with regulatory T cells (Tregs). Both Sato and Milne observed similar outcomes among patients with or without CD4+ T cell staining within tumors [ 51 , 55 ] while Le Page observed that increased CD4+ cells in 199 high grade serous cancers were associated with improved survival [ 56 ]. Kryczek and colleagues found that high levels of IL-17 ascites were associated with greatly improved outcome suggesting that Th17 cells, a subset of CD4 helper T cells that produce IL-17, may have a direct role in eradicating tumor [ 57 ]. Given the abundant expression of IL-17 among innate immune effectors, it is difficult to draw a conclusion as to whether intratumoral Th17 cells might impact the clinical course of the disease [ 58 ]. However, the fact that levels of Th17 cells have a strong inverse correlation with Tregs, suggests that they might be associated with tumor eradication [ 57 ]. Other specific subsets of potential antitumor immune effectors have also been studied but with less than clear results. The generation of antibody responses to ovarian cancer is a common observation suggesting a role for B cells in disease prognosis [ 59 - 61 ]. Although antibody-secreting B cells do not need to be at the tumor site to exert antitumor activity, studies evaluating whether B cell infiltration is associated with improved survival show mixed results [ 55 , 62 ]. Another recent evaluation of infiltrating cells constructed from tumors of women with high grade serous ovarian cancer from 3 patient cohorts, found that infiltration of tumors by both CD8+ and CD20+ (B cell marker) lymphocytes (the two subsets were correlated and often found near each other within the tumor) was associated with increased survival [ 63 ]. The CD20+ B cells did not express CD27, a memory B cell marker, but IgG sequencing suggested they had been exposed to antigen. The authors hypothesized that these cells are likely involved in modulating CTL recruitment or activity [ 63 ]. Natural killer (NK) cells are also found in the ovarian cancer microenvironment. NK cells are a group of lymphocytes that have two different mature phenotypes, either CD16+ CD56 dim NK cells which are highly cytolytic and found in the periphery, or CD16-CD56 bright NK cells which are less cytolytic and found in the secondary lymphoid tissue [ 64 ]. CD16+ CD56 dim NK cells have two types of surface receptors: activating (i.e., NKG2D) receptors and inhibitory (i.e., KIR) receptors, and can lyse cells without having to recognize specific antigens [ 65 ]. The balance between inhibitory and activating signals through the different NK receptors is important in the NK cell activation process. NK cells can also be activated in an antigen-dependent manner to mediate antigen-dependent cellular cytotoxicity, which requires antibody binding to Fc receptors on the NK cells. The activating NKG2 receptors bind to stress ligands such as MICA, MICB, and ULBP1-3, while KIR receptors bind to major histocompatibility complex class I and class I associated ligands [ 65 , 66 ]. Nearly all ovarian cancers express the NK ligands MICA, MICB and ULBP2 [ 67 ]. Despite the potential antitumor activity, increased numbers of NK cells in peritoneal and pleural effusions of metastatic ovarian carcinoma has been associated with poor rather than good prognosis [ 62 ]. Immune evasion in ovarian tumors involves a complex array of immune suppressive factors and cells that effectively halt the generation and clonal expansion of anti-tumor immunity ( Figure 2 ). Genetic changes also occur, permitting the tumor cells to be ignored by the immune response. Immune suppression is mediated by factors released from the tumor or by infiltration of the tumors by a variety of either lymphoid or myeloid derived suppressor or regulatory cells. The role of CD4 + Tregs in ovarian cancers is well characterized. Tregs are a heterogeneous T cell subpopulation whose primary function is suppression of immune responses. CD4 + Tregs are either thymically-matured or induced from naïve CD4 T cells [ 68 , 69 ]. While induced Tregs produce immune-suppressive soluble mediators such as TGF-β and IL-10 to mediate their inhibitory activities and block T cell proliferation, thymically-matured Treg cells use cytokine-dependent, cell contact-dependent, or a cytokine/cellular contact-dependent mechanisms to halt T cells response [ 68 ]. Tumors can increase the numbers of Tregs in the peripheral blood of cancer patients as reported for several tumor types, including ovarian [ 70 - 73 ]. Tumors can also recruit or induce Treg tumor infiltration. The connection between pathogenesis of Tregs and prognosis in ovarian cancer was first suggested by Curiel who showed that infiltration of Tregs (as assessed by CD4 and CD25 staining) was associated with poor patient survival [ 74 ]. Several other manuscripts subsequently published, also demonstrated the importance of Tregs in ovarian cancer pathogenesis and outcome. Wolf and colleagues showed that patients with low levels of intratumoral Foxp3 message had substantially improved survival compared to patients with high levels [ 75 ]. Our group recently demonstrated that high grade serous ovarian cancer is infiltrated with CD4 + CD25 + Foxp3 + Tregs and that a low ratio of infiltrating CD8 T cells and these triple stained Tregs is associated with poor survival [ 73 ]. In addition to lymphoid-derived regulatory cells, the myeloid arm of the immune system can give rise to multiple regulatory species including macrophages, dendritic cells, and myeloid derived suppressor cells (MDSCs). Macrophages normally infiltrate healthy tissue where they carry out various functions such as clearing dead cells and surveying for infectious agents. When faced with an infectious challenge, macrophages typically activate and differentiate into an activating phenotype often referred to as an M1 phenotype. Macrophages that infiltrate tumors, in contrast, differentiate into an alternative phenotype referred to as an M2 phenotype which is characterized by tissue regeneration responses and local immune suppression [ 76 ]. Hagemann first demonstrated that ovarian cancer cells can direct macrophages to become M2 [ 77 ]. Ko later showed that this was due to the high level expression of the homeobox transcription factor, HOXA9 [ 78 ]. A few studies have assessed, using pan macrophage CD68 staining, whether there is an association between macrophages and ovarian cancer survival, finding no effect [ 55 , 79 ]. However, one study that assessed M2 marker, CD206, found that patients with tumors that had a higher proportion of CD206+ relative to total CD68+ cells also had an increased risk of disease progression [ 56 ]. In agreement, Lan and colleagues reported a similar finding using CD163, another M2 macrophage marker [ 80 ]. Thus, strategies aimed at selectively depleting M2 macrophages may aid in immune-mediated tumor regression. Indeed, we have recently shown in murine models that co-immunization targeting both oncogenes and M2 macrophages can lead to regression of established tumors [ 81 ]. DCs are classified into two general types: plasmacytoid DCs (pDCs) which are characterized as CD123 + , CD45RA + CD8 + CD11c − ILT3 + ILT1 − Lin − , and myeloid DCs (mDCs) which are CD11c + , CD33 + CD45RA − CD123 − [ 82 ]. Under normal conditions, DCs are present in the body in an immature form and are responsible for detecting danger and sampling of antigens in various tissues. Upon detection of danger through their pathogen-associated molecular pattern (PAMP) receptors or danger-associated molecular pattern (DAMP) receptors, DC mature and migrate to the lymph nodes to activate T helper cells and CTL [ 82 ]. Although tumors are able to produce danger signals, they are ineffective in inducing DC maturation and trafficking to lymph nodes which is thought to be due to tumor-induced alterations in DC differentiation, thus reducing the number of functional cells available for effective T cell activation [ 83 ]. For example, ovarian cancers secrete large amounts of IL-10 which promotes redifferentiation of mDCs to an aberrant phenotype with reduced T cell activation properties [ 83 ]. In murine modeling studies, our group has found that suppressor mDCs within ovarian cancers are induced to express high levels of PD-1 which reversibly suppresses expression of co-stimulatory molecules, reduces migration, and blocks responsiveness to danger signals [ 84 ]. More recent work also shows that PD-1 ligation of infiltrating mDCs suppresses IL-7 receptor expression on infiltrating T cells, thereby limiting persistence of T cells in the tumor bed [ 85 ]. Blockade of PD-1 on DCs results in reversal of immune suppression. These mDCS also express high levels of PD-L1 which is the primary means by which these DCs suppress T cell activation. Interestingly, PD-L1 is not downregulated on mDCs when DC-bound PD-1 is blocked even though the immune suppressive properties of the mDCs are reversed suggesting that the immunogenic properties of tumor infiltrating mDCs is regulated by a balance of immune suppressive or activating molecules. mDCs in their immature states are also known promoters of angiogenesis and vasculogenesis during tumor growth [ 86 - 88 ]. Human ovarian cancers have also been observed to recruit immature pDCS which induce T cells to release large amounts of IL-10 preventing local T cell activation [ 89 ]. This recruitment is thought to involve SDF-1 (produced by the tumor) ligation of CXCR4. CXCR4 plasmacytoid precursor cells (preDC2s) are attracted into the tumor microenvironment by tumor derived stromal cell-derived factor 1 (SDF-1). Induction of IL-10 is due, at least in part, to local induction of IL-10 + regulatory CD8 T cells [ 90 ]. Overall, the DC component of ovarian cancers remains an intriguing and underexplored area of ovarian cancer research and could represent a therapeutic target. This concept is borne out of recent murine modeling studies demonstrating improved antitumor immunity following specific depletion of DCs [ 91 ]. Apart from pDCs and mDCs, there is another subset of myeloid-derived cells, called myeloid-derived suppressor cells (MDSCs), that have an increased ability to block local and systemic immune activation [ 92 ]. This is a heterogeneous population consisting of macrophages, DCs and granulocytes at early stages of differentiation [ 87 ]. MDSCs are classified into two subpopulations, granulocytic and monocytic MDSCs which, in mice but not humans, are uniquely identified by co-expression of CD11b and Gr1 [ 93 ]. MDSCs are known to expand dramatically under various health conditions such as tumor growth, inflammation, and infection [ 87 , 94 , 95 ], and may have a role in immune suppression of ovarian cancer in murine models; however, their relevance in human ovarian cancers remains uncertain [ 96 ]. Aside from the recruitment of suppressive cells into the tumor microenvironment, tumor cells themselves express a variety of molecules that directly block immune responses. PD-L1 is expressed on the surface of several gynecological cancers including ovarian and is associated with poor overall survival in ovarian cancer [ 97 - 99 ]. Its ligand, PD-1 is expressed on, not only on infiltrating mDCs but also on various other adaptive immune effectors, notably CD4 and CD8 T cells, where it negatively regulates cell activation in cancers including ovarian [ 100 - 103 ]. Another suppressive molecule is MUC16 (CA-125). MUC16 augments the immune suppressive tumor microenvironment by inhibiting the activity of NK cells [ 104 - 107 ]. A recent study by Gubbels and colleagues sheds more light into the role and importance of MUC16 on cancer cells [ 106 ]. They showed that MUC16 acts as an inhibitor of the NK-tumor conjugation. NK cells killed cells expressing low levels of MUC16 more effectively, with ~20% more NK cell lysis and a 2-3 fold increase in NK leukemia cells lysis when compared with lysis of cells with high MUC16 expression [ 106 ]. Furthermore, a recent study by Krockenberger and colleagues showed that macrophage migration inhibitory factor (MIF, inducer of inflammation and promoter of tissue repair) inhibits the antitumor immune response against ovarian cancer cells by down regulating NKG2D receptor in NK cells [ 108 ]. Another mechanism by which several different types of immune cells are suppressed in the tumor microenvironment is through the production of indoleamine 2, 3-dioxygenase (IDO) [ 109 , 110 ]. IDO mediates immune tolerance in T cells and natural killer cells by consuming tryptophan and forming kynurenine resulting in inhibition of proliferation and functional impairment [ 111 - 115 ]. It also promotes a regulatory T-cell phenotype [ 116 - 118 ]. IDO is expressed by many tumor types, including ovarian cancer [ 119 - 121 ]. Takao and colleagues reported protein expression of IDO in serous (57.5%), clear cell (49.2%), and endometrioid (73.3%) ovarian adenocarcinomas [ 121 ]. They additionally found an association between IDO staining in ovarian cancer tumors and lower overall survival for serous ovarian cancer patients with advanced stage disease who had optimal surgery and were given first-line paclitaxel-carboplatin chemotherapy [ 121 ]. Inaba and colleagues reported a similar association between high compared to low expression of IDO in ovarian cancer tumors of mixed histology, stage and grade and lower overall and progression-free survival. In addition, they found reduced numbers of CD8+ TILs in the tumors expressing high IDO [ 119 ]. IDO plays an important role in immune suppression and tumor immune evasion and may have potential as a therapeutic target. Indeed, several studies have investigated the efficacy of blocking IDO in tumor models.[ 119 , 122 - 126 ]. Several matters complicate targeting IDO, however. For example, there are two IDO genes( IDO1 and IDO2 ) which produce products that contribute to tryptophan metabolism (reviewed in Soliman [ 127 ]). Additionally, two isomers of the most commonly studied inhibitor, 1-methyl-tryptophan (1MT), exist [ 128 , 129 ]. Many early studies used a racemic mixture of the two, however, a study by Metz found that the L isomer of 1MT, preferentially inhibits IDO1 while the D isomer preferentially inhibits IDO2 [ 130 ]. In vitro studies demonstrated that the L isoform was superior in restoring T cell and antigen presenting functions in vitro . However, the D isomer, in conjunction with conventional chemotherapy drugs, was more potent than the L isomer in in vivo studies [ 130 ]. Currently, which isoform to target is not well understood. Third, in addition to IDO1 and IDO2, increased expression of TDO (tryptophan 2, 3-dioxygenase), normally expressed in the liver, has been reported in several different tumor types, including ovarian cancer [ 131 ]. Kynurenin, one of the metabolites of TDO binds aryl hydrocarbon receptor, causing this transcription factor to translocate to the nucleus and activate several genes, including those activating Tregs [ 131 , 132 ]. The structure of TDO differs greatly from IDO1 and IDO2 and IMT is unable to inhibit the enzyme. Pilotte and colleagues found that a derivative (LM10) of a known TDO inhibitor (680C91) was able to prevent the growth of TDO expressing tumors [ 133 ].