Hannah Goyne

Dendritic cell vaccination against ovarian cancer--tipping the Treg/TH17 balance to therapeutic advantage?

The pathology of ovarian cancer is characterized by profound immunosuppression in the tumor microenvironment. Mechanisms that contribute to the immunosuppressed state include tumor infiltration by regulatory T cells (Treg), expression of B7-H1 (PDL-1), which can promote T cell anergy and apoptosis through engagement of PD-1 expressed by effector T cells, and expression of indoleamine 2,3-dioxygenase (IDO), which can also contribute to effector T cell anergy. Expression of both B7-H1 and IDO has been associated with differentiation and recruitment of Treg, and clinical studies have shown that each of these mechanisms correlates independently with increased morbidity and mortality in patients with ovarian cancer. In a remarkable counterpoint to these observations, ovarian tumor infiltration with TH17 cells correlates with markedly improved clinical outcomes. In this Future Perspectives review, we argue that dendritic cell (DC) vaccination designed to drive tumor-antigen-specific TH17 T cell responses, combined with adjuvant treatments that abrogate immunosuppressive mechanisms operative in the tumor microenvironment, offers the potential for clinical benefit in the treatment of ovarian cancer. We also discuss pharmacological approaches to modulation of MAP kinase signaling for manipulation of the functional plasticity of DC, such that they may be directed to promote TH17 responses following DC vaccination.

Modulation of p38 MAPK signaling enhances dendritic cell activation of human CD4+ Th17 responses to ovarian tumor antigen

The recent finding that Th17 infiltration of ovarian tumors positively predicts patient outcomes suggests that Th17 responses play a protective role in ovarian tumor immunity. This observation has led to the question of whether Th17 cells could be induced or expanded to therapeutic advantage by tumor vaccination. In this study, we show that treatment of ovarian tumor antigen-loaded, cytokine-matured human dendritic cells (DC) with a combination of IL-15 and a p38 MAP kinase inhibitor offers potent synergy in antagonism of CD4+ Treg induction and redirection toward CD4+ Th17 responses that correlate with strong CD8+ cytotoxic T lymphocyte (CTL) activation. Ovarian tumor antigen-specific CD4+ T cells secrete high levels of IL-17 and show reduced expression of CTLA-4, PD-1, and Foxp3 following activation with IL-15/p38 inhibitor-treated DC. We further show that modulation of p38 MAPK signaling in DC is associated with reduced expression of B7-H1 (PD-L1), loss of indoleamine 2,3-dioxygenase activity, and increased phosphorylation of ERK 1/2 MAPK. These observations may allow the development of innovative DC vaccination strategies to boost Th17 immunity in ovarian cancer patients.

Ovarian tumor ascites CD14+ cells suppress dendritic cell-activated CD4+ T-cell responses through IL-10 secretion and indoleamine 2,3-dioxygenase.

The observation that Th17 infiltration in ovarian cancer correlates with markedly improved survival has prompted the question of whether ovarian tumor antigen–specific Th17 responses could be stimulated by tumor vaccination. Dendritic cells (DCs) treated with IL-15 and an inhibitor of p38 MAPK signaling (DCIL-15/p38inhib) bias T-cell responses toward a Th1/Th17 phenotype, raising the prospect of therapeutic vaccination; however, significant barriers remain. Tumor vaccines, including DC vaccination, usually stimulate immune responses, but the lack of clinical responses in cancer patients has been disappointing. Possible reasons may include an inability of antitumor T cells to migrate into the tumor microenvironment, and an inability of T cells to retain effector function in the face of tumor-associated immune suppression. We found that ovarian tumor antigen–specific CD4+ T cells induced by DCIL-15/p38inhib migrated in response to CXCL12 and CCL22 (both highly expressed in ovarian cancer) and to ascites CD14+ myeloid cells. Cocultures showed that ascites CD14+ cells markedly suppressed antigen-specific CD4+ T responses, but suppression could be alleviated by treatment with anti-IL-10 or inhibition of indoleamine 2,3-dioxygenase. These results suggest that the efficacy of DC vaccination against ovarian cancer may be boosted by agents that inhibit tumor-associated CD14+ myeloid cell suppression or indoleamine 2,3-dioxygenase activity.

Dendritic Cell Vaccination, Immune Regulation, and Clinical Outcomes in Ovarian Cancer

Clinical optimism for dendritic cell vaccination against ovarian cancer has been tempered by the knowledge that tumors avail themselves of multiple mechanisms of immune evasion, thus blunting the efficacy of therapeutic vaccination. Mechanisms of immune suppression include infiltration by regulatory T cells (Treg) and myeloid suppressor cell populations, expression of co-inhibitory receptors, and expression of indoleamine 2,3-dioxygenase (IDO). Expression of both B7-H1 and IDO are associated with differentiation and recruitment of Treg, and clinical studies have shown that each of these mechanisms correlates independently with increased morbidity and mortality in ovarian cancer patients. In sharp contrast, recent studies have indicated that Th17 cell infiltration in ovarian cancer correlates with improved patient outcomes and prolonged overall survival. Given that IDO plays a pivotal role in the balance between Treg and Th17 immunity, elucidation of the mechanisms that regulate IDO activity and immune suppression may lead to novel adjuvants to boost the clinical efficacy of dendritic cell vaccination against ovarian cancer and other malignancies.

Cystoisospora belli Infection of the Gallbladder in Immunocompetent Patients: A Clinicopathologic Review of 18 Cases

Cystoisospora belli, previously known as Isospora belli, is an obligate intracellular coccidian parasite that is most often associated with gastrointestinal disease in immunocompromised patients. In this study, we detail the clinicopathologic features of 18 cases of Cystoisospora infection affecting the gallbladder in immunocompetent individuals and compare them with a control group. Each case was reviewed for cholecystitis (none, acute, chronic), epithelial disarray, presence of intraepithelial lymphocytes (none, rare [⩽5 per 20 epithelial cells], present [>5 per 20 epithelial cells]), architectural distortion, intramucosal eosinophilia, and mural thickening/serositis. The mean age of patients with Cystoisospora infection was 33 years and the male to female ratio 1:4.3. Cholecystectomy was performed for biliary dyskinesia (n=7), abdominal pain (n=7), suspected cholelithiasis (n=5), and cholecystitis (n=3). In 2 cases, Cystoisospora was found in donor gallbladders resected at the time of liver transplantation. Each case was characterized by eosinophilic, oval or banana-shaped intraepithelial parasites within perinuclear parasitophorous vacuoles. Most cases showed epithelial disarray and minimal intraepithelial lymphocytosis. Of the 11 cases with an average follow-up of 15 months, none had evidence of disease related to Cystoisospora infection within the biliary tract or elsewhere in the gastrointestinal tract. We present the largest series of gallbladder cystoisosporiasis in immunocompetent patients to date. Cystoisospora infection is underrecognized in the gallbladders of immunocompetent patients, in part due to the subtle findings in routine cholecystectomy specimens. On the basis of the clinical follow-up, gallbladder cystoisosporiasis in immunocompetent individuals appears to be a self-limited infection.

Combinatorial strategies for alleviation of tumor‑associated immune suppression and therapeutic vaccination against ovarian cancer

The prospects for development of therapeutic cancer vaccines have enjoyed a renaissance in recent years, with further encouragement accruing from the US FDA’s approval in 2010 of Provenge® (Dendreon Corporation, Seattle, WA, USA), an autologous dendritic cell (DC) vaccine for prostate cancer. Provenge was the first active cellular therapy to reach the market, breaking through what was widely perceived to be a glass ceiling for cellular therapeutics and cancer vaccines. This renewed optimism has also been fuelled by advances in our knowledge of the mechanisms of immune regulation in the tumor microenvironment. This is particularly true for ovarian cancer, which represents a paradigm for self-defense against immunological attack. Although ovarian cancer recruits multiple mechanisms of immune suppression, thus presenting a formidable barrier to effective tumor vaccination, an understanding of these mechanisms may yield opportunities for adjuvant treatments that counterbalance immune suppression and promote vaccine-induced antitumor immunity. The various mechanisms of immune suppression employed by ovarian cancer are of more than academic interest to tumor immunologists, since many parameters of immune function have a direct impact on the course of disease. A groundbreaking study from Curiel and colleagues showed that ovarian tumors recruit immuno suppressive Tregs, thus conferring immune privilege, and that Treg infiltration was associated with poor prognosis and increased mortality [1]. These observations demonstrated that immune regulation has a direct and profound relationship with the pathogenesis of disease. Other parameters of immune regulation in ovarian cancer also influence patient outcomes, including expression of B7-H1 (PD-L1) by ovarian tumor cells and infiltrating macrophages or DCs, resulting in T-cell anergy and apoptosis through the engagement of PD-1 expressed by effector T cells [2,3]. Expression of B7-H1 has been associated with differentiation and recruitment of Treg [4], and clinical studies have shown that B7-H1 expression correlates with increased morbidity and mortality in ovarian cancer patients [5]. Expression of B7-H4 (a related member of the family of immunomodulatory accessory receptors) by ovarian tumor-associated macrophages has also been correlated with Treg infiltration and adverse patient outcomes [6]. Two independent studies have further shown that ovarian tumor expression of indoleamine 2,3-dioxygenase (IDO) is associated with poor prognosis [7,8]. IDO first came to prominence for its role in immune suppression and fetal protection at the maternal–fetal interface, and is now widely acknowledged to be an important mediator of T-cell anergy through tryptophan catabolism. IDO is known to be expressed by ovarian tumor cells, and most probably by infiltrating myeloid-derived suppressor cells, including DC and macrophages. Of particular note, IDO expression drives the differentiation and recruitment of Treg by DCs [9], and blocks the conversion of Treg to p roinflammatory Th17 cells [10,11]. In contrast to the evidence that Treg infiltration is associated with poor outcomes in ovarian cancer, a counterpoint is furnished by the recent observation that Th17 cell infiltration correlates with more favorable clinical outcomes [12]. Tumor-infiltrating Th17 cells were positively associated with effector cells and negatively associated with Treg infiltration, leading to the question of whether Th17 cells could be induced or expanded to therapeutic advantage, either by tumor vaccines or adoptive immunotherapy [13]. There is a substantial body of evidence suggesting that Th17 responses can be induced in vitro with proinflammatory cytokines, notably IL-1β. Such an approach may be viable for adoptive T-cell immunotherapy of ovarian cancer, but the adjuvant use of cytokines in support of tumor vaccination is likely to carry a significant risk of off-target toxicities. Of the alternatives, DC vaccination designed to drive ovarian tumor antigen-specific Th17 responses appears to be the strongest option [14]. In comparison with other approaches to tumor vaccination, the plasticity of DC function allows greater scope for fine-tuning T-cell responses, and currently available evidence suggests that modulation of MAPK signaling in DCs may provide the cornerstone for stimulation of Th17 immunity [14]. If we accept the premise that tumor antigen-specific Th17 responses will be of therapeutic benefit for ovarian cancer patients, either through DC vaccination or alternative approaches to tumor vaccination, we still have to confront the myriad mechanisms of immune suppression in the ovarian tumor microenvironment, which remain the greatest barrier to effective antitumor immunity. The history of tumor vaccination has shown that stimulation of tumor antigen-specific T-cell responses is relatively facile, yet translation to clinical efficacy has remained stubbornly elusive. The judicious use of adjuvants designed to alleviate tumor-associated immunosuppression may furnish the key to successful therapeutic vaccination. Several chemotherapeutic agents have shown evidence of activity against Treg, including fludarabine, imatinib mesylate (Gleevec®, Novartis, Basel, Switzerland) and, most notably, cyclophosphamide, which has long been known for its ability to inhibit Treg suppression and potentiate cellular immune responses against tumors [15]. Cyclophosphamide is gaining popularity as an adjuvant in clinical trials of tumor vaccines, and has the advantages of being cheap, well tolerated and easy to administer orally. In a remarkable twist to the story, a recent report has shown that cyclophosphamide may also promote Th17 cell differentiation in cancer patients [16], further adding to its appeal as an adjuvant for clinical trials of ovarian tumor vaccines. We should also consider the root causes of Treg recruitment in the ovarian tumor microenvironment. Treg infiltration is driven by expression of CCL22 [1], of which the most prominent sources are likely to be tumor-associated macrophages and DCs. Treg differentiation and expansion may be promoted by multiple mechanisms, including local expression B7-H1 and B7-4, both of which correlate with Treg infiltration in ovarian tumors, and expression of IDO, which may also antagonize Th17 differentiation. A notable feature is that all of these mechanisms are independent prognostic indicators of poor outcomes, and all may be mediated by tumor-associated macrophages or DCs. Accordingly, adjuvant treatments that modify the function of tumor-infiltrating myeloid suppressor cells may have a profound impact on Treg recruitment, local immunity and clinical responses to vaccination. There is some evidence from animal studies that gemcitabine, used as a single agent [17] or in combination with a cyclooxygenase inhibitor [18] has activity against myeloid suppressor cells, preferentially depleting this population in tumor-bearing mice. However, absolute depletion of myeloid cells may not be the most favorable course of action, since tumor-associated macrophages and, to a lesser extent, myeloid DCs also have the ability to stimulate Th17 responses [12]. An alternative course of action may reside with the use of agents that modulate myeloid suppressor cell activity, a function that could be fulfilled by adjuvant chemotherapy with aminobisphosphonates. This class of drugs, which includes zoledronate and pamidronate, inhibits metastatic spread to the bone, and can also impair angiogenesis by blocking VEGF signaling and MMP-9 expression by tumor-associated macrophages. Zoledronate can block myeloid-derived suppressor cell expansion and proangiogenic activity [19], and recent evidence points to a proinflammatory shift in myeloid cell function, favoring polarization from a protumor M2 phenotype to an antitumor M1 phenotype with reduced expression of VEGF and IL-10 and increased production of type I cytokines such as IFN-γ [20,21]. Although the available clinical evidence provides a strong rationale for the design of tumor vaccines that promote Th17 immunity in ovarian cancer patients [12,13], this is likely to be a contentious issue, since the various studies that show therapeutic benefit from Th17 immunity are counterbalanced by reports that indicate a role for IL-17 in tumor progression [14]. We believe that Th17-based therapeutic vaccines for ovarian cancer should be tested in clinical trials, but there remains the question of whether vaccination alone would be sufficient to provide clinical benefit in the face of continuing local immune suppression in the tumor microenvironment. The long history of clinical disappointment from tumor vaccination has clearly shown that stimulation of antitumor immunity is not in itself sufficient, and that there is a clear need for adjuvant treatments that create a more favorable environment for vaccine efficacy. Cyclophosphamide has obvious appeal, since it may help to support vaccine-driven Th17 responses in addition to abrogating Treg activity. We also feel that there is a strong case for targeting tumor-associated macrophages/myeloid suppressor cells, since these populations are likely the drivers that fuel Treg infiltration, differentiation and expansion in ovarian tumors.

The case for HER2/neu as a therapeutic target for gynecologic malignancies

Evaluation of: Guzzo F, Bellone S, Buza N et al. HER2/neu as a potential target for immunotherapy in gynecological carcinosarcomas. Int. J. Gynecol. Pathol. 31, 211-221 (2012). Trastuzumab (Herceptin(®)) is a human monoclonal antibody that is US FDA-approved for the treatment of HER2/neu-overexpressing breast cancer and metastatic gastric cancer. HER2/neu expression has also been observed in other malignancies, notably in subsets of endometrial and ovarian cancers, raising the possibility that trastuzumab may be a viable treatment option in these settings. The current paper reveals that HER2/neu may also be expressed in gynecologic carcinosarcomas, which are rare but aggressive tumors with a high rate of resistance to chemotherapy. Cell lines derived from ovarian carcinosarcomas expressed HER2/neu and were sensitive to trastuzumab ADCC. By contrast, cell lines derived from uterine carcinosarcomas were HER2/neu-negative and insensitive to trastuzumab ADCC. These observations indicate that at least a subset of gynecologic carcinosarcoma patients may benefit from trastuzumab treatment. However, trastuzumab monotherapy typically has relatively low response rates, which in part may be related to impaired NK cell function in patients with advanced disease. Accordingly, treatments that boost NK cell activity may improve response rates to trastuzumab treatment, not only for gynecologic carcinosarcomas, but also for other HER2/neu-positive malignancies.

Platform-independent gene expression signature differentiates sessile serrated adenomas/polyps and hyperplastic polyps of the colon

BackgroundSessile serrated adenomas/polyps are distinguished from hyperplastic colonic polyps subjectively by their endoscopic appearance and histological morphology. However, hyperplastic and sessile serrated polyps can have overlapping morphological features resulting in sessile serrated polyps diagnosed as hyperplastic. While sessile serrated polyps can progress into colon cancer, hyperplastic polyps have virtually no risk for colon cancer. Objective measures, differentiating these types of polyps would improve cancer prevention and treatment outcome.MethodsRNA-seq training data set and Affimetrix, Illumina testing data sets were obtained from Gene Expression Omnibus (GEO). RNA-seq single-end reads were filtered with FastX toolkit. Read mapping to the human genome, gene abundance estimation, and differential expression analysis were performed with Tophat-Cufflinks pipeline. Background correction, normalization, and probe summarization steps for Affimetrix arrays were performed using the robust multi-array method (RMA). For Illumina arrays, log2-scale expression data was obtained from GEO. Pathway analysis was implemented using Bioconductor package GSAR. To build a platform-independent molecular classifier that accurately differentiates sessile serrated and hyperplastic polyps we developed a new feature selection step. We also developed a simple procedure to classify new samples as either sessile serrated or hyperplastic with a class probability assigned to the decision, estimated using Cantelli’s inequality.ResultsThe classifier trained on RNA-seq data and tested on two independent microarray data sets resulted in zero and three errors. The classifier was further tested using quantitative real-time PCR expression levels of 45 blinded independent formalin-fixed paraffin-embedded specimens and was highly accurate. Pathway analyses have shown that sessile serrated polyps are distinguished from hyperplastic polyps and normal controls by: up-regulation of pathways implicated in proliferation, inflammation, cell-cell adhesion and down-regulation of serine threonine kinase signaling pathway; differential co-expression of pathways regulating cell division, protein trafficking and kinase activities.ConclusionsMost of the differentially expressed pathways are known as hallmarks of cancer and likely to explain why sessile serrated polyps are more prone to neoplastic transformation than hyperplastic. The new molecular classifier includes 13 genes and may facilitate objective differentiation between two polyps.

Mo1973 Immunohistochemical Markers Differentiating Colonic Sessile Serrated Adenomas/Polyps (SSA/Ps) From Hyperplastic Polyps

Differentiating SSA/Ps from hyperplastic polyps (HPs), with little or no risk for progression to cancer, can be a major challenge using conventional endoscopic and histological examinations. SSA/Ps are frequently right sided, flat and have a mucus cap. The goal of this study was identifying potential immunohistochemical (IHC) markers that help to differentiate SSA/ Ps from HPs. Methods: We compared RNA sequencing transcriptional signatures of SSA/Ps (n=15) and control (n=20) colonic mucosa from patients undergoing screening colonoscopy. Reads from the analysis were aligned to the GRCh37/Hg19 human reference genome and filtered for high quality alignments (http://novocraft.com). Hierarchical clustering of log2 ratios comparing SSA/Ps and controls were performed and mined for candidate IHC targets. IHC analysis was performed on formalin-fixed paraffin-embedded sections of 12 SSA/Ps, 10 HPs, 5 adenomas, 5 colon cancers and 5 control colon specimens to assess staining for selected antigens. Expression of MUC5AC and TFF1 protein were quantified with ImageJ software. Results: Bioinformatics analysis identified 1,294 (≥1.5-fold change) differentially expressed annotated genes (false discovery rate (FDR) <0.05) in SSA/Ps as compared to normal controls. The dataset was mined for the most highly expressed genes and genes with possible biological functions related to SSA/Ps. A total of 249 genes were overexpressed ≥5fold in SSA/Ps compared to controls. SSA/Ps markedly overexpressed MUC5AC (582-fold), a component of the gastric mucus layer, and a secretory protein that may stabilize mucin layers, trefoil factor 1 (TFF1, 79-fold). Neither protein is typically expressed in normal colonic mucosa. SSA/Ps exhibited strong co-localized staining for both proteins (Fig. 1 and 2). Intense MUC5AC staining was primarily seen in the serrated crypts, whereas expression in adjacent non-serrated mucosa was identical to controls. Conversely, HPs showed only weak patchy staining of MUC5AC and variable TFF1 staining that was infrequently colocalized. Normal colon had no detectable MUC5AC staining and minimal TFF1 staining. Conventional adenomas showed trace MUC5AC and TFF1 staining. Conclusions: RNA-seq and IHC analysis showed markedly increased expression of two mucin-associated genes/ proteins in SSA/Ps, MUC5AC and TFF1, which are not typically expressed in normal colonic mucosa or adenomas. There was also intense co-localization of MUC5AC and TFF1 in SSA/ Ps but not in HPs. These results identify intense co-localized expression of MUC5AC and TFF1 as a possible diagnostic marker that would aid differentiating SSA/Ps from HPs. Further studies will be needed with larger numbers of SSA/Ps. Since MUC5AC has been linked to signaling related to bacteria, these findings suggest that luminal factors may play a role in the formation or progression of SSA/Ps.