Dr Nicola Annels

Abstract Background The immune system is highly responsive and positively adapts to exercise. A single bout of exercise results in the mobilisation of highly functional effector CD8+ T cells and NK-cells into the circulation. Murine cancer models have shown that exercise reduces tumour burden by increasing the frequency of tumour-infiltrating lymphocytes (TILs). There are no studies assessing the impact of an exercise programme on the levels of TILs in patients’ solid tumours in any cancer. Methods We recently completed a 16-week randomised prehabilitation exercise program (NCT02950324) in esophageal cancer patients before esophagogastric cancer resection. Exercise training was a low-to-moderate intensity twice supervised, thrice home-based weekly program. Tumour specimens obtained at the time of resection were formalin fixed paraffin embedded (FFPE) for multispectral immunohistochemical analysis. Tumour tissues were stained with primary antibodies for CD68, CD57, CD8, CD4, FoxP3, Granzyme B, PDL1 and pancytokeratin. Cell populations and spatial relationships were analysed using the Phenoimager HT (Akoya Biosciences) and QuPath. Results Although our exercise program was ~33% of the physical activity guidelines for cancer patients, physical fitness and well-being were maintained rather than significantly reduced in the intervention group compared to the control group. Multispectral analysis observed that 3.2% ± 1.1% of cells in the tumours were CD8+ T cells compared to 1.4% ± 0.5% in the control group (p < 0.001). Furthermore, we observed positive associations between increased frequencies of CD8 + TILs (Fig 1C: r = 0.562, p = 0.016), Granzyme B+/CD8 + TILs (r = 0.637, p = 0.003) and larger increases in exercise induced aerobic capacity. This data suggests that the more exercise can increase aerobic fitness, the greater the likelihood of increasing functional TILs. Conclusion New approaches to improve outcomes following surgery for esophageal adenocarcinoma are required. One such approach is immunotherapy. However, immunotherapy is relatively ineffective in esophageal adenocarcinoma due to the lack of CD8+ T cells and NK cells in the tumours. Increasing TILs through exercise programmes that are designed to focus on maintaining or improving aerobic capacity may improve patients’ response to immunotherapy and positively impact prognosis and survival.

Renal cell carcinoma is an immunogenic tumour with a prominent dysfunctional immune cell infiltrate, unable to control tumour growth. Although tyrosine kinase inhibitors and immunotherapy have improved the outlook for some patients, many individuals are non-responders or relapse despite treatment. The hostile metabolic environment in RCC affects the ability of T-cells to maintain their own metabolic programme constraining T-cell immunity in RCC. We investigated the phenotype, function and metabolic capability of RCC TILs correlating this with clinicopathological features of the tumour and metabolic environment at the different disease stages. Flow cytometric analysis of freshly isolated TILs showed the emergence of exhausted T-cells in advanced disease based on their PD-1high and CD39 expression and reduced production of inflammatory cytokines upon in vitro stimulation. Exhausted T-cells from advanced stage disease also displayed an overall phenotype of metabolic insufficiency, characterized by mitochondrial alterations and defects in glucose uptake. Nanostring nCounter cancer metabolism assay on RNA obtained from 30 ccRCC cases revealed significant over-expression of metabolic genes even at early stage disease (pT1-2), while at pT3-4 and the locally advanced thrombi stages, there was an overall decrease in differentially expressed metabolic genes. Notably, the gene PPARGC1A was the most significantly down-regulated gene from pT1-2 to pT3-4 RCC which correlated with loss of mitochondrial function in tumour-infiltrating T-cells evident at this tumour stage. Down-regulation of PPARGC1A into stage pT3-4 may be the 'tipping-point' in RCC disease progression, modulating immune activity in ccRCC and potentially reducing the efficacy of immunotherapies in RCC and poorer patient outcomes.

Glioblastoma multiforme (GBM) is the most common high-grade malignant brain tumour in adults and arises from the glial cells in the brain. The prognosis of treated GBM remains very poor with 5-year survival rates of 5%, a figure which has not improved over the last few decades. Currently, there is a modest 14-month overall median survival in patients undergoing maximum safe resection plus adjuvant chemoradiotherapy. HOX gene dysregulation is now a widely recognised feature of many malignancies. In this study we have focused on HOX gene dysregulation in GBM as a potential therapeutic target in a disease with high unmet need. We show significant dysregulation of these developmentally crucial genes and specifically that HOX genes A9, A10, C4 and D9 are strong candidates for biomarkers and treatment targets for GBM and GBM cancer stem cells. We evaluated a next generation therapeutic peptide, HTL-001, capable of targeting HOX gene over-expression in GBM by disrupting the interaction between HOX proteins and their co-factor, PBX. HTL-001 induced both caspase-dependent and -independent apoptosis in GBM cell lines. In vivo biodistribution studies confirmed that the peptide was able to cross the blood brain barrier. Systemic delivery of HTL-001 resulted in improved control of subcutaneous murine and human xenograft tumours and improved survival in a murine orthotopic model.

Prostate cancers are considered “cold” tumors characterized by minimal T cell infiltrates, absence of a type I interferon (IFN) signature, and the presence of immunosuppressive cells. This non-inflamed phenotype is likely responsible for the lack of sensitivity of prostate cancer patients to immune checkpoint blockade (ICB) therapy. Oncolytic virus therapy can potentially overcome this resistance to immunotherapy in prostate cancers by transforming cold tumors into “hot,” immune cell-infiltrated tumors. We investigated whether the combination of intratumoral oncolytic reovirus, followed by targeted blockade of Programmed cell death protein 1 (PD-1) checkpoint inhibition and/or the immunomodulatory CD73/Adenosine system can enhance anti-tumor immunity. Treatment of subcutaneous TRAMP-C2 prostate tumors with combined intratumoral reovirus and anti-PD-1 or anti-CD73 antibody significantly enhanced survival of mice compared with reovirus or either antibody therapy alone. Only combination therapy led to rejection of pre-established tumors and protection from tumor re-challenge. This therapeutic effect was dependent on CD4 + T cells and natural killer (NK) cells. NanoString immune profiling of tumors confirmed that reovirus increased tumor immune cell infiltration and revealed an upregulation of the immune-regulatory receptor, B- and T-lymphocyte attenuator (BTLA). This expression of BTLA on innate antigen-presenting cells (APCs) and its ligand, Herpesvirus entry mediator (HVEM), on T cells from reovirus-infected tumors was in keeping with a role for the HVEM-BTLA pathway in promoting the potent anti-tumor memory response observed. Immunotherapy in prostate cancer is limited because of a lack of immune cells within the tumor. Annels et al. used oncolytic viruses to recruit immune cells into the tumor and showed that in combination with checkpoint inhibitors, which take the brake off of the immune system, established tumors were rejected.

It is now well-recognized that the tumor microenvironment (TME) is not only a key regulator of cancer progression but also plays a crucial role in cancer treatment responses. Recently, several high-profile publications have demonstrated the importance of particular immune parameters and cell types that dictate responsiveness to immunotherapies. With this increased understanding of TME-mediated therapy, approaches that increase therapeutic efficacy by remodeling the TME are actively being pursued. A classic example of this, in practice by urologists for over 40 years, is the manipulation of the bladder microenvironment for the treatment of non-muscle invasive bladder cancer (NMIBC) by instillation of intravesical bacillus Calmette-Guerin (BCG). The success of BCG treatment is thought to be due to its ability to induce a massive influx of Th1-polarized inflammatory cells, production of Th1 inflammatory cytokines and the generation of tumor-targeted Th1-mediated cytotoxic responses. Whilst BCG immunotherapy is currently the best treatment for NMIBC, similar to 30% of patients show no response to this treatment. Here we present a review highlighting a variety of promising alternative immunotherapies being developed that remodel the bladder tumor microenvironment. These include (1) the use of oncolytic viruses which selectively replicate within cancer cells whilst also modifying the immunological components of the TME, (2) manipulation of the bladder microbiome to augment the response to BCG or other immunotherapies (3) utilizing Toll-like Receptor agonists as anti-tumor agents due to their potent stimulation of innate and adaptive immunity and (4) the growing recognition that immunotherapeutic strategies that will have the largest impact on patients may require multiple therapeutic approaches combined together. The accumulating knowledge on TME remodeling holds promise for providing an alternative therapy for patients with BCG-unresponsive NMIBC.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is lethal. It is projected that by 2030, PDAC will become the 2nd leading cause of cancer-related death. The intra-tumoural microbiome can influence pancreatic tumourigenesis and chemoresistance, and therefore patient survival. The role played by bile microbiota in PDAC is unknown. We aimed to define bile microbiome signatures in patients presenting with obstructive jaundice caused by benign and malignant pancreaticobiliary disease to develop novel cancer biomarkers. Method: Prospective bile samples were obtained from 37 patients who underwent either endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC). Variable regions (V3–V4) of the 16S rRNA genes were amplified by PCR and next generation sequencing was performed. The cohort consisted of 12 PDAC, 6 cholangiocarcinoma, 10 choledocholithiasis, 7 gallstone pancreatitis, and 2 primary sclerosing cholangitis patients. Bile samples from 8 patients were excluded from the analysis because of low read count. Results: Using the 16S rRNA method, we identified a total of 108 genera from 29 individuals (12 PDAC and 17 benign). Bile microbial diversity significantly differed between patients with PDAC vs. benign disease (p=0.0173). The separation of PDAC from benign samples is clearly seen through unsupervised clustering based on Canberra distances shown in Figure 1. We found 4 genera to be of significantly different abundance between PDAC vs. benign groups by association p-value and supported by false discovery rate (fdr). These were Escherichia (p=6.5x10-6, fdr=0.0002), Rothia (p=0.011, fdr=0.074), Streptococcus (p=0.012, fdr=0.074) and Prevotella (p=0.015, fdr=0.079). Conclusion: We show that patients with obstructive jaundice caused by PDAC have an altered microbiome composition in the bile, compared to those with benign disease. These bile-based microbes could be developed into potential diagnostic and prognostic biomarkers for PDAC and warrant further investigation.

This fully updated volume explores recently improved avenues to study urothelial carcinomas. Beginning with several novel chapters on molecular characterization and urothelial carcinogenesis, the book continues with sections on cellular and animal models, biomarkers, and approaches for targeted therapy. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Urothelial Carcinoma: Methods and Protocols, Second Edition serves as a valuable resource to further increase our knowledge on urothelial carcinoma and also to aid research on numerous other cancers.

Colorectal cancer (CRC) is the 2nd leading cause of cancer-related deaths worldwide, primarily due to the development of metastatic disease. The liver is the most frequently affected site. The metastatic cascade relies on a complex interaction between the immune system, tumor, and distant organs. Communication between the tumor and the metastatic site can be mediated by tumor-derived extracellular vesicles (EVs) and their cargo. The mechanisms underlying this process are starting to be understood through research that has rapidly expanded over the past 15 years. One crucial aspect is the remodeling of the microenvironment at the site of metastasis, which is essential for the formation of a premetastatic niche and the subsequent establishment of metastatic deposits. In the evaluated study, the authors use cellular experiments and a mouse model to investigate how tumour derived extracellular vesicles and their microRNA contents interact with hepatic stellate cells (HSCs). They demonstrate how this may lead to remodelling of the microenvironment and the formation of colorectal liver metastasis using their experimental model. In this mini review, we examine the current evidence surrounding tumour derived EVs and their effect on the tumour microenvironment to highlight potential areas for future research in CRC and other malignancies.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor survival. The intra-tumoural microbiome can influence pancreatic tumourigenesis and chemoresistance and, therefore, patient survival. The role played by bile microbiota in PDAC is unknown. We aimed to define bile microbiome signatures that can effectively distinguish malignant from benign tumours in patients presenting with obstructive jaundice caused by benign and malignant pancreaticobiliary disease. Prospective bile samples were obtained from 31 patients who underwent either Endoscopic Retrograde Cholangiopancreatography (ERCP) or Percutaneous Transhepatic Cholangiogram (PTC). Variable regions (V3–V4) of the 16S rRNA genes of microorganisms present in the samples were amplified by Polymerase Chain Reaction (PCR) and sequenced. The cohort consisted of 12 PDAC, 10 choledocholithiasis, seven gallstone pancreatitis and two primary sclerosing cholangitis patients. Using the 16S rRNA method, we identified a total of 135 genera from 29 individuals (12 PDAC and 17 benign). The bile microbial beta diversity significantly differed between patients with PDAC vs. benign disease (Permanova p = 0.0173). The separation of PDAC from benign samples is clearly seen through unsupervised clustering of Aitchison distance. We found three genera to be of significantly lower abundance among PDAC samples vs. benign, adjusting for false discovery rate (FDR). These were Escherichia (FDR = 0.002) and two unclassified genera, one from Proteobacteria (FDR = 0.002) and one from Enterobacteriaceae (FDR = 0.011). In the same samples, the genus Streptococcus (FDR = 0.033) was found to be of increased abundance in the PDAC group. We show that patients with obstructive jaundice caused by PDAC have an altered microbiome composition in the bile compared to those with benign disease. These bile-based microbes could be developed into potential diagnostic and prognostic biomarkers for PDAC and warrant further investigation.

Oncolytic viruses are anticancer agents that selectively target and kill cancer cells by direct lysis, while at the same time stimulating a tumor antigen-specific adaptive immune response. These promising therapeutic agents target multiple cancers and have already proven to be an effective treatment option for solid malignancies. One such agent, T-Vec (Talimogene laherparepvec) has been licensed and is in routine clinical use for treatment of malignant melanoma.Non-muscle invasive bladder cancer (NMIBC) is an ideal potential target for oncolytic immunotherapy as locally instilled live biological therapy using Bacille Calmette-Guerin (BCG) is already well established in the clinical setting. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)-targeted immunotherapeutic virus. We have investigated CVA21-induced cytotoxicity in a panel of human bladder cancer cell lines, revealing a range of sensitivities largely correlating with expression of the viral receptor ICAM-1. CVA21 in combination with low doses of mitomycin-C enhanced CVA21 viral replication and oncolysis by increasing surface expression levels of ICAM-1. In addition to cell lines and an animal model a key component of our studies into oncolytic immunotherapy for bladder cancer was the use of a bladder tumor precision slice preclinical model system which represents tumor architecture, heterogeneity, and the complexity of a tumor in vitro. Results seen in cell lines were reflected in the tumor slice model whereby levels of virus protein expression and induction of apoptosis were enhanced with prior exposure to mitomycin-C. In this chapter we demonstrate the utility of the precision cut tumor slice model as a unique organotypic model to test oncolytic viruses. We will describe how to prepare and slice the tumor using a vibrating microtome together with the optimum culture and conditions for treatment.

Lymphocyte-activation gene 3 (LAG-3) is a coreceptor found on activated T-lymphocytes activated B-lymphocytes and natural killer (NK) cells. It is closely related to CD4 where it shares multiple common and divergent features. It contains specific binding sites with high affinity to major histocompatibility complex (MHC) Class II and functions as an inhibitor of T-cell signalling. Tumour-infiltrating lymphocytes with high LAG-3 expression have been found in many solid tumours including ovarian cancer, melanoma, colorectal cancer and haematological malignancies including Hodgkin and diffuse large B-cell lymphoma. LAG-3 antagonism has been demonstrated to restore the anti-tumourigenic function of T-cells in vivo, however, mechanistic knowledge remains relatively poorly defined. As other immune checkpoint inhibitors have transformed the management of difficult to treat cancers, such as melanoma, it is hoped that LAG-3 might have the same potential. This review will explore LAG-3 modulation as an anticancer therapy, highlighting recent clinical developments.

Background10%–15% of early-stage colon cancers harbour either deficient mismatch repair (dMMR), microsatellite instability high (MSI-H) or POLE exonuclease domain mutations, and are characterised by high tumour mutational burden and increased lymphocytic infiltrate. Metastatic dMMR colon cancers are highly sensitive to immune checkpoint inhibition, and recent data show POLE-mutant tumours are similarly responsive. We are conducting a phase III randomised trial to determine if the addition of the anti-PD-L1 antibody avelumab following adjuvant chemotherapy improves disease-free survival (DFS) in patients with stage III dMMR/MSI-H or POLE mutant colon cancer and is a cost-effective approach for the UK National Health Service (NHS).MethodsWe are recruiting patients with completely resected, stage III colon cancer confirmed to have dMMR/MSI-H, locally or POLE exonuclease domain mutation on central testing. Eligible patients are randomised in a 1:1 ratio to standard fluoropyrimidine-based chemotherapy (capecitabine, oxaliplatin for 12 weeks or capecitabine for 24 weeks) or chemotherapy, followed by avelumab (10 mg/kg, 2 weekly for 24 weeks). Stratification is by chemotherapy received and MMR/MSI-H status. The primary endpoint is DFS. Secondary endpoints include overall survival, toxicity, quality of life and health resource use. The 3-year DFS rate in the control arm is expected to be ~75%. Avelumab is expected to improve the 3-year DFS rate by 12% (ie, 87%). Target accrual is 402 patients, which provides 80% power to detect an HR of 0.48 for DFS at a two-sided alpha of 0.05. This national, multicentre phase III trial is sponsored by the Royal Marsden NHS Foundation Trust and it is anticipated that approximately 40 centres in the UK will participate. This study opened to recruitment in August 2018.Trial registration numberNCT03827044

Oncolytic viruses are biological agents which can easily be delivered at high doses directly to the bladder through a catheter (intravesical), with low risk of systemic uptake and toxicity. To date, a number of viruses have been delivered intravesically in patients and in murine models with bladder cancer and antitumour effects demonstrated. Here, we describe in vitro methods to evaluate Coxsackie virus, CVA21, as an oncolytic virus for the treatment of human bladder cancer by determining the susceptibility of bladder cancer cell lines expressing differing levels of ICAM-1 surface receptor to CVA21.

Simple Summary This review summarizes the current literature related to the microbiome and pancreatic ductal adenocarcinoma (PDAC). The aim of this review is to explore the current role of the microbiome in the disease process, screening/diagnostics and to postulate the future role with regards to therapeutic strategies including chemotherapy, immunotherapy and surgery. We further explore the future of microbiome modulation (faecal microbiome transplants, bacterial consortiums, anti-microbials and probiotics), their applications and how we can improve the future of microbiome modulation in a bid to improve PDAC outcomes. Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second most common cause of cancer death in the USA by 2030, yet progress continues to lag behind that of other cancers, with only 9% of patients surviving beyond 5 years. Long-term survivorship of PDAC and improving survival has, until recently, escaped our understanding. One recent frontier in the cancer field is the microbiome. The microbiome collectively refers to the extensive community of bacteria and fungi that colonise us. It is estimated that there is one to ten prokaryotic cells for each human somatic cell, yet, the significance of this community in health and disease has, until recently, been overlooked. This review examines the role of the microbiome in PDAC and how it may alter survival outcomes. We evaluate the possibility of employing microbiomic signatures as biomarkers of PDAC. Ultimately this review analyses whether the microbiome may be amenable to targeting and consequently altering the natural history of PDAC.

Simple Summary Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, responsible for the majority of cases and ranking seventh as a leading cause of cancer-related deaths. It is difficult to treat because is it often detected at advanced stages, there are no effective screening tests available, and patients can develop resistance to standard treatments like chemotherapy and radiation. Recent interest has involved immunotherapy, which stimulate the immune system to recognise and attack cancer cells. Therefore, our paper aims to summarise the findings of studies investigating immunotherapies in PDAC and we discuss the limitations of such therapies and avenues of future research. In a bid to address the outcomes associated with the disease.Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for up to 95% of all pancreatic cancer cases and is the seventh-leading cause of cancer death. Poor prognosis is a result of late presentation, a lack of screening tests and the fact some patients develop resistance to chemotherapy and radiotherapy. Novel therapies like immunotherapeutics have been of recent interest in pancreatic cancer. However, this field remains in its infancy with much to unravel. Immunotherapy and other targeted therapies have yet to yield significant progress in treating PDAC, primarily due to our limited understanding of the disease immune mechanisms and its intricate interactions with the tumour microenvironment (TME). In this review we provide an overview of current novel immunotherapies which have been studied in the field of pancreatic cancer. We discuss their mechanisms, evidence available in pancreatic cancer as well as the limitations of such therapies. We showcase the potential role of combining novel therapies in PDAC, postulate their potential clinical implications and the hurdles associated with their use in PDAC. Therapies discussed with include programmed death checkpoint inhibitors, Cytotoxic T-lymphocyte-associated protein 4, Chimeric Antigen Receptor-T cell therapy, oncolytic viral therapy and vaccine therapies including KRAS vaccines, Telomerase vaccines, Gastrin Vaccines, Survivin-targeting vaccines, Heat-shock protein (HSP) peptide complex-based vaccines, MUC-1 targeting vaccines, Listeria based vaccines and Dendritic cell-based vaccines.

Immune modulators play a crucial role in carcinogenesis and cancer progression by impairing cancer cell-targeted immune responses. T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) regulates T-cell function and cancer cell recognition and was therefore identified as a promising target for cancer immunotherapy. TIGIT is expressed in T cells and natural killer (NK) cells and has three ligands: CD155, CD112 and CD113. CD155 binds TIGIT with the highest affinity and promotes direct and indirect downregulation of T-cell response. TIGIT signalling further inhibits NK function and secretion of proinflammatory cytokines. An association between TIGIT expression and poor survival was identified in multiple cancer entities. Blocking TIGIT with monoclonal antibodies, and a combination of TIGIT and programmed cell death protein 1 blockade in particular, prevented tumour progression, distant metastasis and tumour recurrence in in vivo models. Inhibition of TIGIT is currently evaluated in first clinical trials.

Background The most important insights into mechanisms of tumour rejection, and how these could be exploited therapeutically, is likely to come from patients displaying the best responses. Those individuals with complete and sustained antitumour responses, without maintenance therapy, will provide the best evidence of genuine disease modification. Despite much speculation as to how these exceptional responses are generated there is increasing evidence for an immune basis which may be influenced by the tumour microbiome underlying the mechanism for sustained tumour rejection. Methods To focus on individuals with complete and sustained metastatic disease resolution, we designed a pilot study, the Continuum Long-Term Survivor study, to evaluate patients with the best outcomes, where disease modification may have occurred. The study targeted only those patients (n=50) with a sustained (>5 years) complete clearance of metastatic cancers, without requiring maintenance therapy. Matched controls comprised patients unable to generate an initial response, or those who relapsed within 12 months. DNA extracted from tumour samples was analyzed by 16S BENCHMARK™ microbial amplicon sequencing (Diversigen) to profile the tumour microbiome, whilst the microbial composition of stool samples was determined using BoosterShot Shotgun Sequencing. In parallel mRNA expression from the tumour tissue was evaluated using NanoString’s PanCancer IO360 gene expression panel. From a homogeneous subgroup of bowel cancer long-term survivors and their matched controls, multiplex IHC using a panel of 8 immune markers to identify tertiary lymphoid structures (TLS) was performed on the tumour tissues and imaged using a PhenoImager (AkoyaBiosciences). Results Results will be presented comparing tumour and gut-derived microbial species diversity between long-term vs. short-term survivor matched controls using alpha diversity estimates as well as differential abundance analysis. This will define the microbial species that are more likely to be associated with long-term survivorship. Characterization of the immune gene transcriptional patterns within the tumour microenvironment (TME) from long-term survivors vs controls will also be reported along with any observed differences from the multiplex IHC analysis of immune infiltrates in corresponding patient tissue. To explore any connections of the tumour-microbiome on the TME, an integrated analysis of the microbiome and tumour immune transcriptome will be presented. Conclusions This study will reveal whether changes in TME influenced by the tumour microbiota may be important factors associated with long-term survivorship. Understanding the precise mechanisms of total tumour rejection in patients, and their evaluation may be game-changing in terms of design of new molecular, biological and immune therapies. Ethics Approval This study was approved by the University of Surrey Ethics Board; approval number 266581.

© 2013 Springer-Verlag London. All rights are reserved.The approval in April 2010 by the FDA in the USA of a prostate cancer vaccine (Sipuleucel-T, Provenge, Dendreon Inc.) may herald a new era in T-cell-directed cancer therapies. The magnitude of scientific effort to reach this point should not be underestimated. The key has been unraveling the complex mechanisms between the recognition of tumor antigen, breaking immune tolerance, and generation of a long-lasting and clinically meaningful cellular response. Therapeutic vaccines (i.e., vaccines for patients with ongoing disease) have two objectives: priming Ag-specific T cells and reprogramming memory T cells (i.e., a transformation from one type of immunity to another, for example, regulatory to cytotoxic). Numerous therapeutic approaches have been tested in prostate cancer patients, including autologous and allogeneic tumor cells modified to express various cytokines, peptides, proteins, and DNA vaccines. The evolution of cellular vaccines includes addressing the local immunosuppressive tumor microenvironment, modulating immune response through checkpoint blockade and, importantly, combining cellular immunotherapy with other treatment modalities such as chemotherapy exploiting their intrinsic immunomodulatory properties.

In 2010, the US FDA approved the first therapeutic cancer vaccine for the treatment of castration refractory prostate cancer - sipuleucel-T. Prostate cancer is an ideal model for cancer vaccine development based on the ready demonstration of humoral and cellular immunity to a range of cancer antigens as well as often slow progression which means that patients who are otherwise well may have a radiologically evaluable minor progression, after conventional treatment and can undergo vaccine therapy over sufficient periods of time, so as to allow the generation of a robust antitumor response. The association of prostate cancer with one of the few serum cancer biomarkers in general use has also allowed assessment of response and risk stratification of patients. In this review, we will examine key aspects of the evolution of prostate cancer vaccines, which provides an accurate prototype for other cancers, and the challenges we face.

We undertook a comprehensive analysis of circulating myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) in pancreatic, esophageal and gastric cancer patients and investigated whether MDSCs are an independent prognostic factor for survival. We evaluated a series of plasma cytokines and in particular re-evaluated the Th2 cytokine interleukin-13 (IL-13). Peripheral blood was collected from 131 cancer patients (46 pancreatic, 60 esophageal and 25 gastric) and 54 healthy controls. PBMC were harvested with subsequent flow cytometric analysis of MDSC (HLADR(-) Lin1(low/-) CD33(+) CD11b(+)) and Treg (CD4(+) CD25(+) CD127(low/-) FoxP3(+)) percentages. Plasma IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL-17, G-CSF, IFN-γ, TNF-α and VEGF levels were analyzed by the Bio-Plex cytokine assay. Plasma arginase I levels were analyzed by ELISA. MDSCs and Tregs were statistically significantly elevated in pancreatic, esophageal and gastric cancer compared with controls, and MDSC numbers correlated with Treg levels. Increasing MDSC percentage was associated with increased risk of death, and in a multivariate analysis, MDSC level was an independent prognostic factor for survival. A unit increase in MDSC percentage was associated with a 22% increased risk of death (hazard ratio 1.22, 95% confidence interval 1.06-1.41). Arginase I levels were also statistically significantly elevated in upper gastrointestinal cancer patients compared with controls. There was Th2 skewing for cytokine production in all three diseases, and importantly there were significant elevations of the pivotal Th2 cytokine interleukin-13, an increase that correlated with MDSC levels.

Purpose: The CANON (CAVATAK in NON-muscle invasive bladder cancer) study evaluated a novel ICAM-1-targeted immunotherapeutic-coxsackievirus A21 as a novel oncolytic agent against bladder cancer. Experimental Design: Fifteen patients enrolled on this 'window of opportunity' phase 1 study, exposing primary bladder cancers to CAVATAK prior to surgery. The first nine patients received intravesical administration of monotherapy CAVATAK; in the second stage, six patients received CAVATAK with a sub-therapeutic dose of mitomycinC, known to enhance expression of ICAM-1 on bladder cancer cells. The primary endpoint was to determine patient safety and maximum tolerated dose. Secondary endpoints were evidence of viral replication, induction of inflammatory cytokines, anti-tumour activity and viral-induced changes in resected tissue. Results: Clinical activity of CAVATAK was demonstrated by induction of tumour inflammation and haemorrhage following either single or multiple administrations of CAVATAK in multiple patients, and a complete resolution of tumour in one patient. Whether used alone or in combination with mitomycinC, CAVATAK caused marked inflammatory changes within NMIBC tissue biopsies by up-regulating interferon-inducible genes including both immune checkpoint-inhibitory genes (PD-L1 and LAG3) and Th1-associated chemokines as well as induction of the innate activator RIG-I, compared to bladder cancer tissue from untreated patients. No significant toxicities were reported in any patient, from either virus or combination therapy. Conclusions: The acceptable safety profile of CAVATAK, proof of viral targeting, replication and tumour cell death together with the virus-mediated increases in "immunological heat" within the tumour microenvironment all indicate that CAVATAK may be potentially considered as a novel therapeutic for NMIBC.

From a therapeutic perspective, the bourgeoning literature on Th17 cells should allow us to decide whether to rationally pursue the manipulation of Th17 cells in cancer. The purpose of this review is to attempt a synthesis of a number of contradictory conclusions as to the role that these cells are playing in the process of tumourigenesis in order to provide guidance as to whether our current understanding is sufficient to safely pursue Th17-targeted therapy in cancer at this time. Th17 cells are a highly plastic population and the cytokine drivers for Th17 cell generation and skewing will vary between various cancers and importantly between different sites of tumour involvement in any individual patient. The net impact of the pro-angiogenic IL-17 produced not only by Th17 cells but by other cells particularly macrophages and the anti-tumour effects of Th1/Th17 cells will in turn be determined by the complex interplay of diverse chemokines and cytokines in any tumour microenvironment. Th17 cells that fail to home to tumours may be immunosuppressive. The complexity of IL-17 and Th17 dynamics makes easy prediction of the effects of either enhancing or suppressing Th17 cell differentiation in cancer problematic.

Bladder cancer (BCa) is the most frequent urinary tract neoplasm. BCa results in significant mortality when the disease presents as muscle invasive. Around 75% to 80% of patients present with nonmuscle invasive bladder cancer (NMIBC), but recurrence and progression are significant issues, compelling current guidelines to recommend long-term surveillance. There is therefore an urgent and unmet need to identify and validate accurate biomarkers for the detection of disease recurrence to improve quality of life for the patients and reduce costs for health care providers, while maintaining or improving current outcomes. In this review, 38 publications on immunohistochemistry prognostic biomarkers, that were studied may be related in nonmuscle invasive bladder cancer, have been analyzed. The studies were organized according to the evaluated marker and their findings. It was demonstrated that the combination of independent complementary biomarkers could allow a more accurate prognosis than an isolated marker. Biomarkers, including p53, Ki-67, and CK20, with classic and prognostic factors with recurrence and novel markers such as EN2 may provide a more accurate prediction of outcome compared with any single marker, improving risk stratification and clinical management of patients with BCa.

As a clinical setting in which local live biological therapy is already well established, non-muscle invasive bladder cancer (NMIBC) presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)-targeted immunotherapeutic virus. This study investigated CVA21-induced cytotoxicity in a panel of human bladder cancer cell lines, revealing a range of sensitivities largely correlating with expression of the viral receptor ICAM-1. CVA21 in combination with low doses of mitomycin-C enhanced CVA21 viral replication and oncolysis by increasing surface expression levels of ICAM-1. This was further confirmed using 300-μm precision slices of NMIBC where levels of virus protein expression and induction of apoptosis were enhanced with prior exposure to mitomycin-C. Given the importance of the immunogenicity of dying cancer cells for triggering tumor-specific responses and long-term therapeutic success, the ability of CVA21 to induce immunogenic cell death was investigated. CVA21 induced immunogenic apoptosis in bladder cancer cell lines, as evidenced by expression of the immunogenic cell death (ICD) determinant calreticulin, and HMGB-1 release and the ability to reject MB49 tumors in syngeneic mice after vaccination with MB49 cells undergoing CVA21 induced ICD. Such CVA21 immunotherapy could offer a potentially less toxic, more effective option for the treatment of bladder cancer.

BACKGROUND: Epithelial ovarian cancer is a common malignancy, with no clinically approved diagnostic biomarker. Engrailed-2 (EN2) is a homeodomain-containing transcription factor, essential during embryological neural development, which is dysregulated in several cancer types. We evaluated the expression of EN2 in Epithelial ovarian cancer, and reviewed its role as a biomarker. METHODS We evaluated 8 Epithelial ovarian cancer cell lines, along with >100 surgical specimens from the Royal Surrey County Hospital (2009-2014). In total, 108 tumours and 5 normal tissue specimens were collected. En2 mRNA was evaluated by semi-quantitative RT-PCR. Histological sub-type, and platinum-sensitive/-resistant status were compared. Protein expression was assessed in cell lines (immunofluorescence), and in >150 tumours (immunohistochemistry). RESULTS En2 mRNA expression was elevated in serous ovarian tumours compared with normal ovary (p

In pre-clinical models, the only two chemotherapy drugs which have been demonstrated to directly reduce the number of myeloid-derived suppressor cells (MDSCs) are gemcitabine and 5-fluorouracil. Here we analyze the dynamics of MDSCs, phenotyped as Lin-DR-CD11b+, in patients with advanced pancreatic cancer receiving the combination of gemcitabine and capecitabine, a 5-FU pro-drug. We found no evidence that gemcitabine and capecitabine directly reduce MDSC% in patients. Gemcitabine and capecitabine reduced MDSCs in 42 % of patients (n = 19) and MDSC% fell in only 3/9 patients with above-median baseline MDSCs. In 5/8 patients with minimal tumour volume change on treatment, the MDSC% went up: increases in MDSC% in these patients appeared to correlate with sustained cancer-related inflammatory cytokine upregulation. In a separate cohort of 21 patients treated with gemcitabine and capecitabine together with concurrently administered GV1001 vaccine with adjuvant GM-CSF, the MDSC% fell in 18/21 patients and there was a significant difference in the trajectory of MDSCs between those receiving GV1001 and GM-CSF in combination with chemotherapy and those receiving chemotherapy alone. Thus, there was no evidence that the addition of low-dose adjuvant GM-CSF increased Lin-DR-CD11b+ MDSC in patients receiving combination chemoimmunotherapy. 9/21 patients developed an immune response to GV1001 and the MDSCs fell in 8 of these 9 patients, 6 of whom had above-median pre-vaccination MDSC levels. A high pre-vaccination MDSC% does not preclude the development of immunity to a tumour-associated antigen.

Background: Ovarian cancer remains the most lethal gynaecologic tumour in the Western world. Stimulation of the immune system to consolidate response to chemotherapy can potentially be beneficial however so far none of the vaccination strategies have offered survival advantage. Thus identifying and targeting clinically relevant antigens for immunotherapy continues to be an important research strategy. We have evaluated Engrailed-2 (EN2) as a potential target for vaccine strategy. EN2 is a homeodomain-containing transcription factor with a multifunctional role in neural development. There is evidence that over-expression of EN2 protein maybe linked to tumour development. Methods: Ovarian cancer cell lines were analysed by FACS for EN2 cell surface expression. EN2 expression in ovarian cancer tissue arrays were done by immunohistochemistry. A serum analysis (ELISA) was done to evaluate the presence of antibodies to EN2 in ovarian cancer patients and age-matched controls. A set of potentially immunogenic HLA-A2 restricted epitopes from the EN2 protein was identified using a computer algorithm SYFPEITHI. These peptides have been tested on HLA-A2 positive ovarian cancer patients’ PBMC using an in vitro culture method. The specificity of these T cell lines was analysed against T2 target cells loaded with or without EN2 peptides Results: Cell surface expression of EN2 was observed in ovarian cancer cell lines OVCAR3, OV90, CaOV-3, ES-2 and SKOV-3 of which ES-2 and SKOV3 showed strong expression. EN2 was also present in approximately 80% of ovarian cancer tissues whereas EN-2 expression was very low (