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Purpose:The prevention and treatment of metastatic sarcoma are areas of significant unmet need. Immune checkpoint inhibitor monotherapy has shown little activity in sarcoma and there is great interest in identifying novel treatment combinations that may augment responses. In vitro and in vivo, we investigated the potential for an oncolytic vaccinia virus (GLV-1h68) delivered using isolated limb perfusion (ILP) to promote antitumor immune responses and augment response to PD-1 blockade in sarcoma.Experimental Design: In an established animal model of extremity sarcoma, we evaluated the potential of locoregional delivery of a vaccinia virus (GLV-1h68) alongside biochemotherapy (melphalan/TNFα) in ILP. Complementary in vitro assays for markers of immunogenic cell death were performed in sarcoma cell lines.Results:PD-1 monotherapy had minimal efficacy in vivo, mimicking the clinical scenario. Pretreatment with GLV-1h68 delivered by ILP (viral ILP) significantly improved responses. Furthermore, when performed prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented both local and distant relapse, curing a previously treatment-refractory model. Enhanced therapy was associated with marked modulation of the tumor microenvironment, with an increase in the number and penetrance of intratumoral CD8+ T cells and expansion and activation of dendritic cells. GLV-1h68 was capable of inducing markers of immunogenic cell death in human sarcoma cell lines.Conclusions:Viral ILP augments the response to PD-1 blockade, transforming this locoregional therapy into a potentially effective systemic treatment for sarcoma and warrants translational evaluation.

Supplementary tables and figures

Purpose:The CANON [CAVATAK in NON–muscle-invasive bladder cancer (NMIBC)] study evaluated a novel ICAM-1–targeted immunotherapeutic-coxsackievirus A21 as a novel oncolytic agent against bladder cancer.Patients and Methods:Fifteen patients enrolled in this “window of opportunity” phase I study, exposing primary bladder cancers to CAVATAK prior to surgery. The first 9 patients received intravesical administration of monotherapy CAVATAK; in the second stage, 6 patients received CAVATAK with a subtherapeutic dose of mitomycin C, known to enhance expression of ICAM-1 on bladder cancer cells. The primary endpoint was to determine patient safety and maximum tolerated dose (MTD). Secondary endpoints were evidence of viral replication, induction of inflammatory cytokines, antitumor activity, and viral-induced changes in resected tissue.Results:Clinical activity of CAVATAK was demonstrated by induction of tumor inflammation and hemorrhage following either single or multiple administrations of CAVATAK in multiple patients, and a complete resolution of tumor in 1 patient. Whether used alone or in combination with mitomycin C, CAVATAK caused marked inflammatory changes within NMIBC tissue biopsies by upregulating IFN-inducible genes, including both immune checkpoint inhibitory genes (PD-L1 and LAG3) and Th1-associated chemokines, as well as the induction of the innate activator RIG-I, compared with 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 tumor cell death together with the virus-mediated increases in “immunological heat” within the tumor microenvironment all indicate that CAVATAK may be potentially considered as a novel therapeutic for NMIBC.

Supplementary data - revised

Cytoplasmic pattern recognition receptors (PRRs) for double-stranded RNA (RIG-I/MDA5) are key mediators of anti-viral responses. PRR agonists, such as dsRNA oncolytic Reovirus type 3 Dearing (Rt3D), potently activate RNA sensors. We used an unbiased cytotoxicity screen to reveal synergistic drug-virotherapy combinations and found potent effects of Rt3D combined with the CDK4/6 inhibitor, palbociclib. The combination augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and the expression and activation/signaling of RNA sensors. Combined Rt3D-palbociclib treatment potently increased interferon production and signaling, and knockdown studies implicated key UPR proteins and the RNA sensor, RIG-I, as essential to the phenotype observed. Further experiments, using canonical RIG-I agonists and an ER stress inducer, thapsigargin, confirmed cross-talk between RNA sensing and ER stress pathways that augmented cancer cell death and interferon production. Combined Rt3D-palbociclib also increased innate immune activation within tumour cells and IFN-induced HLA expression. Analysis of the immunopeptidome revealed changes to HLA-captured peptides with Rt3D-palbociclib, including altered expression of peptides from cancer/testis antigens (CTA) and endogenous retroviral elements (ERVs). Our findings highlight cross-talk between UPR signaling and RNA-mediated PRR activation as a means of enhancing anti-cancer efficacy with potential pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, and broadens the therapeutic remit of CDK4/6 inhibitors to include roles as both ER stress and dsRNA PRR sensitizers.

Summary Background The WHO VISION 2020 global initiative against blindness, launched in 2000, prioritised childhood visual disability by aiming to end avoidable childhood blindness by 2020. However, progress has been hampered by the global paucity of epidemiological data concerning childhood visual disability. The British Childhood Visual Impairment and Blindness Study 2 (BCVIS2) was done to address this evidence gap. Methods BCVIS2 was a prospective UK-wide, cross-sectional, observational study to establish an inception cohort of children newly diagnosed with visual impairment. Ophthalmologists and paediatricians reported cases from 89 hospitals and community centres across the UK. We included children aged 18 years or younger who were newly diagnosed with any condition causing impaired visual acuity to a level of 0·5 logMAR or worse (worse than 6/18 Snellen) in each eye, or equivalent vision as assessed by standard qualitative measures, between Oct 1, 2015, and Nov 1, 2016. Eligible children were notified simultaneously but independently by their managing ophthalmologists and paediatricians via the two national active surveillance schemes, the British Ophthalmological Surveillance Unit and the British Paediatric Surveillance Unit. Standardised detailed demographic, socioeconomic, and clinical data about detection, management, and treatment were collected at diagnosis and 1 year later. We calculated incidence estimates and relative rates by key sociodemographic factors. We did descriptive analyses of underlying ophthalmic disorders and non-ophthalmic comorbidities. Findings 61 (7%) of 845 eligible children initially notified were ineligible at follow-up because of improved vision after treatment. Thus, the study sample comprised 784 children with permanent newly-diagnosed all-cause visual impairment, severe visual impairment, or blindness. 559 (72%) of 778 children had clinically significant non-ophthalmic impairments or conditions. 28 (4%) of 784 children died within a year after diagnosis of visual disability (all had underlying systemic disorders). Incidence of visual disability in the first year of life was 5·19 per 10 000 children (95% CI 4·71–5·72), almost ten times higher than among 1-to-4-year-olds and between 20 times and 100 times higher than in the older age groups. The overall cumulative incidence (or lifetime risk) of visual impairment, severe visual impairment, or blindness was 10·03 per 10 000 children (9·35–10·76). Incidence rates were higher for those from any ethnic minority group, the lowest quintile of socioeconomic status, and those born preterm or with low birthweight. 345 (44%) of 784 children had a single affected anatomical site. Disorders of the brain and visual pathways affected 378 (48%) of 784 children. Interpretation BCVIS2 provides a contemporary snapshot of the heterogeneity, multi-morbidity, and vulnerability associated with childhood visual disability in a high-income country. These findings could facilitate developing and delivering health care and planning of interventional research. Our findings highlight the importance of including childhood visual disability as a sentinel event and metric in global child health initiatives. Funding Fight for Sight, National Institute for Health Research, and Ulverscroft Foundation.

Background Gastric and gastro-esophageal junction cancers (GCs) frequently recur after resection, but markers to predict recurrence risk are missing. T-cell infiltrates have been validated as prognostic markers in other cancer types, but not in GC because of methodological limitations of past studies. We aimed to define and validate the prognostic role of major T-cell subtypes in GC by objective computational quantification. Methods Surgically resected chemotherapy-naïve GCs were split into discovery (n = 327) and validation (n = 147) cohorts. CD8 (cytotoxic), CD45RO (memory), and FOXP3 (regulatory) T-cell densities were measured through multicolor immunofluorescence and computational image analysis. Cancer-specific survival (CSS) was assessed. All statistical tests were two-sided. Results CD45RO-cell and FOXP3-cell densities statistically significantly predicted CSS in both cohorts. Stage, CD45RO-cell, and FOXP3-cell densities were independent predictors of CSS in multivariable analysis; mismatch repair (MMR) and Epstein–Barr virus (EBV) status were not statistically significant. Combining CD45RO-cell and FOXP3-cell densities into the Stomach Cancer Immune Score showed highly statistically significant (all P ≤ .002) CSS differences (0.9 years median CSS to not reached). T-cell infiltrates were highest in EBV-positive GCs and similar in MMR-deficient and MMR-proficient GCs. Conclusion The validation of CD45RO-cell and FOXP3-cell densities as prognostic markers in GC may guide personalized follow-up or (neo)adjuvant treatment strategies. Only those 20% of GCs with the highest T-cell infiltrates showed particularly good CSS, suggesting that a small subgroup of GCs is highly immunogenic. The potential for T-cell densities to predict immunotherapy responses should be assessed. The association of high FOXP3-cell densities with longer CSS warrants studies into the biology of regulatory T cells in GC.

Background Accumulation of multiple pockets of fluid at the fovea, as a complication of poor blood glucose control in diabetes, causes impairment of central vision. A new ability to demonstrate a pre-clinical phase of this maculopathy could be valuable, enabling diabetic individuals to be alerted to the need to improve their glycaemic control. This study aimed to use swept-source optical coherence tomography (SS-OCT) to measure foveal thickness and macular volume in diabetic individuals without cystoid macular oedema, and in non-diabetic individuals, and relate these measures to participants’ glycaemic control. Methods Centre point thickness (CPT) and total macular volume (TMV) were measured using SS-OCT (DRI OCT Triton™, Topcon, Tokyo, Japan). Participants’ glycosylated haemoglobin (HbA1c) level was also assessed (A1cNow®+ System, PTS Diagnostics, Indianapolis, IN, USA). The diabetic (n = 27) and non-diabetic (n = 27) groups were matched for age (p = 0.100) and sex (p = 0.414), and HbA1c level differed between diabetic and non-diabetic groups (p n = 7) and type 2 (n = 20) diabetic individuals who were matched for duration of diabetes (p = 0.617) and whose glycaemic control was similar (p = 0.814). Results Diabetic individuals had significantly higher CPT (t(37) = 3.859, p 1c level (β = 0.501, t(21) = 3.139, p = 0.005): there was a 19-μm increase in CPT for each 1% increase in HbA1c level. This relationship was not present in the non-diabetic group (β = − 0.068, t(23) = − 0.373, p = 0.712). Conclusions SS-OCT is the only way to measure macular thickness in vivo. Diabetic individuals en bloc had higher CPT compared with non-diabetic individuals. Moreover, in the diabetic group, HbA1c level significantly predicted CPT. Our results suggest that, in diabetes, sub-clinical thickening may occur at the fovea before cystoid macular oedema becomes clinically evident. This could provide diabetic individuals with an early warning of disease progression and motivate them to improve control of their diabetes, with a view to avoiding the need of intra-vitreal injections with their attendant risks.

Background Prostate-specific membrane antigen (PSMA) is a prostatic epithelial protein that is used as a radiotracer (68Ga-PSMA-11) for prostate cancer staging. PSMA-PET/CT (positron emission tomography/computed tomography) performed for prostate cancer has been observed to detect melanoma metastases. The aim of this study was to investigate the performance of PSMA immunohistochemistry on resected melanoma metastases to explore its use as a diagnostic imaging biomarker for melanoma. Methods A total of 41 specimens with stage III/IV melanoma were stained with PSMA immunohistochemistry. All specimens required both disease and control regions. Two pathologists scored the specimens and a receiver operating characteristic (ROC) curve was plotted. Western blot and multiplex immunofluorescence were also performed. Results The area under the ROC curve was 0.82, suggesting that PSMA has excellent discriminatory power in melanoma metastases. Sensitivity is 82.9% and specificity 73.2%. Immunohistochemistry and Western blot reveal that PSMA staining in melanoma consistently and most intensely occurs in tumor neovasculature. Multiplex immunofluorescence shows that melanocytes may also weakly express PSMA. Conclusion The performance of PSMA immunohistochemistry in melanoma metastases contrasts with that reported in prostate cancer studies. This study indicates that PSMA shows promise for use as a novel biomarker in melanoma and justifies further research in the clinical setting with potential as a PET/CT radiotracer and intraoperative fluorescence marker for melanoma.

Cancer-associated fibroblasts (CAFs) perform diverse roles and can modulate therapy responses1. The inflammatory environment within tumours also influences responses to many therapies, including the efficacy of oncolytic viruses2; however, the role of CAFs in this context remains unclear. Furthermore, little is known about the cell signalling triggered by heterotypic cancer cell-fibroblast contacts and about what activates fibroblasts to express inflammatory mediators1,3. Here, we show that direct contact between cancer cells and CAFs triggers the expression of a wide range of inflammatory modulators by fibroblasts. This is initiated following transcytosis of cytoplasm from cancer cells into fibroblasts, leading to the activation of STING and IRF3-mediated expression of interferon-β1 and other cytokines. Interferon-β1 then drives interferon-stimulated transcriptional programs in both cancer cells and stromal fibroblasts and ultimately undermines the efficacy of oncolytic viruses, both in vitro and in vivo. Further, targeting IRF3 solely in stromal fibroblasts restores oncolytic herpes simplex virus function.

Introduction: Immune checkpoint inhibitors (ICI) have dramatically improved the outcome for cancer patients across multiple tumor types. However the response rates to ICI monotherapy remain relativ...

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or “cancer-killing” viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

Introduction Diabetes is a global issue that currently affects 425 million people worldwide. One observable microvascular complication of this condition is a change in the foveal avascular zone (FAZ). In this study, we used optical coherence tomography angiography (OCT-A) to investigate the effect of diabetes on the FAZ. Materials and methods Eleven diabetic and eleven non-diabetic participants took part in this study. Participants in both groups were matched for age (p = 0.217) and sex (p = 0.338), and had no history of ocular disease. Macular OCT-A scans of participants' right and left eyes were taken. Glycosylated haemoglobin (HbA1c ) and blood glucose levels were also measured. The FAZ area was manually segmented at the levels of the superficial capillary plexus (FAZSCP ) and deep capillary plexus (FAZDCP ). Results There was a strong relationship between the FAZ area of participants' right and left eyes (p ≤ 0.001) in both diabetic and non-diabetic groups. In the diabetic group, the FAZSCP (p = 0.047) and FAZDCP (p = 0.011) areas was significantly larger than in the non-diabetic group. Moreover, multiple linear regression analysis predicted a 0.07-mm2 increase in diabetic individuals' FAZSCP and FAZDCP areas for every 1% increase in their HbA1c level. Conclusions Our findings indicate that there is enlargement of the FAZ in diabetic individuals compared with non-diabetic individuals. In the diabetic group, this enlargement appears to be correlated with HbA1c level. OCT-A imaging could, therefore, be a useful tool to monitor the FAZ and identify potential early microvasculopathy in diabetes.

BackgroundOncolytic reovirus therapy for cancer induces a typical antiviral response to this RNA virus, including neutralizing antibodies. Concomitant treatment with cytotoxic chemotherapies has been hypothesized to improve the therapeutic potential of the virus. Chemotherapy side effects can include immunosuppression, which may slow the rate of the antiviral antibody response, as well as potentially make the patient more vulnerable to viral infection.MethodReovirus neutralizing antibody data were aggregated from separate phase I clinical trials of reovirus administered as a single agent or in combination with gemcitabine, docetaxel, carboplatin and paclitaxel doublet or cyclophosphamide. In addition, the kinetics of individual antibody isotypes were profiled in sera collected in these trials.ResultsThese data demonstrate preserved antiviral antibody responses, with only moderately reduced kinetics with some drugs, most notably gemcitabine. All patients ultimately produced an effective neutralizing antibody response.ConclusionPatients’ responses to infection by reovirus are largely unaffected by the concomitant drug treatments tested, providing confidence that RNA viral treatment or infection is compatible with standard of care treatments.

Purpose: The prevention and treatment of metastatic sarcoma are areas of significant unmet need. Immune checkpoint inhibitor monotherapy has shown little activity in sarcoma and there is great interest in identifying novel treatment combinations that may augment responses. In vitro and in vivo, we investigated the potential for an oncolytic vaccinia virus (GLV-1h68) delivered using isolated limb perfusion (ILP) to promote antitumor immune responses and augment response to PD-1 blockade in sarcoma. Experimental Design: In an established animal model of extremity sarcoma, we evaluated the potential of locoregional delivery of a vaccinia virus (GLV-1h68) alongside biochemotherapy (melphalan/TNFα) in ILP. Complementary in vitro assays for markers of immunogenic cell death were performed in sarcoma cell lines. Results: PD-1 monotherapy had minimal efficacy in vivo, mimicking the clinical scenario. Pretreatment with GLV-1h68 delivered by ILP (viral ILP) significantly improved responses. Furthermore, when performed prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented both local and distant relapse, curing a previously treatment-refractory model. Enhanced therapy was associated with marked modulation of the tumor microenvironment, with an increase in the number and penetrance of intratumoral CD8+ T cells and expansion and activation of dendritic cells. GLV-1h68 was capable of inducing markers of immunogenic cell death in human sarcoma cell lines. Conclusions: Viral ILP augments the response to PD-1 blockade, transforming this locoregional therapy into a potentially effective systemic treatment for sarcoma and warrants translational evaluation.

BackgroundRectal cancers show a highly varied response to neoadjuvant radiotherapy/chemoradiation (RT/CRT) and the impact of the tumor immune microenvironment on this response is poorly understood. Current clinical tumor regression grading systems attempt to measure radiotherapy response but are subject to interobserver variation. An unbiased and unique histopathological quantification method (change in tumor cell density (ΔTCD)) may improve classification of RT/CRT response. Furthermore, immune gene expression profiling (GEP) may identify differences in expression levels of genes relevant to different radiotherapy responses: (1) at baseline between poor and good responders, and (2) longitudinally from preradiotherapy to postradiotherapy samples. Overall, this may inform novel therapeutic RT/CRT combination strategies in rectal cancer.MethodsWe generated GEPs for 53 patients from biopsies taken prior to preoperative radiotherapy. TCD was used to assess rectal tumor response to neoadjuvant RT/CRT and ΔTCD was subjected to k-means clustering to classify patients into different response categories. Differential gene expression analysis was performed using statistical analysis of microarrays, pathway enrichment analysis and immune cell type analysis using single sample gene set enrichment analysis. Immunohistochemistry was performed to validate specific results. The results were validated using 220 pretreatment samples from publicly available datasets at metalevel of pathway and survival analyses.ResultsΔTCD scores ranged from 12.4% to −47.7% and stratified patients into three response categories. At baseline, 40 genes were significantly upregulated in poor (n=12) versus good responders (n=21), including myeloid and stromal cell genes. Of several pathways showing significant enrichment at baseline in poor responders, epithelial to mesenchymal transition, coagulation, complement activation and apical junction pathways were validated in external cohorts. Unlike poor responders, good responders showed longitudinal (preradiotherapy vs postradiotherapy samples) upregulation of 198 immune genes, reflecting an increased T-cell-inflamed GEP, type-I interferon and macrophage populations. Longitudinal pathway analysis suggested viral-like pathogen responses occurred in post-treatment resected samples compared with pretreatment biopsies in good responders.ConclusionThis study suggests potentially druggable immune targets in poor responders at baseline and indicates that tumors with a good RT/CRT response reprogrammed from immune “cold” towards an immunologically “hot” phenotype on treatment with radiotherapy.

How did narcotics become the largest industry in Afghanistan? When the war started in late 1970s, most of the opium and heroin production in the world was in the Golden Triangle—Myanmar, Thailand, and Laos—with smaller levels of production in the Golden Crescent—Pakistan, Iran, and...

Systemic relapse, after treatment of a localised primary tumour with neo-adjuvant radiotherapy and surgery, is the major cause of disease related mortality in patients with sarcoma. As with other cancers, many sarcoma patients derive no benefit from anti-PD-1 treatment. Combining radiotherapy and immunotherapy is under investigation as a means to improve response rates and control metastatic disease. Here, we use a retrospective cohort of sarcoma patients, treated with neoadjuvant radiotherapy, and TCGA data to explore patient stratification for immunotherapy and therapeutic targets of relevance to sarcoma. We show a group of patients with immune-hot undifferentiated pleomorphic sarcoma as one of the highest-ranking candidates for emerging 4-1BB targeting agents. A binary hot/cold classification method indicates 4-1BB-high hot sarcomas share many characteristics with immunotherapy responsive cancers of other pathologies. Hot tumours in sarcoma are however substantially less prevalent. Patient stratification, of intense interest for immunotherapies, is therefore even more important in sarcoma.

Aims Diabetes mellitus affects about 6% of the world’s population, and the chronic complications of the disease may result in macro- and micro-vascular changes. The purpose of the current study was to shed light on visual cortical oxygenation in diabetic individuals. We then aimed to compare the haemodynamic response (HDR) to visual stimulation with glycaemic control, given the likelihood of diabetic individuals suffering from such macro- and micro-vascular insult. Methodology Thirty participants took part in this explorative study, fifteen of whom had diabetes and fifteen of whom were non-diabetic controls. The HDR, measured as concentrations of oxyhaemoglobin [HbO] and deoxyhaemoglobin [HbR], to visual stimulation was recorded over the primary visual cortex (V1) using a dual-channel oximeter. The stimulus comprised a pattern-reversal checkerboard presented in a block design. Participants’ mean glycated haemoglobin (HbA1c) level (± SD) was 7.2 ± 0.6% in the diabetic group and 5.5 ± 0.4% in the non-diabetic group. Raw haemodynamic data were normalised to baseline, and the last 15 s of data from each ‘stimulus on’ and ‘stimulus off’ condition were averaged over seven duty cycles for each participant. Results There were statistically significant differences in ∆[HbO] and ∆[HbR] to visual stimulation between diabetic and non-diabetic groups (p

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. Keywords: bladder cancer, coxsackievirus A21, intercellular adhesion molecule-1

Simple Summary Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer that has a poor response to chemotherapy. Here, we assessed the immunological features of a series of 33 OCCCs and identified an immune-related gene expression signature that correlated with a patient’s risk of recurrence. Additionally, using multiplex immunofluorescence, we assessed the spatial distribution and abundance of immune cell populations at the protein level and identified that tumour-associated macrophages (TAM) and regulatory T cells are excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A mutated OCCCs, suggesting that the exclusion of these immune effectors could determine the host response in ARID1A mutant OCCCs. Abstract Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer characterised by a high frequency of loss-of-function ARID1A mutations and a poor response to chemotherapy. Despite their generally low mutational burden, an intratumoural T cell response has been reported in a subset of OCCC, with ARID1A purported to be a biomarker for the response to the immune checkpoint blockade independent of micro-satellite instability (MSI). However, assessment of the different immune cell types and spatial distribution specifically within OCCC patients has not been described to date. Here, we characterised the immune landscape of OCCC by profiling a cohort of 33 microsatellite stable OCCCs at the genomic, gene expression and histological level using targeted sequencing, gene expression profiling using the NanoString targeted immune panel, and multiplex immunofluorescence to assess the spatial distribution and abundance of immune cell populations at the protein level. Analysis of these tumours and subsequent independent validation identified an immune-related gene expression signature associated with risk of recurrence of OCCC. Whilst histological quantification of tumour-infiltrating lymphocytes (TIL, Salgado scoring) showed no association with the risk of recurrence or ARID1A mutational status, the characterisation of TILs via multiplexed immunofluorescence identified spatial differences in immunosuppressive cell populations in OCCC. Tumour-associated macrophages (TAM) and regulatory T cells were excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A-mutated OCCCs compared to ARID1A wild-type tumours, suggesting that the exclusion of these immune effectors could determine the host response of ARID1A-mutant OCCCs to therapy. Overall, our study has provided new insights into the immune landscape and prognostic associations in OCCC and suggest that tailored immunotherapeutic approaches may be warranted for different subgroups of OCCC patients.

Cytoplasmic nucleic acid sensors for double-stranded (ds) RNA (RIG-I/MDA5) and DNA (cGAS-STING) are pattern recognition receptors (PRRs) key to intracellular anti-viral responses. Recent research has highlighted roles for PRR agonists, including oncolytic virotherapy agents, in anti-tumor immunotherapy. Reovirus type 3 Dearing (Rt3D) is an oncolytic dsRNA virus with limited single-agent activity in clinical studies, but potential for use in combination regimens. We sought synergistic drug-virotherapy combinations using an unbiased screening approach that highlighted the CDK4/6 inhibitor, palbociclib, as a leading hit. We found that, when combined with Rt3D, palbociclib augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) signaling. Combined Rt3D-palbociclib treatment potently increased interferon signaling and endogenous retroviral transcripts. Knockdown (siRNA) studies indicated key UPR proteins and the RNA sensor, RIG-I, were essential to the phenotype observed. Mechanistically independent experiments, using canonical RIG-I agonists and the ER stress inducer (thapsigargin), confirmed cross-talk between RNA sensing and ER stress pathways that augment cancer cell death and interferon production. Combined Rt3D-palbociclib increased innate immune activation and effector function. Our findings demonstrate that UPR signaling and innate immune RNA sensor crosstalk can be exploited to enhance anti-cancer efficacy with pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, as well as broadening the therapeutic remit of CDK4/6 inhibitors to include their role as ER stress sensitizers. Citation Format: Victoria Roulstone, Joan Kyula, Richard Elliot, Christopher J. Lord, Nik Matthews, Vicki Jennings, Harriet Whittock, David Mansfield, Jyoti Choudhary, James Wright, Lu Yu, Alan Melcher, Richard Vile, Matt Coffey, Martin McLaughlin, Kevin Harrington. Mechanisms of therapeutic synergy between pattern recognition response agonists and cdk4 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1960.

Background Oncolytic viruses preferentially replicate in tumors as compared to normal tissue and promote immunogenic cell death and induction of host systemic anti-tumor immunity. HSV-1 was chosen for further development as an oncolytic immunotherapy in this study as it is highly lytic, infects human tumor cells broadly, kills mainly by necrosis and is a potent activator of both innate and adaptive immunity. HSV-1 also has a large capacity for the insertion of additional, potentially therapeutic, exogenous genes. Finally, HSV-1 has a proven safety and efficacy profile in patients with cancer, talimogene laherparepvec (T-VEC), an oncolytic HSV-1 which expresses GM-CSF, being the only oncolytic immunotherapy approach that has received FDA approval. As the clinical efficacy of oncolytic immunotherapy has been shown to be further enhanced by combination with immune checkpoint inhibitors, developing improved oncolytic platforms which can synergize with other existing immunotherapies is a high priority. In this study we sought to further optimize HSV-1 based oncolytic immunotherapy through multiple approaches to maximize: (i) the extent of tumor cell killing, augmenting the release of tumor antigens and danger-associated molecular pattern (DAMP) factors; (ii) the immunogenicity of tumor cell death; and (iii) the resulting systemic anti-tumor immune response. Methods To sample the wide diversity amongst clinical strains of HSV-1, twenty nine new clinical strains isolated from cold sores from otherwise healthy volunteers were screened across a panel of human tumor cell lines to identify the strain with the most potent tumor cell killing ability, which was then used for further development. Following deletion of the genes encoding ICP34.5 and ICP47 to provide tumor selectivity, the extent of cell killing and the immunogenicity of cell death was enhanced through insertion of a gene encoding a truncated, constitutively highly fusogenic form of the envelope glycoprotein of gibbon ape leukemia virus (GALV-GP-R−). A number of further armed derivatives of this virus were then constructed intended to further enhance the anti-tumor immune response which was generated following fusion-enhanced, oncolytic virus replication-mediated cell death. These viruses expressed GMCSF, an anti-CTLA-4 antibody-like molecule, CD40L, OX40L and/or 4-1BB, each of which is expected to act predominantly at the site and time of immune response initiation. Expression of these proteins was confirmed by ELISA and/or western blotting. Immunogenic cell death was assessed by measuring the levels of HMGB1 and ATP from cell free supernatants from treated cells, and by measuring the surface expression of calreticulin. GALV-GP-R− mediated cell to cell fusion and killing was tested in a range of tumor cell lines in vitro. Finally, the in vivo therapeutic potential of these viruses was tested using human A549 (lung cancer) and MDA-MB-231(breast cancer) tumor nude mouse xenograft models and systemic anti-tumor effects tested using dual flank syngeneic 4434 (melanoma), A20 (lymphoma) mouse tumor models alone and in combination with a murine anti-PD1 antibody, and 9 L (gliosarcoma) tumors in rats. Results The twenty nine clinical strains of HSV-1 isolated and tested demonstrated a broad range of tumor cell killing abilities allowing the most potent strain to be identified which was then used for further development. Oncolytic ability was demonstrated to be further augmented by the expression of GALV-GP-R− in a range of tumor cell lines in vitro and in mouse xenograft models in nude mice. The expression of GALV-GP-R− was also demonstrated to lead to enhanced immunogenic cell death in vitro as confirmed by the increased release of HMGB1 and ATP and increased levels of calreticulin on the cell surface. Experiments using the rat 9 L syngeneic tumor model demonstrated that GALV-GP-R− expression increased abscopal uninjected (anenestic) tumor responses and data using mouse 4434 tumors demonstrated that virus treatment increased CD8+ T cell levels both in the injected and uninjected tumor, and also led to increased expression of PD-L1. A combination study using varying doses of a virus expressing GALV-GP-R− and mGM-CSF and an anti-murine PD1 antibody showed enhanced anti-tumor effects with the combination which was most evident at low virus doses, and also lead to immunological memory. Finally, treatment of mice with derivatives of this virus which additionally expressed anti-mCTLA-4, mCD40L, m4-1BBL, or mOX40L demonstrated enhanced activity, particularly in uninjected tumors. Conclusion The new HSV-1 based platform described provides a potent and versatile approach to developing new oncolytic immunotherapies for clinical use. Each of the modifications employed was demonstrated to aid in optimizing the potential of the virus to both directly kill tumors and to lead to systemic therapeutic benefit. For clinical use, these viruses are expected to be most effective in combination with other anti-cancer agents, in particular PD1/L1-targeted immune checkpoint blockade. The first virus from this program (expressing GALV-GP-R− and hGM-CSF) has entered clinical development alone and in combination with anti-PD1 therapy in a number of tumor types (NCT03767348). Electronic supplementary material The online version of this article (10.1186/s40425-019-0682-1) contains supplementary material, which is available to authorized users.

In the past four decades, much of the contemporary narrative of Afghanistan has been defined by opium. However, underneath the veil of the opium economy, the cannabis trade remains an enduring component of Afghanistan’s political economy and culture. Much of this stems from the long history of cannabis cultivation and hashish production in the region. During the 1960s, the growing demand from Western nations for Afghan hashish helped forge key global trafficking networks, as well as significant changes to the cultivation of cannabis and production of hashish. Since then, production and trade evolved, with cultivation of cannabis more widespread. Ultimately, analyzing the cannabis trade and its historical antecedents, reveals how the cannabis trade, like the opium economy, transformed in response to local, regional, and global factors, remaining an important piece of the rural Afghan economy today.

The aim was to compare efficacy of treatments for diabetic macular oedema (DMO) from changes in visual acuity (VA) and central macular thickness (CMT).Peer-reviewed articles from 2004 to 2014 reporting intravitreal injections of bevacizumab (IVB), ranibizumab (IVR) or triamcinolone acetonide (IVTA) or laser photocoagulation therapy (LPT) provided data on pre-treatment (baseline) and final outcome measures. Net changes and relative changes (percentage) were assessed by linear regression analyses.From 88 data sets the overall net change of VA was -0.10 ± 0.12 logMAR (mean ± standard deviation), being -0.13 ± 0.11 logMAR for IVB, 0 ± 0.08 logMAR for IVR and -0.12 ± 0.08 logMAR for IVTA as compared to 0.01 ± 0.14 logMAR for LPT. For CMT, the overall net change was -103 ± 71 microns, being -108 ± 64 microns for IVB, -182 ± 73 microns for IVR, and -102 ± 57 microns for IVTA and was -49 ± 60 microns for LPT. Overall, modest correlations were found between the absolute central retinal (macular) thickness change and the VA change, and the relative changes in these measures (p0.001, r = 0.522 or 0.457). The predicted visual outcome from a 100 microns reduction in CMT was -0.083 logMAR units, an effect not substantially influenced by the CMT measurement method.Pharmacological treatment of DMO can be expected to result in a predictable decrease in CMT with an accompanying increase in VA, with the overall outcome being better than laser treatment.

Advanced extremity melanoma and sarcoma present a significant therapeutic challenge, requiring multimodality therapy to treat or even palliate disease. These aggressive tumours are relatively chemo-resistant, therefore new treatment approaches are urgently required. We have previously reported on the efficacy of oncolytic virotherapy (OV) delivered by isolated limb perfusion. In this report, we have improved therapeutic outcomes by combining OV with radiotherapy. In vitro, the combination of oncolytic vaccinia virus (GLV-1h68) and radiotherapy demonstrated synergistic cytotoxicity. This effect was not due to increased viral replication, but mediated through induction of intrinsic apoptosis. GLV-1h68 therapy downregulated the anti-apoptotic BCL-2 proteins (MCL-1 and BCL-XL) and the downstream inhibitors of apoptosis, resulting in cleavage of effector caspases 3 and 7. In an in vivo ILP model, the combination of OV and radiotherapy significantly delayed tumour growth and prolonged survival compared to single agent therapy. These data suggest that the virally-mediated down-regulation of anti-apoptotic proteins may increase the sensitivity of tumour cells to the cytotoxic effects of ionizing radiation. Oncolytic virotherapy represents an exciting candidate for clinical development when delivered by ILP. Its ability to overcome anti-apoptotic signals within tumour cells points the way to further development in combination with conventional anti-cancer therapies.

// Grainne McEntee 1 , Joan N. Kyula 1 , David Mansfield 1 , Henry Smith 1, 2 , Michelle Wilkinson 1, 2 , Claire Gregory 1 , Victoria Roulstone 1 , Matt Coffey 3 , Kevin J. Harrington 1 1 Targeted Therapy Team, the Institute of Cancer Research, London, UK 2 Sarcoma/Melanoma Unit, Department of Academic Surgery, Royal Marsden Hospital NHS Foundation Trust, London, UK 3 Oncolytics Biotech Inc., Calgary, Canada Correspondence to: Kevin J. Harrington, email: Kevin.Harrington@icr.ac.uk Keywords: reovirus, radiotherapy, melanoma, CUG2, apoptosis Received: January 18, 2016 Accepted: June 09, 2016 Published: July 1, 2016 ABSTRACT Oncolytic viruses selectively target and replicate in cancer cells, providing us with a unique tool with which to target and kill tumour cells. These viruses come from a diverse range of viral families including reovirus type 3 Dearing (RT3D), a non-pathogenic human double-stranded RNA oncolytic virus, which has been shown to be an effective therapeutic agent, both as a mono-therapy and in combination with traditional chemotherapeutic drugs. This study investigated the interaction between RT3D and radiotherapy in melanoma cell lines with a BRAF mutant, Ras mutant or BRAF/Ras wild type genotype. The data indicates that RT3D combined with radiotherapy significantly increased cytotoxicity relative to either single agent, independent of genotype, both in vitro and in vivo . The mechanism of enhanced cytotoxicity was dependent on an increase in viral replication, mediated by CUG2 up-regulation and subsequent down-regulation of pPKR and p-eIF2α, leading to the activation of mitochondrial apoptotic signalling resulting in increased cell death.

Introduction: Globally Intensive Care Unit (ICU) mortality rates vary, ranging from 10-30% with Australian rates at 8%. End-of-life care (EOLC) in ICU is an under researched area despite ICU clinicians being at the forefront of transitioning critically ill patients from receiving life-sustaining treatment to commencing EOLC. Objectives/Aims: To examine ICU clinicians’ perspective of EOLC in a tertiary referral Australian ICU. Methods: A convergent parallel mixed-method design was conducted at a 28-bed tertiary adult ICU. ICU clinicians were invited to complete Hansen and colleagues’ 30 item, 5 sub-scale EOLC questionnaire examining knowledge and ability, work environment, support for patient/family, work stress, and staff support. The 4-point Likert scale ranged from 1 = very good to 4 = poor. Demographic data were analysed using descriptive statistics and mean scores were calculated for sub-scales. Focus groups were held with nurses to elicit their perceptions of EOLC. Field notes were made, and inductive thematic analysis was used to identify emerging themes. Ethical approval was obtained. Results: 143 questionnaires were returned (response rate 64%). The mean overall score was 2.7 (SD 0.9), with scores > 2 indicating a need for improvement. Exploratory analysis identified that nurses rated all domains of EOLC as having potential for improvement, with support for staff 2.6 (SD 0.48), and work stress work stress 2.9 (SD 0.54) emerging as the prominent area requiring improvement. Focus groups identified themes of workload, ICU ward acuity, clinician inexperience, lack of education, role ambiguity, person-centered care, and effective interprofessional team work. Respondents suggested areas for improvement including increasing EOLC resources, education, an EOLC guideline and improved collaboration, including with EOLC specialists. Conclusion: Nurses perceived that EOLC can be improved in this ICU with key barriers and enablers identified. Questionnaire results identified that interventions targeting all five domains is needed.

Purpose: The CANON [CAVATAK in NON–muscle-invasive bladder cancer (NMIBC)] study evaluated a novel ICAM-1–targeted immunotherapeutic-coxsackievirus A21 as a novel oncolytic agent against bladder cancer. Patients and Methods: Fifteen patients enrolled in this “window of opportunity” phase I study, exposing primary bladder cancers to CAVATAK prior to surgery. The first 9 patients received intravesical administration of monotherapy CAVATAK; in the second stage, 6 patients received CAVATAK with a subtherapeutic dose of mitomycin C, known to enhance expression of ICAM-1 on bladder cancer cells. The primary endpoint was to determine patient safety and maximum tolerated dose (MTD). Secondary endpoints were evidence of viral replication, induction of inflammatory cytokines, antitumor activity, and viral-induced changes in resected tissue. Results: Clinical activity of CAVATAK was demonstrated by induction of tumor inflammation and hemorrhage following either single or multiple administrations of CAVATAK in multiple patients, and a complete resolution of tumor in 1 patient. Whether used alone or in combination with mitomycin C, CAVATAK caused marked inflammatory changes within NMIBC tissue biopsies by upregulating IFN-inducible genes, including both immune checkpoint inhibitory genes (PD-L1 and LAG3) and Th1-associated chemokines, as well as the induction of the innate activator RIG-I, compared with 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 tumor cell death together with the virus-mediated increases in “immunological heat” within the tumor microenvironment all indicate that CAVATAK may be potentially considered as a novel therapeutic for NMIBC.