Immune landscape, evolution, hypoxia-mediated viral mimicry pathways and therapeutic potential in molecular subtypes of pancreatic neuroendocrine tumours.

OBJECTIVE: A comprehensive analysis of the immune landscape of pancreatic neuroendocrine tumours (PanNETs) was performed according to clinicopathological parameters and previously defined molecular subtypes to identify potential therapeutic vulnerabilities in this disease. DESIGN: Differential expression analysis of 600 immune-related genes was performed on 207 PanNET samples, comprising a training cohort (n=72) and two validation cohorts (n=135) from multiple transcriptome profiling platforms. Different immune-related and subtype-related phenotypes, cell types and pathways were investigated using different in silico methods and were further validated using spatial multiplex immunofluorescence. RESULTS: The study identified an immune signature of 132 genes segregating PanNETs (n=207) according to four previously defined molecular subtypes: metastasis-like primary (MLP)-1 and MLP-2, insulinoma-like and intermediate. The MLP-1 subtype (26%-31% samples across three cohorts) was strongly associated with elevated levels of immune-related genes, poor prognosis and a cascade of tumour evolutionary events: larger hypoxic and necroptotic tumours leading to increased damage-associated molecular patterns (viral mimicry), stimulator of interferon gene pathway, T cell-inflamed genes, immune checkpoint targets, and T cell-mediated and M1 macrophage-mediated immune escape mechanisms. Multiplex spatial profiling validated significantly increased macrophages in the MLP-1 subtype. CONCLUSION: This study provides novel data on the immune microenvironment of PanNETs and identifies MLP-1 subtype as an immune-high phenotype featuring a broad and robust activation of immune-related genes. This study, with further refinement, paves the way for future precision immunotherapy studies in PanNETs to potentially select a subset of MLP-1 patients who may be more likely to respond.

Sub-clinical thickening of the fovea in diabetes and its relationship to glycaemic control: a study using swept-source optical coherence tomography.

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 < 0.0005). The diabetic group comprised type 1 (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 < 0.0005) than non-diabetic individuals. In the diabetic group, multiple linear regression analysis revealed a conspicuous relationship between CPT and HbA1c level (ß = 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 (ß = - 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.

Prostate-specific membrane antigen expression in melanoma metastases.

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.

HSP90 inhibition sensitizes head and neck cancer to platin-based chemoradiotherapy by modulation of the DNA damage response resulting in chromosomal fragmentation.

BACKGROUND: Concurrent cisplatin radiotherapy (CCRT) is a current standard-of-care for locally advanced head and neck squamous cell carcinoma (HNSCC). However, CCRT is frequently ineffective in patients with advanced disease. It has previously been shown that HSP90 inhibitors act as radiosensitizers, but these studies have not focused on CCRT in HNSCC. Here, we evaluated the HSP90 inhibitor, AUY922, combined with CCRT. METHODS: The ability of AUY922 to sensitize to CCRT was assessed in p53 mutant head and neck cell lines by clonogenic assay. Modulation of the CCRT induced DNA damage response (DDR) by AUY922 was characterized by confocal image analysis of RAD51, BRCA1, 53BP1, ATM and mutant p53 signaling. The role of FANCA depletion by AUY922 was examined using shRNA. Cell cycle checkpoint abrogation and chromosomal fragmentation was assessed by western blot, FACS and confocal. The role of ATM was also assessed by shRNA. AUY922 in combination with CCRT was assessed in vivo. RESULTS: The combination of AUY922 with cisplatin, radiation and CCRT was found to be synergistic in p53 mutant HNSCC. AUY922 leads to significant alterations to the DDR induced by CCRT. This comprises inhibition of homologous recombination through decreased RAD51 and pS1524 BRCA1 with a corresponding increase in 53BP1 foci, activation of ATM and signaling into mutant p53. A shift to more error prone repair combined with a loss of checkpoint function leads to fragmentation of chromosomal material. The degree of disruption to DDR signalling correlated to chromosomal fragmentation and loss of clonogenicity. ATM shRNA indicated a possible rationale for the combination of AUY922 and CCRT in cells lacking ATM function. CONCLUSIONS: This study supports future clinical studies combining AUY922 and CCRT in p53 mutant HNSCC. Modulation of the DDR and chromosomal fragmentation are likely to be analytical points of interest in such trials.

Isolated limb perfusion with biochemotherapy and oncolytic virotherapy combines with radiotherapy and surgery to overcome treatment resistance in an animal model of extremity soft tissue sarcoma.

The management of locally advanced or recurrent extremity sarcoma often necessitates multimodal therapy to preserve a limb, of which isolated limb perfusion (ILP) is a key component. However, with standard chemotherapeutic agents used in ILP, the duration of response is limited. Novel agents or treatment combinations are urgently needed to improve outcomes. Previous work in an animal model has demonstrated the efficacy of oncolytic virotherapy when delivered by ILP and, in this study, we report further improvements from combining ILP-delivered oncolytic virotherapy with radiation and surgical resection. In vitro, the combination of radiation with an oncolytic vaccinia virus (GLV-1h68) and melphalan demonstrated increased cytotoxicity in a panel of sarcoma cell lines. The effects were mediated through activation of the intrinsic apoptotic pathway. In vivo, combinations of radiation, oncolytic virotherapy and standard ILP resulted in delayed tumour growth and prolonged survival when compared with standard ILP alone. However, local disease control could only be secured when such treatment was combined with surgical resection, the timing of which was crucial in determining outcome. Combinations of oncolytic virotherapy with surgical resection and radiation have direct clinical relevance in extremity sarcoma and represent an exciting prospect for improving outcomes in this pathology.

BRAF- and MEK-Targeted Small Molecule Inhibitors Exert Enhanced Antimelanoma Effects in Combination With Oncolytic Reovirus Through ER Stress.

Reovirus type 3 (Dearing) (RT3D) infection is selective for cells harboring a mutated/activated RAS pathway. Therefore, in a panel of melanoma cell lines (including RAS mutant, BRAF mutant and RAS/BRAF wild-type), we assessed therapeutic combinations that enhance/suppress ERK1/2 signaling through use of BRAF/MEK inhibitors. In RAS mutant cells, the combination of RT3D with the BRAF inhibitor PLX4720 (paradoxically increasing ERK1/2 signaling in this context) did not enhance reoviral cytotoxicity. Instead, and somewhat surprisingly, RT3D and BRAF inhibition led to enhanced cell kill in BRAF mutated cell lines. Likewise, ERK1/2 inhibition, using the MEK inhibitor PD184352, in combination with RT3D resulted in enhanced cell kill in the entire panel. Interestingly, TCID50 assays showed that BRAF and MEK inhibitors did not affect viral replication. Instead, enhanced efficacy was mediated through ER stress-induced apoptosis, induced by the combination of ERK1/2 inhibition and reovirus infection. In vivo, combined treatments of RT3D and PLX4720 showed significantly increased activity in BRAF mutant tumors in both immune-deficient and immune-competent models. These data provide a strong rationale for clinical translation of strategies in which RT3D is combined with BRAF inhibitors (in BRAF mutant melanoma) and/or MEK inhibitors (in BRAF and RAS mutant melanoma).

Isolated limb perfusion with melphalan, tumour necrosis factor-alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma.

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in-transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer-selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour-targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor-alpha (TNF-α) and Lister strain vaccinia virus (GLV-1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF-α, melphalan and GLV-1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV-1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF-α/GLV-1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF-α/melphalan-based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.

Reliability and reproducibility of several methods of arthroscopic assessment of femoral tunnel position during anterior cruciate ligament reconstruction.

PURPOSE: To assess interobserver and intraobserver agreement of estimating anterior cruciate ligament (ACL) femoral tunnel positioning arthroscopically using circular and linear (noncircular) estimation methods and to determine whether overlay template visual aids improve agreement. METHODS: Standardized intraoperative pictures of femoral tunnel pilot holes (taken with a 30° arthroscope through an anterolateral portal at 90° of knee flexion with horizontal being parallel to the tibial surface) in 27 patients undergoing single-bundle ACL reconstruction were presented to 3 fellowship-trained arthroscopists on 2 separate occasions. On both viewings, each surgeon estimated the femoral tunnel pilot hole location to the nearest half-hour mark using a whole clock face and half clock face, to the nearest 15° using a whole compass and half compass, in the top or bottom half of a linear quadrant, and in the top or bottom half of a linear trisector. Evaluations were performed first without and then with an overlay template of each estimation method. RESULTS: The average difference among reviewers was quite similar for all 4 circular methods with the use of visual aids. Without overlay template visual aids, pair-wise κ statistic values for interobserver agreement ranged from -0.14 to 0.56 for the whole clock face and from 0.16 to 0.42 for the half clock face. With overlay visual guides, interobserver agreement ranged from 0.29 to 0.63 for the whole clock face and from 0.17 to 0.66 for the half clock face. The quadrant method's interobserver agreement ranged from 0.22 to 0.60, and that of the trisection method ranged from 0.17 to 0.57. Neither linear estimation method's reliability uniformly improved with the use of overlay templates. Intraobserver agreement without overlay templates ranged from 0.17 to 0.49 for the whole clock face, 0.11 to 0.47 for the half clock face, 0.01 to 0.66 for the quadrant method, and 0.20 to 0.57 for the trisection method. Use of overlay templates did not uniformly improve intraobserver agreement for any estimation method. CONCLUSIONS: There does not appear to be any advantage of using a half clock face or compass for estimating femoral tunnel position compared with a whole clock-face analogy. Visual reference aids appear to improve interobserver agreement (reliability) of circular analogies. The linear quadrant appears to be the most reliable method (fair to moderate agreement) for estimating femoral tunnel position without a visual aid for reference, but even better reliability, ranging from fair to good agreement, may be obtained by using the whole clock-face analogy with a visual aid. CLINICAL RELEVANCE: Increasing femoral tunnel position reliability may improve outcomes of ACL reconstruction surgery.

A survey on autonomous environmental monitoring approaches: towards unifying active sensing and reinforcement learning.

The environmental pollution caused by various sources has escalated the climate crisis making the need to establish reliable, intelligent, and persistent environmental monitoring solutions more crucial than ever. Mobile sensing systems are a popular platform due to their cost-effectiveness and adaptability. However, in practice, operation environments demand highly intelligent and robust systems that can cope with an environment's changing dynamics. To achieve this reinforcement learning has become a popular tool as it facilitates the training of intelligent and robust sensing agents that can handle unknown and extreme conditions. In this paper, a framework that formulates active sensing as a reinforcement learning problem is proposed. This framework allows unification with multiple essential environmental monitoring tasks and algorithms such as coverage, patrolling, source seeking, exploration and search and rescue. The unified framework represents a step towards bridging the divide between theoretical advancements in reinforcement learning and real-world applications in environmental monitoring. A critical review of the literature in this field is carried out and it is found that despite the potential of reinforcement learning for environmental active sensing applications there is still a lack of practical implementation and most work remains in the simulation phase. It is also noted that despite the consensus that, multi-agent systems are crucial to fully realize the potential of active sensing there is a lack of research in this area.

Modelling bronchial epithelial-fibroblast cross-talk in idiopathic pulmonary fibrosis (IPF) using a human-derived in vitro air liquid interface (ALI) culture.

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3-4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air-liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFß-2, ZO-1 and DN12. When stimulated with exogenous TGFß, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFß-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1ß, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFß isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFß signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

Durable responses to ATR inhibition with ceralasertib in tumors with genomic defects and high inflammation.

BACKGROUNDPhase 1 study of ATRinhibition alone or with radiation therapy (PATRIOT) was a first-in-human phase I study of the oral ATR (ataxia telangiectasia and Rad3-related) inhibitor ceralasertib (AZD6738) in advanced solid tumors.METHODSThe primary objective was safety. Secondary objectives included assessment of antitumor responses and pharmacokinetic (PK) and pharmacodynamic (PD) studies. Sixty-seven patients received 20-240 mg ceralasertib BD continuously or intermittently (14 of a 28-day cycle).RESULTSIntermittent dosing was better tolerated than continuous, which was associated with dose-limiting hematological toxicity. The recommended phase 2 dose of ceralasertib was 160 mg twice daily for 2 weeks in a 4-weekly cycle. Modulation of target and increased DNA damage were identified in tumor and surrogate PD. There were 5 (8%) confirmed partial responses (PRs) (40-240 mg BD), 34 (52%) stable disease (SD), including 1 unconfirmed PR, and 27 (41%) progressive disease. Durable responses were seen in tumors with loss of AT-rich interactive domain-containing protein 1A (ARID1A) and DNA damage-response defects. Treatment-modulated tumor and systemic immune markers and responding tumors were more immune inflamed than nonresponding.CONCLUSIONCeralasertib monotherapy was tolerated at 160 mg BD intermittently and associated with antitumor activity.TRIAL REGISTRATIONClinicaltrials.gov: NCT02223923, EudraCT: 2013-003994-84.FUNDINGCancer Research UK, AstraZeneca, UK Department of Health (National Institute for Health Research), Rosetrees Trust, Experimental Cancer Medicine Centre.

Mutations in CNNM4 cause Jalili syndrome, consisting of autosomal-recessive cone-rod dystrophy and amelogenesis imperfecta.

The combination of recessively inherited cone-rod dystrophy (CRD) and amelogenesis imperfecta (AI) was first reported by Jalili and Smith in 1988 in a family subsequently linked to a locus on chromosome 2q11, and it has since been reported in a second small family. We have identified five further ethnically diverse families cosegregating CRD and AI. Phenotypic characterization of teeth and visual function in the published and new families reveals a consistent syndrome in all seven families, and all link or are consistent with linkage to 2q11, confirming the existence of a genetically homogenous condition that we now propose to call Jalili syndrome. Using a positional-candidate approach, we have identified mutations in the CNNM4 gene, encoding a putative metal transporter, accounting for the condition in all seven families. Nine mutations are described in all, three missense, three terminations, two large deletions, and a single base insertion. We confirmed expression of Cnnm4 in the neural retina and in ameloblasts in the developing tooth, suggesting a hitherto unknown connection between tooth biomineralization and retinal function. The identification of CNNM4 as the causative gene for Jalili syndrome, characterized by syndromic CRD with AI, has the potential to provide new insights into the roles of metal transport in visual function and biomineralization.

Measuring the foveal avascular zone in diabetes: A study using optical coherence tomography angiography.

AIMS/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 to investigate the effect of diabetes on the FAZ. MATERIALS AND METHODS: A total of 11 participants with diabetes and 11 participants without diabetes 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 optical coherence tomography angiography (OCT-A) scans of participants' right and left eyes were taken. Glycosylated hemoglobin (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 diabetes and non-diabetes groups. In the diabetes group, the FAZSCP (P = 0.047) and FAZDCP (P = 0.011) areas was significantly larger than in the non-diabetes group. Moreover, multiple linear regression analysis predicted a 0.07-mm2 increase in the FAZSCP and FAZDCP areas of individuals with diabetes for every 1% increase in their HbA1c level. CONCLUSIONS: Our findings show that there is enlargement of the FAZ in individuals with diabetes compared with individuals without diabetes. In the diabetes 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.

Virally programmed extracellular vesicles sensitize cancer cells to oncolytic virus and small molecule therapy.

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.

Antiviral antibody responses to systemic administration of an oncolytic RNA virus: the impact of standard concomitant anticancer chemotherapies.

BACKGROUND: Oncolytic 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. METHOD: Reovirus 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. RESULTS: These 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. CONCLUSION: Patients' 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.

Proteomics of REPLICANT perfusate detects changes in the metastatic lymph node microenvironment.

In breast cancer (BC), detecting low volumes of axillary lymph node (ALN) metastasis pre-operatively is difficult and novel biomarkers are needed. We recently showed that patient-derived ALNs can be sustained ex-vivo using normothermic perfusion. We now compare reactive (tumour-free; n = 5) and macrometastatic (containing tumour deposits >2 mm; n = 4) ALNs by combining whole section multiplex immunofluorescence with TMT-labelled LC-MS/MS of the circulating perfusate. Macrometastases contained significantly fewer B cells and T cells (CD4+/CD8+/regulatory) than reactive nodes (p = 0.02). Similarly, pathway analysis of the perfusate proteome (119/1453 proteins significantly differentially expressed) showed that immune function was diminished in macrometastases in favour of 'extracellular matrix degradation'; only 'neutrophil degranulation' was preserved. Qualitative comparison of the perfusate proteome to that of node-positive pancreatic and prostatic adenocarcinoma also highlighted 'neutrophil degranulation' as a contributing factor to nodal metastasis. Thus, metastasis-induced changes in the REPLICANT perfusate proteome are detectable, and could facilitate biomarker discovery.

Computational Image Analysis of T-Cell Infiltrates in Resectable Gastric Cancer: Association with Survival and Molecular Subtypes.

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.

Differential and longitudinal immune gene patterns associated with reprogrammed microenvironment and viral mimicry in response to neoadjuvant radiotherapy in rectal cancer.

BACKGROUND: Rectal 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. METHODS: We 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. RESULTS: ΔTCD 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. CONCLUSION: This 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.

Quantitative Assessment and Prognostic Associations of the Immune Landscape in Ovarian Clear Cell Carcinoma.

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.