Hepatocellular carcinoma (HCC) is a leading cause of global cancer-related deaths and cases are predicted to rise in the coming decades. In the vast majority of patients, HCC develops in the context of chronic liver disease, which provides the setting for a complex tumour microenvironment characterised by constant induction of cell death with compensatory hyperproliferation, chronic inflammation, maladaptive wound healing, and fibrosis. While this inflammatory microenvironment provides an overall pro-carcinogenic milieu, the hepatic immune system also participates in tumour surveillance and anti-tumour immune responses. Targeting the tumour microenvironment is therefore a critical strategy in both treating advanced HCC and preventing tumour recurrence in patients undergoing curative therapies. The recent approval for the use of immunotherapy for treating HCC, specifically the combination of immune checkpoint blockers with anti-VEGF agents, has helped to confirm the long held belief that targeting the immune microenvironment could be an effective approach to treating this tumour. While immuno-oncological therapeutic options generally provided survival benefit for advanced, non-resectable HCC with manageable side effects compared to previous medical approaches, treatment efficacy is still not satisfying and patient stratification is not well defined. The aim of this article collection is to highlight new pathways that may help in developing novel immunotherapeutic approaches for HCC and to explore the optimal use of immunotherapies in the context of the expanding arsenal of therapies that are now becoming available for advanced HCC.Lu et al. report that the Na+/K+-ATPase, ATP1B3, is upregulated in HCC tissue and HCC cell lines and proteomic analysis of publically available databases confirmed a correlation with tumour immune infiltrates. Further functional assays demonstrate that ATP1B3 contributes to key tumourigenic pathways including cell proliferation and migration, and raise the possibility of targeting ATP1B3 in patients with HCC. In another article, Jiang et al. focus on the contribution of Tank-binding kinase 1 (TBK1) in regulating CD8 T cell infiltration in HCC. Using a model of HCC developed on the background of chronic liver inflammation, they demonstrate that an inhibitor of TBK1 alters the cytokine milieu within the tumour and promotes CD8 T cell infiltration leading to suppression of tumour growth. The need to take into account other characteristics of the tumour microenvironment, specifically hypoxia, is highlighted by the analysis carried out by Mo et al. Based on large HCC genomic datasets, they identified a link between hypoxia-associated genes and immunosuppressive features in tumour samples. The hypoxia gene signature may be a potential tool for stratifying patients for immunotherapy but also sheds light on the need to overcome alternative TME pathways in order to boost the efficacy of immunotherapy.The last few years have seen a major interest in the study of the tumour microenvironment (TME). It is now clear that tumours not only have intrinsic effects that promote cell survival/proliferation but can also influence the extrinsic tissue microenvironment and drive a programme of immune evasion. Giraud et al. cover the factors that initiate tumour development in the liver and subsequent tumour progression. They highlight the complex cross-talk between the immune system and hepatic microenvironment and summarise the clinical trials that are now taking place building on the knowledge that has been gathered in the field. Lurje et al. focus on the critical role of antigen presentation in the cross-talk between immune cells and how promoting effective antigen presentation could switch the TME from an immunosuppressive to an immunostimulatory state. They review the rationale for in situ vaccination and cover the mechanisms of antigen presentation and the range of approaches that are being undertaken to harness the TME.Whilst T cells are the critical effector arm of the immune system in attacking and preventing tumours, they are educated by a range of innate and adaptive immune populations. In the context of HCC, the liver microenvironment itself has a vital role in influencing T cell function by way of its resident non-parenchymal cell populations including Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells. In their review detailing the landscape of HCC, Sharma and Motedayen Aval highlight the issues surrounding second line agents in patients with advanced HCC. They provide a summary of approaches being considered for novel combination therapies to overcome the resistance to – or lack of efficacy – of immune checkpoint inhibitors. Looking at the experience with renal cell carcinoma they also discuss the sequential use of Tyrosine Kinase inhibitors following immunotherapy. In another article, Mohr et al. review the journey towards the use of immune checkpoint inhibitors in HCC but also highlight the questions that still need answering regarding the optimal use of these therapies in the setting of current treatment algorithms. Such open questions include optimal treatment options in non-viral induced HCC or for patients with advanced stage cirrhosis (e.g. Child B or C). Moreover, they summarise the future directions with immune checkpoint inhibitors including combinations with novel immunotherapies as well as elucidating the role of immunotherapy in the neoadjuvant setting and combining with loco-regional therapy and trans-arterial chemoembolization.The advent of immunotherapy has opened up new options for patients with HCC, yet the outlook in patients with advanced disease remains poor and there still remain significant questions regarding the stratification of patients for immunotherapy. Immunotherapy has been an exciting breakthrough in HCC yet the tumour microenvironment of HCC still remains a major challenge to therapeutic success. Understanding the optimal use of immunotherapies in the clinical setting and the identification of new therapies to boost the efficacy of current strategies will hopefully lead to major improvements in survival for patients with HCC.All authors made a contribution to writing the editorial and approved it for publication.SS is funded by a Cancer Research UK Advanced Clinician Scientist fellowship C53575/A29959 and supported by HUNTER funded through a partnership between Cancer Research United Kingdom, Fondazione AIRC and Fundación Científica de la Asociación Española Contra el Cáncer. AL is funded by NIH/NCI R37CA230636 and Damon Runyon-Rachleff Innovator Award.SS receives consultancy fees from Faron Pharmaceuticals. Work in the lab of FT has received funding by Allergan, Inventiva, Gilead and BMS. Work in the lab of AL has received funding from Pfizer and Genentech for unrelated projects.


Sporadic Legionnaires’ disease is frequently detected in commercial truck drivers. We report 2 sporadic cases of this disease in Barcelona, Spain, that occurred during December 2019 and September 2020. Laboratory findings were consistent with windshield wiper fluid without added screen wash as a possible source of infection for both cases. Legionella bacteria. Most Legionella infections are related to contaminated artificial water systems. Systems with warm water (35°C), stagnation, and lack of disinfection and maintenance can lead to proliferation of Legionella spp.  received a case report of Legionnaires’ disease in a 59-year-old man. Onset of symptoms had begun a week before diagnosis. On December 13, the patient sought care at a hospital, and a diagnosis of Legionnaires’ disease was made by UAT. A respiratory sample was not available because of the lack of productive secretions. The patient was hospitalized briefly, and his clinical course was unremarkable. After discharge, he completed the remaining course of antimicrobial drug therapy and proceeded favorably to cure.After the case was reported, public health nurses contacted the patient to complete a structured epidemiologic questionnaire that included demographic data, personal risk factors, activities, and potential exposures during the 14 days before illness onset. The patient smoked and had a medical history of hypertension and type 2 diabetes. He worked as a commercial truck driver, and his driving route included merchandise pickup at an industrial area once a day. The truck used for work was self-owned; the patient had purchased it secondhand ≈1.5 years before illness. He reported that the vehicle had been unused for several months before the purchase and denied using screen wash in the windshield wiper fluid.https://www.deltalab.es) treated with sodium thiosulfate. The PHAB laboratory analyzed the sample for L. pneumophila. Culture results using Legionella selective media, according to the ISO 11731:2017 protocol (https://www.iso.org), were negative. However, the sample was also analyzed by using real-time PCR  because both patients were given a diagnosis by UAT, which detects only Lp1. DNA of L. pneumophila (unknown serogroup) was found in the windshield wiper fluid for the first case, and Legionella spp. was cultured from the fluid for the second case. Although the presence of Lp1 was not confirmed in the windshield wiper fluid for either case, laboratory findings indicated that the flid was a potential source for both cases.We report 2 sporadic cases of Legionnaires’ disease in commercial truck drivers in which the laboratory findings were consistent with, but not exclusive to, the windshield wiper fluid as the source of infection. The etiologic agent was presumed to be Legionella spp. in windshield wiper fluid (Previous studies have identified windshield wiper fluid without screen wash as a potential risk factor for Legionnaires’ disease (L. pneumophila in road puddles (Legionella spp. could reach the tank after a car wash at a contaminated installation or through a contaminated water source used to fill the tank. Warm temperatures or heat radiated from the motor of the vehicle to the water tank, along with lower or absent methanol levels in screen wash fluid, and water stagnation could favor bacterial proliferation in the windshield wiper fluid tank.Several vehicle-related sources have been described as confirmed or potential sources of Legionnaires’ disease, although these sources were infrequently considered and difficult to investigate (Legionella spp. growth through a bactericidal effect of screen wash components, such as propanol/methanol (Beyond routinely investigating this source, especially in commercial truck drivers, a simple measure of adding screen wash to the fluid can be recommended. This action has the potential benefit of decreasing the risk for infection by inhibiting growth of In summary, our results indicate that windshield wiper fluid is a potential source of sporadic Legionnaires’ disease, especially in commercial truck drivers, and should be routinely investigated. A simple recommendation of adding screen wash to windshield wiper fluid or emptying the tank when the vehicle is unused for several months is a preventive measure likely to be effective and should be adopted by drivers.Additional information on vehicle windshield wiper fluid as potential source of sporadic Legionnaire’s disease in commercial truck drivers.


Enterobacterales (CNSE) produce carbapenemases, but some isolates do not carry these enzymes. Ceftazidime-avibactam (CAZ-AVI), meropenem-vaborbactam (MBV), and imipenem-relebactam (IMR) have activity against CNSE isolates producing serine-carbapenemases, but these agents might have variable activity against isolates that do not produce these enzymes. We evaluated the activity of these agents against a collection of 170 carbapenemase-negative CNSE collected during 5 years in US hospitals.Most carbapenem-nonsusceptible Enterobacterales isolates  collected in US hospitals from 2016-2020 were susceptibility (S) tested by reference broth microdilution methods. Results were interpreted using CLSI 2020 breakpoints. CNSE displayed nonsusceptible (NS) MICs for imipenem or meropenem. CNSE isolates were screened for carbapenemase genes using whole genome sequencing.K. aerogenes (n=42), K. pneumoniae (32), and E. cloacae (32), but 13 other species also were carbapenemase negative. CAZ-AVI inhibited all carbapenemase-negative CNSE isolates . MBV and IMR inhibited 97.6% and 92.3% of the isolates. Amikacin (AMK) and tigecycline (TIG) inhibited 97.1% and 93.5%, while levofloxacin and meropenem (MEM) inhibited 63.5% and 64.7%. A total of 141 (82.9%) isolates were nonsusceptible (NS) to imipenem. When MEM NS isolates (n=60) were analysed separately, the S rates were lower for all agents except CAZ-AVI. CAZ-AVI inhibited all MEM NS carbapenemase-negative CNSE isolates. MBV and IMR inhibited 93.1% and 89.7% of these isolates, respectively. AMK and TIG inhibited 93.3% and 88.3%, respectively. Only 8.3% of the isolates were resistant to colistin.Among 685 (1.4% of the isolates) CNSE, 170 (24.8% of the CNSE) isolates did not produce carbapenemases. Most of these isolates were CAZ-AVI displayed good activity against carbapenemase-negative CNSE isolates, including all MEM NS that displayed greater resistance rates against all comparator agents. High-risk patients with infections caused by CNSE have an increased mortality rate compared to isolates susceptible to these agents. Implementation of appropriate therapy for these isolates is critical.Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Helio S. Sader, MD, PhD, FIDSA, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)


Here we describe the presentation, identification, operation details and subsequent histological analysis of an onychomatricoma, a benign rare subungual tumour that is often misidentified and diagnosed.No public involvement to declare, patient was consented for surgical excision and provided verbal consent to photography and its use in publishing.One 44-year-old female.One time surgical excision.Not applicable.Complete excision and histopathological report of specimen provided.Complete surgical excision is an effective means of treatment for onychomatricoma. A 44-year-old right-handed female was referred for a dystrophic nail affecting her left middle finger for the past four years. There was no associated pain or loss of function. The patient was on regular fluoxetine and was otherwise fit and well. Examination revealed yellow discolouration and a subtle fullness of the medial nail plate on the left middle finger.Initial working differential diagnoses included myxoid cyst, ganglion, or glomus tumour. MRI of the left hand detected a small, ovoid, enhancing 2 × 1 mm lesion with no underlying bony erosion, suggestive of a small glomus tumour.Operatively, the nail plate was removed, revealing a peculiar frond-like tumour occupying the germinal matrix. The tumour had invaded the proximal soft tissues locally and was completely excised in continuity.Histological examination of the 4 × 3 mm specimen on microscopy showed an exophytic fibroepithelial tumour formed from tall and broad papillary structures covered by a thin layer of matrical type epithelium. The core of the papillary structures consisted of uniform fibroblast-like cells with collagen fibres arranged in a vertical orientation. A few capillaries were noted within the stroma. The overall configuration resembled that of a sea anemone.Sections of the deep edge of the nail plate showed jigsaw-like variations, likely due to invasion by the finger-like projections of the tumour. Immunohistochemistry revealed background stromal staining with CD34, with negative staining of: S100, actin, desmin, EMA, AE1/3, CK8/18.No dysplasia or malignancy was identified, and the overall picture favoured an onychomatricoma. The operative site has healed well and there has been no recurrence at latest follow-up. 2Onychomatricoma is a rare benign subungual tumour affecting the germinal nail matrix, first reported by Baran and Kint in 1992.Differentials for onychomatricoma include Bowen's disease, fibrokeratoma, onychomycosis, squamous cell carcinoma, and subungual verruca vulgaris amongst others.Complete excision remains the treatment modality of choice. We have no competing interests to declare.


Gall bladder perforation (GBP) is a rare and life-threatening complication of acute cholecystitis that requires immediate intervention. The diagnosis itself poses a diagnostic challenge, if the patient presents after the perforation of the gall bladder, especially if the initial imaging techniques such as ultrasonogram (US), computed tomography (CT) scan, hepatobiliary iminodiacetic acid (HIDA) scan and magnetic resonance cholangiopancreatography (MRCP) are inconclusive. Subtle clues such as free fluid around gall bladder and contracted gall bladder should warrant the clinician as these might be the only clues suggestive of gall bladder perforation. Here we describe a case of GBP successfully diagnosed by peritoneal drainage and analysis and subsequently managed by endoscopic retrograde cholangiopancreatography (ERCP) and open cholecystectomy. Gall bladder perforation (GBP) is a rare and life-threatening complication of acute cholecystitis that requires immediate intervention . The oveHere we describe a case of GBP successfully diagnosed by peritoneal fluid drainage and analysis and subsequently managed by endoscopic retrograde cholangiopancreatography (ERCP) and open cholecystectomy.An 80-year-old Hispanic lady with past medical history of diabetes mellitus type 2 and hypertension presented to the Intensive Care Unit (ICU) with fever, hypotension and leukocytosis. Her vitals on presentation were: Pulse- 88/min, Temperature- 102.5F, BP- 86/49 mmHg, SpO2 87% on room air. On evaluation, she was found to have severe sepsis, acute respiratory distress and a high sequential organ failure assessment score (SOFA score). She immediately developed pulseless ventricular tachycardia with return of spontaneous circulation within 5 minutes. She was then intubated, started on intravenous fluids as per SEPSIS protocol, vasopressor (Norepinephrine) to maintain target mean arterial pressure (MAP) of 65 and broad-spectrum antibiotics. Chest X-ray was negative for acute pathology. Peripheral blood culture and urine culture did not show any growth. No focus of infection was found. Lab was unremarkable except for elevated blood urea nitrogen (BUN) and creatinine Table  which deThe presumptive diagnosis of gall bladder perforation is a challenge when patient presents with features of sepsis and imaging modalities such as ultrasonography, CT scan, MRCP are also inconclusive . The HIDUltrasound is the best screening modality for biliary diseases . Usual uCT abdomen may offer complementary information and can reveal more accurate signs of free intraperitoneal fluid, pericholecystic fluid, abscess, a distended gallbladder with wall thickening and the defect on the wall due to perforation ,9. GallsGallbladder perforation is usually managed with drainage of fluid, and cholecystectomy. Cholecystectomy can be performed after the infection is relieved by US-guided percutaneous drainage .The mortality rate of gall bladder perforation was as high as 42% in a study by Glenn and Moore  while otWhen in diagnostic dilemma, subtle clues such as free fluid in the abdomen and contracted gall bladder should raise suspicion of gall bladder perforation. Percutaneous cholecystostomy is usually practiced in unstable patient presenting with cholecystitis. However, in case of gall bladder perforation, percutaneous cholecystostomy alone may not be sufficient and drainage of intraperitoneal fluid pocket may act as a bridging therapy until definitive surgical management is done. It is vital to optimize the hemodynamics to prevent the downward spiral of end organ dysfunction until the source control is addressed. Every effort should be made to drain the fluid as even a minor intervention such as peritoneal fluid drainage with pigtail placement can reverse the trajectory of SOFA worsening as in our case . The galGBP is a rare complication of cholecystitis with high morbidity and mortality. However, it is often difficult to diagnose when imaging modalities such as CT scan, ultrasonogram and HIDA scan are inconclusive and patients present late with sepsis and hemodynamic stability. In such cases, subtle clues such as free fluid in the abdomen with contracted gall bladder should be enough to raise suspicion of gall bladder perforation. Timely recognition and prompt intervention, as minor as peritoneal drainage, can result in unremarkable recovery and reduction in mortality.


Angiogenesis is an active process, regulating new vessel growth, and is crucial for the survival and growth of tumours next to other complex factors in the tumour microenvironment. We present possible molecular imaging approaches for tumour vascularisation and vitality, focusing on radiopharmaceuticals (tracers). Molecular imaging in general has become an integrated part of cancer therapy, by bringing relevant insights on tumour angiogenic status. After a structured PubMed search, the resulting publication list was screened for oncology related publications in animals and humans, disregarding any cardiovascular findings. The tracers identified can be subdivided into direct targeting of angiogenesis  and indirect targeting . Presenting pre-clinical and clinical data of most tracers proposed in the literature, the indirect targeting agents are not 1:1 correlated with angiogenesis factors but do have a strong prognostic power in a clinical setting, while direct targeting agents show most potential and specificity for assessing tumour vascularisation and vitality. Within the direct agents, the combination of multiple targeting tracers into one agent (multimers) seems most promising. This review demonstrates the present clinical applicability of indirect agents, but also the need for more extensive research in the field of direct targeting of angiogenesis in oncology. Although there is currently no direct tracer that can be singled out, the RGD tracer family seems to show the highest potential therefore we expect one of them to enter the clinical routine. Angiogenesis is an active process, which regulates the growth of new blood vessels from a pre-existing vascular bed and is crucial for any regenerative mechanisms and for the survival and growth of tumours. Under physiological conditions a fine-tuned regulation of pro- and anti-angiogenic factors expands the surrounding vascular network in the target tissue. However, in the case of solid cancers, there is a pathological misbalance in the favour of the pro-angiogenetic factors, which results in abnormal, leaky, new capillary formation, required for its rapid growth and metastasis . Beyond Concentration gradient via diffusion may achieve a steady nutrient supply and waste product elimination only in tissues smaller than 2 mm in diameter. Henceforth, any tumour larger than this requires vascularisation for its subsequent growth. This is an important aspect, which is frequently overlooked, as both chemotherapy and imaging tracers that directly target the process of angiogenesis have a limited effect (if any) on primary tumours and metastases smaller than 2 mm. Thus, the detection capabilities of imaging probes targeting angiogenesis are restricted both by the spatial resolution of the scanner and by the tumour volume. Nonetheless, as most solid tumours are prone to acquire resistance to anti-angiogenic therapies ,4, thereComputed tomography (CT) and magnetic resonance imaging (MRI) are in principle not ideal for assessing tumour angiogenesis. These techniques mainly evaluate parameters such as changes in tumour volume, morphology, or even tumour blood flow/blood volume and vascular permeability, but they cannot quantify changes in the tumour vasculature. However, there are significant advances being made, especially with MRI, that allow imaging of angiogenesis-correlated parameters. This is beyond the scope of the current review, but interesting applications may be found in the following reviews ,6,7.Molecular imaging, either via positron-emitting isotopes for imaging with PET or via single photon emitting isotopes for imaging with SPECT, makes use of radioactively labelled tracers that bind to specific targets. The technique of molecular imaging is significantly more sensitive compared to CT and MRI, allowing for the visualisation and even quantification of sub-nanomolar concentrations of targets. Both PET and SPECT imaging techniques rely on small amounts of “tracers”, which bind a specific molecular target. Both modalities comes with a long list of tracer parameters that need to be met, including good target to background ratio, fast clearance , suitable biodistribution, no toxicity, and high specificity for the specific target. Therefore, the field of molecular imaging is ever developing with improvements in the tracer, either via incremental improvements within a given class of compounds; or via “new” tracers that are used for targets that previously could not be detected.The possible molecular mechanisms to combat angiogenesis in oncology have been previously described in many papers ,4,8,9,10The key question is to identify a suitable target for the PET and SPECT tracers. In adult tissues, under physiological conditions, there is a balance between the pro-and anti-angiogenic factors . In situAngiogenesis is directed towards the hypoxic tissue mainly by integrins, which promote endothelial cell migration and survival and pro-angiogenic macrophage trafficking to tumours . Many tuAs angiogenesis is a multi-factorial process, there are a multitude of potential targets for molecular imaging. These targets can be subdivided into direct targets (specific receptors on or near the cell that are directly related to angiogenesis) and indirect targets  .The discovery of VEGF has revolutionised the understanding of vasculogenesis and angiogenesis during embryogenesis and physiological homeostasis. It is the key mediator of angiogenesis and binds two VEGF receptors (VEGF receptor-1 and VEGF receptor-2), which are expressed on vascular endothelial cells ,42. It iv integrins, which are overexpressed on the surface of endothelial cells during angiogenesis. Different types of integrins can also imply different metastatic progresses: Hoshino et al. report that a high content of exosomal integrin αvβ5 was associated with liver metastasis, whereas exosomal integrins α6β4 and α6β1 correlates with lung metastasis -FDG), it is certainly worthwhile to investigate GLUTs as an angiogenesis marker. PSMA, a target well known for visualising prostate cancer, also seems to play a role in angiogenesis in solid tumours -FDG is a well-known and widely used tracer in the field of oncology (and beyond). Given the correlation between [18F]-FDG’s target GLUT1 expression and angiogenesis, several attempts have been made to use [18F]-FDG as a tool to monitor angiogenesis in vivo. Mirus et al. showed that both [18F]-FDG PET/CT and contrast enhanced CT are capable to detect parameters closely connected to the degree of tumour vascularization, and could therefore be used beyond its original role of quantifying glucose metabolism [18F]-FDG kinetics were shown to be modulated by angiogenesis-related genes: the transport rate for [18F]-FDG (k1) is higher in tumours with a higher expression of VEGF-A and angiopoietin-2 [[tabolism . Straussoietin-2 . More reoietin-2 .18F]-FDG seems to be inferior. Provost et al. demonstrated this in a preclinical mouse tumour model using bevacizumab and temozolomide, where tumour uptake of [68Ga]-RGD, an integrin-specific angiogenesis tracer, was concordant with tumour growth in controls and in treated groups but [18F]-FDG was not -NODAGA-RGD has a different spatial distribution than [18F]-FDG bringing different tumour information [18F]-FDG with [18F]-FPPRGD2 [18F]-FDG uptake using immunohistochemical staining measurements of angiogenesis in NSCLC [18F]-FDG as a true angiogenesis marker.Clinically, Durante et al. showed in a limited study of 10 patients that -FDG does have a clinical use however, as it remains a tracer with strong prognostic power—even when used in reference to angiogenic treatment. Several studies provided evidence that pre-treatment [18F]-FDG PET can serve as an imaging biomarker for predicting survival following anti-angiogenic therapy with bevacizumab -FMISO). [18F]-FMISO is a nitroimidazole compound, which may be intracellularly oxidised in normoxic cells. On the other hand, in hypoxic cells, FMISO cannot be oxidised, and it permanently binds to cellular components. The correlation of angiogenesis and hypoxia (imaging) was further investigated by several independent groups [18F]-FMISO PET uptake was closely linked to tumour grade, with high uptake in glioblastomas [18F]-FMISO uptake group. Most importantly, this last study showed that patients without [18F]-FMISO uptake had a longer survival time than uptake positive patients—indicating a true clinical application.Angiogenesis is recognised as an outcome of hypoxia, where signalling of proliferative markers such as VEGF is upregulated . The degt groups ,66. Uedat groups . Similarlastomas . ExpressHowever, even though VEGF is a major factor implicated in angiogenesis, evidence for VEGF expression in tumours correlating with hypoxia is conflicting . For exa18F]-FMISO is a well-known hypoxia marker that has been shown to be useful as a prognostic marker, in glioma but also in other tumours -diacetyl-bis-(N4-methylthiosemicarbazone) ([64Cu]-ATSM) has been shown to be superior in terms of imaging performance, and may even play a role as an add-on for anti-angiogenic tumour treatment -FAZA, which has improved pharmacokinetics and biodistribution -Flortanidazole ([18F]-HX4) was developed and validated both preclinically and clinically as a third-generation nitroimidazole-based tracer, with an even faster clearance and improved distribution [18F]-HX4 and angiogenesis markers, its improved biodistribution and tumour visualization as demonstrated in several clinical trials make this a strong candidate for future (attempts at) imaging of angiogenesis in hypoxia-rich environments Fluorohex-1-ynyl)-benzenesulfonylamino]-3-methylbutyric acid ([18F]-SAV03) was developed to visualise MMP-2 -VEGF121, [111In]-VEGF165, [64Cu]-DOTA-VEGF121 and [64Cu]-VEGF121 have been investigated, as well as two radiolabelled versions of Bevacizumab: [111In]-Bevacizumab and [89Zr]-Bevacizumab.VEGF consists of at least six isoforms of a various number of amino acids  produced through alternative splicing, and are active as homodimers linked by disulphide bonds . VEGF121123I]-VEGF165 binds to a variety of human tumour cells/tissues compared with the corresponding normal tissues or normal peripheral blood cells [123I]-VEGF121, although to a lesser extent. In the following years, several authors continued this work in preclinical models using non-invasive imaging with iodinated VEGF [64Cu]-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA)-scVEGF ([64Cu]-DOTA-scVEGF) or [99mTc]-hydrazinonicotinic acid (HYNIC)-scVEGF ([99mTc]-HYNIC-scVEGF) -VEGF165 scans were compared with CT and MRI, demonstrating the usefulness of the [123I]-VEGF165 scan to visualise the tumour angiogenesis, despite the superiority of CT and MRI for the visualization of the gastrointestinal tumours and metastasis [123I]-VEGF165 in nine patients with pancreatic carcinomas. They demonstrated that [123I]-VEGF165 visualised the primary tumour and their metastasis, but also noticed severe deiodination thus hampering routine clinical applicability [123I]-VEGF165 in three patients but failed to visualise the tumours. More recently, the World Health Organization (WHO) grade IV glioma lesions were shown to have significant [123I]-VEGF uptake 18 h after the injection, whereas other brain tumours of grade II or III showed negative results in a study with 18 patients [123I]-VEGF scans yielded prognostic information as patients with [123I]-VEGF T/N ratio above a particular threshold showed significantly longer survival than patients below that threshold (2680 vs. 295 days).Clinical trials with labelled VEGF so far are limited. One example in particular involving staging and follow-up of 18 patients with solid gastrointestinal tumours, [tastasis . Li et acability . Ubl et patients . The mos89Zr]-bevacizumab PET was able to visualise tumours in renal carcinoma patients -VEGF scans [89Zr]-bevacizumab may prove useful as a prognostic tool, even in children -labelled dimeric L19 [L19(scFv)(2)] in 20 patients with lung, colorectal, or brain cancer -Galacto-RGD did not show a high uptake in normal brain tissue, especially when compared to [18F]-FDG [18F]-Fluciclatide developed by GE Healthcare and [18F]-RGD-K5 developed by Siemens Molecular Imaging Inc. Although they all showed suitable properties in preclinical studies, their long and sometimes complicated synthesis made them unattractive as commercially viable tracers [Several variants of RGD have been produced by adding groups to improve the biodistribution and pharmacokinetics. The [ of αvβ3 . This tr18F]-FDG . Other v tracers .18F]-FB-RGD, a fluorobenzoyl-labelled RGD peptide with its pegylated analogue [18F]-PEG-RGD, Chen et al. found that pegylation resulted in an improved tumour uptake, but also a slowed pharmacokinetics and clearance [18F]-FPRGD2, an RGD-variant with a mini-pegylation, was found to show improved radiosynthesis yields, equal tumour uptake and reduced renal uptake in a preclinical study [18F]-FDG.Next to glycosylation, pegylation was also attempted. When comparing [learance . [18F]-Fal study . Later cal study ,123,124.18F]-FPPRGD2, a dimeric peptide designed by conjugating [18F]-NFP with pegylated RGD dimeric peptide was also shown to bind to integrin-expressing tumours both in vitro and in vivo. A biodistribution study of [18F]-FPPRGD2 in human revealed promising pharmacokinetic properties [18F]-FPPRGD2 to [18F]-FDG, no difference was noted for primary lesions as well as metastases [18F]-FPPRGD2 was slightly superior, as it detected several small metastases that [18F]-FDG did not, while it proved negative in inflammatory lymph nodes where [18F]-FDG showed a false positive. Similarly, [18F]-FPPRGD2 PET showed a higher detection rate of recurrent glioblastoma multiform than that of brain MRI (100.0% vs. 93.3%) -F. 93.3%) .18F]FB-mini-PEG-E{E[c(RGDyK)]2}2; albeit without significant advantages both in vitro and in vivo [99mTc and 68Ga compounds—again modifying the kinetics but generally maintaining tumour to background ratio [64Cu-labeled octameric RGD-peptide as well as [111In]-labelled monomeric, dimeric and tetrameric analogues -FPPRGD2 provides additional information when compared to [18F]-FDG and may even outperform routine brain MRI [68Ga]Ga-NODAGA-E[(cRGDyK)]2 (RGD) PET and hyperpolarised [1-13C]pyruvate-MRSI (hyperPET) provide supplemental information and made a case for a more widespread use of combining angiogenesis imaging with energy metabolism to optimise patient treatment. A human clinical trial confirmed this case, as [18F]-RGD uptake on PET/CT imaging pre-treatment may predict the response to antiangiogenic therapy, with higher [18F]-RGD uptake in tumours predicting a better response to apatinib therapy -FDG is now emerging, especially in the field of head and neck tumours -NOTAc(NGR) of the primary tumours was significantly higher than that of the accumulation of the commercially available [68Ga]-NODAGA-[c(RGD)]2 2 ,153.vβ3 and Aminopeptidase N Dual-Receptor. [68Ga]-NGR-RGD showed higher binding avidities, targeting efficiency and longer tumour retention time compared with monomeric [68Ga]-NGR and [68Ga]-RGD -RGD . It may 177Lu]-DOTA-TATE and [177Lu]-PSMA, both therapeutic agents developed based upon their 68Ga-predecessor designed for imaging, research is now also focusing to investigate the possible role of angiogenesis markers as potential therapeutic targets.Driven by the successful clinical applications of Re-NGR-VEGI showed excellent tumour inhibition effect with no observable toxicity -EB-RGD was shown to have an inhibitory effect on tumour growth, either on its own or in combination with other drugs -FDG or hypoxia-markers is not always strictly correlated to angiogenesis, they do have strong prognostic power in the clinical setting. Direct imaging with specific markers, targeting specific receptors on the cancer cells or the surrounding vasculature, are more tightly correlated to angiogenesis. Their clinical impact remains to be determined however, as there are a multitude of potentially useful tracers, both preclinical and clinical, but further research is needed to determine which of those will be translated to the clinical setting. In our opinion, the RGD family holds the most potential to enter clinical routine as a tracer for direct quantification of angiogenesis, by providing an improved assessment of tumour characteristics required for personalised therapy approaches.Significant progress has been made over the past decade concerning molecular imaging in angiogenesis. While indirect imaging of angiogenesis using either [


Oesophageal cancer rates are increasing rapidly with patients often presenting at an advanced stage. The current approach to treatment involves radiotherapy, chemotherapy, or combination chemoradiotherapy with surgery; however, only a fraction of these patients will achieve meaningful responses. Therefore, there is a need to better understand the tumour and lymph node microenvironments to inform future treatment strategies. This study measured immune markers including immune checkpoint expression in tumour and lymph node tissue in oesophageal cancer patients and patient clinical outcomes, including survival time, response to treatment, and adverse events. We report herein that nodal metastases is of equal prognostic importance to clinical tumour stage and tumour regression grade in OAC and we observed a more immunosuppressive microenvironment in the tumour compared with the lymph node.n = 702). The expression of inhibitory immune checkpoints by T cells infiltrating tumour-draining lymph nodes (TDLNs) and tumour tissue post-chemo(radio)therapy at surgical resection was assessed by flow cytometry. Nodal metastases is of equal prognostic importance to clinical tumour stage and tumour regression grade (TRG) in OAC. The TME exhibited a greater immuno-suppressive phenotype than the LNME. Our data suggests that blockade of these checkpoints may have a therapeutic rationale for boosting response rates in OAC.Response rates to the current gold standards of care for treating oesophageal adenocarcinoma (OAC) remain modest with 15–25% of patients achieving meaningful pathological responses, highlighting the need for novel therapeutic strategies. This study consists of immune, angiogenic, and inflammatory profiling of the tumour microenvironment (TME) and lymph node microenvironment (LNME) in OAC. The prognostic value of nodal involvement and clinicopathological features was compared using a retrospective cohort of OAC patients ( Multimodal neoadjuvant therapy consisting of chemotherapy or chemoradiotherapy has become the standard-of-care for stage II–III cancer of the oesophagus and the oesophagogastric junction (OGJ). The potential benefits of neoadjuvant therapy include downstaging of the primary tumour, facilitating complete surgical resection and eradicating occult micrometastases . HoweverThe lymphatic system has been recognised as a route of metastasis for over 150 years ,5,6 and + T cells by dendritic cells (DCs) occurs in the lymph node, a critical step in the cancer immunity cycle that is potentiated by anti-CTLA-4 immune checkpoint blockade (ICB) [reg) and myeloid-derived suppressor cell (MDSC) infiltration as well as by a generalized state of enhanced T cell anergy. In addition, lymph node (LN)-resident DC subsets show lower levels of activation compared with that of migratory DC subsets [+ T cells do not differentiate into effector T cells but instead differentiate into peripheral Treg cells that restrain anti-tumour immunity [+ T cell infiltration resulting in reduced ICB efficacy [Along with their role in metastasis and as a prognostic factor, tumour-draining lymph nodes (TDLNs) are important in the anti-tumour immune response . Primingde (ICB) ,21. Howede (ICB) . In mela subsets ,23,24, s subsets . In thisimmunity . The LNMimmunity . These simmunity . Furtherefficacy . This alefficacy . Howeverefficacy ,32,33.In this study, we investigated the prognostic implications of clinical and pathologic nodal status in a large single-centre cohort of patients with locally advanced resectable oesophagogastric cancer. Additionally, we profiled immune checkpoint (IC) expression on T cells residing in the TDLN and infiltrating-tumour tissue of oesophagogastric adenocarcinoma patients. Furthermore, the inflammatory, angiogenic, cytokine, and chemokine profile of the LNME and TME in oesophagogastric cancer patients is investigated and correlated with clinicopathologic outcomes. This information will help provide a fundamental understanding of the LNME and TME in oesophagogastric adenocarcinoma patients in order to better inform future therapeutic approaches.Ethical approval was granted from Tallaght/St James’s Hospital Ethics Committee. Informed written consent was obtained for all the sample and data collection, which was carried out using best clinical practice guidelines. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2008. Patient samples were pseudonymised to protect privacy rights.n = 9) along with matched TDLN tissue biopsies (n = 6) were obtained from oesophageal adenocarcinoma patients at time of surgical resection at St James’s Hospital, Dublin, Ireland. Paraoesophageal lymph nodes were confirmed to be negative for metastatic disease by formal laboratory histological analysis.All patients who consented to fresh specimen collection were enrolled between 2018 and 2020. Tumour biopsies (v/v) foetal bovine serum) at 37 °C and agitated at 1500 rpm on an orbital shaker. Tissue was then filtered using 70 uM nylon mash filter and washed with FACs buffer . The resulting single cell suspensions were then stained for flow cytometry.Biopsies were enzymatically digested to perform single-cell phenotyping. Briefly, tissue was minced using a scalpel and digested in collagenase solution  in Hanks Balanced Saline Solution  supplemented with 4%  and the following cell surface antibodies: PD-L1 PE and CD8 BV421 , TIM-3 Viobright FITC and CD3 APC , TIGIT PE/Cy7 and PD-1 APC/Cy7  and CD4 PerCpCy5.5 . Cells were fixed with 1% paraformaldehyde solution , washed and resuspended in FACs buffer, and acquired using BD FACs CANTO II (BD Biosciences) using Diva software vX and analysed using FlowJo v10 software .n = 6) and tumour tissue (n = 6) biopsies were cultured in a 12-well plate in 1 mL of L-15 (Leibovitz) Lonza™ BioWhittaker™  X-vivo media for 24 h ex vivo at 37 °C, 5% CO2. LN tissue was divided into four equal quadrants to ensure adequate distribution of immune cells. LN were confirmed to be benign through formal histological assessment by the St James’ Hospital Histopathology department. The resulting lymph-node-conditioned media (LNCM) and tumour-conditioned media (TCM) was harvested and stored at −80 °C until required for further experimentation.LN . The following secretions were quantified in the LNCM and TCM: CRP, Eotaxin, Eotaxin-3, bFGF, Flt-1, GM-CSF, ICAM-1, IFN-γ, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17B, IL-17C, IL-17D, IL-1RA, IL-1α, IL-1β, IL-2, IL-21, IL-22, IL-23, IL-27, IL-3, IL-31, IL-4, IL-5, IL-6, IL-7, IL-8, IL-8 (High Abundance), IL-9, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, MIP-3α, PlGF, SAA, TARC, Tie-2, TNF-α, TNF-β, TSLP, VCAM-1, VEGF-A, VEGF-C and VEGF-D. All assays were run as per the manufacturer’s instructions, with an overnight supernatant incubation protocol used for all assays except angiogenesis panel 1 and vascular injury panel 2, which were run according to the same day protocol. TCM and LNCM were run undiluted on all assays except vascular injury panel 2, where a one in four dilution was used, as per previous optimisation experiments. Secretion data for all factors was normalised to cell lysate protein content determined using a BCA assay .® (version 18.0) software  and R 2.13.2. A significance level of p < 0.05 was used for all analyses and all p values reported are two-tailed. The Kaplan–Meier method and the log rank test was used to assess differences in survival between groups. Survival time was measured from the date of first treatment to the date of death or last follow-up. Independent variables were entered into a multivariable Cox proportional hazards model, variables found at univariable analysis to have a p value < 0.05 were entered into the multivariable model. Continuous variables were compared using unpaired t-tests. Association of categorical variables was assessed using χ2 test.Statistical analysis was performed using SPSSWilcoxon rank test was utilized to compare checkpoint, cytokine, and markers of angiogenesis expression between the lymph node and the tumour compartment.p < 0.001). N0 and N1 were significantly associated with Barrett’s oesophagus, 55% and 49%, respectively (p < 0.001), with signet ring and mucinous features more frequent in N2 and N3. N0 was associated with less adverse features of tumour when compared to those with nodal positivity. Adverse features consisted of poor differentiation, perineural, lymphatic, and vascular invasion, with a novel three-grouping stratification of 0, 1–2, and 3–4 adverse features previously described by our department [p < 0.001). Chemoradiation was the most commonly utilised neoadjuvant treatment modality across all nodal status. Mandard TRG after neoadjuvant therapy was significantly better in N0, with 25% either TRG 1 or 2, compared with 14% in N1, 12% in N2, and 8% in N3. The clinical and pathologic demographics per nodal burden are outlined in Data for 702 patients were obtained from a prospectively maintained database between 2001 and 2016, of these 356 were classified as node negative, with a higher proportion of the N0 cohort having a Siewert type I tumour (60%). An en bloc radical oesophagectomy was performed in 50% of N0, 66% N1, 48% of N2, and 53% of N3 . On pathpartment . LinkingAdverse features of tumour biology including perineural invasion, vascular invasion, differentiation nodal positivity, and neoadjuvant treatment were significant by multivariate analysis on overall survival. .p < 0.001). The median overall survival was 58.27 months (CI 46.28–70.26). The five year survival for node negative disease in 67%, 45% for N1 disease, 38% for N2, and 11% for N3 disease  ) but pathologically node-negative had improved survival compared to those who were clinically and pathologically node-positive (< 0.001) . ImportaThe five-year survival for clinically node negative, pathologically node negative disease is 68%, compared to 61% in those with clinically node positive, pathologically node negative disease. In those with clinically and pathologically node positive disease, the five-year survival is 26% .p < 0.001)  . The 5-y< 0.001) .Inhibitory ICs play key roles in restraining anti-tumour immunity and with the recent approval of two anti-PD-1 inhibitors, pembrolizumab and nivolumab, for treating oesophagogastric adenocarcinomas, it is important we develop a deeper understanding of IC expression profiles in adenocarcinoma patients. We profiled IC expression on T cells infiltrating OAC tumour tissue and in the TDLN to determine the level of target expression in multimodal therapy.+ T helper and CD8+ cytotoxic T cell compartments (+CD8+PD-1+ cells were found in tumour tissue compared with TDLNs (median 18.2 (range 7.41–46.49) vs. 5.52 (range 0.75–13.65), p < 0.05). There was also a significantly higher percentage of CD3+TIM-3+ (median 7.1 (range 5.23–15.4) vs. 1.33 (range 0.06–5.28)), CD3+CD4+TIM-3+ (median 7.51 (range 0.35–20.3) vs. 0.36 (range 0.05–2.41)) and CD3+CD8+TIM-3+ (median 6.37 (range 2.31–31.9) vs. 1.3 (range 0.06–4.07)) intratumourally compared to the lymph node (p < 0.05). There was a significantly higher expression of CD3+ PD-L1+ cells (median 12.95 (range 2.19–37)) in the tumour compared to (median 2.07 (range 0.15–8.46)) in the lymph node (p < 0.05). The expression of CD3+ PD-1+TIM-3+ (median 5.91 (range 0.01–10.39) vs. 0.27 (range 0–0.63), p < 0.05), and CD3+CD4+ PD-1+TIM-3+ (median 3.41 (range 0–10.3) vs. 0.33 (range 0–1.6), p < 0.05), was significantly higher in the tumour than in the lymph node  compared to the lymph node (median 0.33 (range 0–1.6) p < 0.05). Similarly, CD3+CD8+ PD-1+PD-L1+ cells were also found at a significantly higher frequency in tumour-infiltrating tissue compared with TDLNs (median 1.62 (range 0.02–34.6) vs. 0.12 (range 0–0.87) p < 0.05), similarly for CD3+CD4+TIM-3+PD-L1+ cells (median 5.03 (range 0.02–42.8) vs. 0.11 (range 0–0.41), p < 0.05) (+ PD-1+TIGIT+TIM-3+ (median 5.63 (0.02–7.58) vs. 0.03 (0–0.09) as well as CD3+ PD-1+TIGIT+ PD-L1+ (median 6.02 (range 0.42–7.99) vs. 1.93 (range 0.01–4.44) (p < 0.05). Significantly higher frequencies of CD3+CD4+PD-1+TIM-3+PD-L1+ cells intratumorally (median 0.68 (range 0.01–10.6) vs. 0.03 (range 0–5.65), p < 0.05),  . There w< 0.05), .The levels of anti-tumour cytokines IFN-γ were lower in the TME (median 13.63 pg/mL (range 1.15–43.83) compared to the LNME (median 41.27 pg/mL (range 18.1–182.2) but did not achieve significance by multivariate analysis . HoweverThe LNME and TME was assessed for pro-angiogenic and vascular damage analytes and the levels of soluble bFGF were higher within the TME 124.8 pg/mL (range 66.06–610.8) compared with the LNME (median 34.97 pg/mL (range 22.35–72.94) but were not significant. Interestingly, PIGF was higher within the LNME 116.3 pg/mL (range 18.78–162.6) than the TME 10.15 pg/mL (range 4.61–82.11) of OAC patients but was not significant. The levels of all other analytes were detected at comparable levels between the LNCM and TCM from OAC patients, and these cytokines, chemokines, and markers of vascular injury and angiogenesis are available as n = 702) that the presence of nodal metastases has equivalent prognostic value to that of clinical tumour stage and TRG in OAC patients. This further highlights that the TDLN may play a pivotal role in orchestrating anti-tumour responses and subsequent treatment response in OAC patients and complements previous studies.While there are limitations to the current study, primarily the cohort size of fresh patient samples, this should be borne in mind when considering the conclusions of the study, as this limits the ability to perform clinical correlative work. We have demonstrated in a very large cohort of OAC patients  and disease-free survival in resected specimens ,13,14,15+ differentiation into regulatory T cells, thus promoting tumour immune escape [+ DC subset, a cellular population that is efficient at cross-presenting neoantigens to CD8+ T cells. This immunosuppressive feature of the LNME was correlated to local tumour recurrence [A greater understanding of the relationship between the immune microenvironments of the tumour and lymph node will be useful in understanding the response to chemoradiotherapy and future immunotherapy. Harnessing the power of TLDNs may also boost the clinical outcomes following ICB. Indeed, ongoing clinical trials are investigating the role of ICB alongside neoadjuvant chemoradiotherapy in locally advanced, operable gastroesophageal adenocarcinoma  . The LNMe escape . In humacurrence ,36, wherOur data is broadly in agreement with these studies; however, we not only analysed the immune profile of the LNME, but also compared it to the immune microenvironment of primary tumour samples, demonstrating that the TME exhibited greater immunosuppression compared to the LNME. We found that the TME had higher levels of infiltrating T cells co-expressing multiple ICs, which typically denote T cell exhaustion and functional impairment, compared to the LNME. The presence of IC expression by T cells in the LNME was previously reported in one study of cervical cancer patients, but it was not compared with the TME . Using cThe levels of immunosuppression in the LNME may also have clinical implications, as the presence of suppressed DC subsets in the TDLNs are negatively associated with recurrence and disease-free survival in melanoma and breast cancer ,25. FurtAlthough cancer cells were traditionally thought to spread to distant organs from lymph nodes through lymphatic vessels, recent preclinical studies suggest that cancer cells can invade local nodal blood vessels to enable a mixed haematogenous metastatic dissemination ,5. Thus,Collectively, these data highlight a potentially detrimental role of tumour-promoting inflammation in OAC pathogenesis. Tumour-promoting inflammation is one of the enabling hallmarks of cancer and plays important roles in therapy resistance and tumour progression . InteresThe role of Th17 cells in cancer is controversial, with studies suggesting both pro- and anti-tumour effects in diverse cancer types . There iThere are a number of limitations in this study in addition to the small cohort size for immunophenotyping, including the fact that fresh lymph node and tumour tissue samples were obtained at surgical resection, which is post-multimodal treatment. The impact of treatment can alter the microenvironment of the tumour and the lymph nodes within in the treatment field. However, understanding how treatment can affect the TME and LNME will have a significant bearing on the development of appropriate immunotherapeutic targeting. Whilst we have shown significant immunological differences between the tumour and the draining lymph node in this small cohort, additional larger studies using treatment naïve samples will be required to determine the real clinical significance of these potential therapeutic targets.In summary, nodal involvement, and clinical and pathological tumour staging are significant prognostic indicators for OAC. These results also highlight the immunosuppressive nature of the TME demonstrated by higher percentages of T cells co-expressing multiple ICs in the TME compared to the LNME. The data are timely with the recently published Checkmate 577 trial advocating the use of immunotherapy in the adjuvant setting in carcinoma of the oeosphagogastric junction. Our data supports the hypothesis that combination therapeutic blockade of these aforementioned checkpoints may have a therapeutic rationale for boosting response rates in OAC.


The treatment of oral cancer remains challenging due to its infiltrative nature and a high tendency for tumour relapse leading to an overall poor prognosis. In the case of early recurrence, the patient’s prognosis deteriorates dramatically, with survival rate dropping to below 30%. Minimal improvements in survival trends in recurrent and advanced stage tumours have been reported in recent decades. Neoadjuvant immunotherapy may represent a new therapeutic approach changing the standard of care in advanced oral cancer therapy.We describe the case of a woman in her late 30’s who presented in mid-2019 with oral squamous cell carcinoma (OSCC) localized to the floor of the mouth. After initial R0 resection, selective neck dissection, and adjuvant brachytherapy, an early recurrence of OSCC located between the hyoid bone and the mandible was diagnosed at the end of 2019. An off-label treatment regimen was performed with neoadjuvant use of Pembrolizumab 19 days prior to salvage surgery. Radiological and histological assessment of T-cell and programmed cell death protein 1 ligand 1 (PD-L1) expression was performed before and after checkpoint inhibitor application. Neoadjuvant immunotherapy resulted in increased T-cell infiltration and PD-L1 expression, as well as a significant tumour necrosis rate. One cycle of Pembrolizumab led to significant regressive tumour changes with increases in immune infiltration, sclerosis, and necrosis of 75% of the tumour mass with only 25% vital tumour cells remaining. By June 2020, the patient remained without recurrence.The case presented outlines the potential effects of neoadjuvant immunotherapy in recurrent or advanced OSCC prior to definitive surgical tumour treatment. The benefit of additional adjuvant treatment after histologic response will be discussed. The case is also analysed considering ongoing clinical trials of neoadjuvant immunotherapy for head and neck malignancies. Oral cancer is a serious and growing problem in many parts of the globe including Europe. Oral and oropharyngeal cancer grouped together is the sixth most common cancer in the world . ConsideTo date, the standard of care is primary surgical tumour resection with concurrent reconstruction and cervical lymph node dissection followed by postoperative adjuvant therapy (radiotherapy ± cisplatin) in high-risk cases . CurrentIn mid-2019, a female patient in her late 30’s presented with a squamous cell carcinoma of the oral cavity localized to the right margin of the tongue Table 1.in situ and pharyngeal wall. Tumour resection also included partial resection of the right mandibular angle, hyoid bone and resection of the hypoglossal nerve because of its attachment to the tumour mass. The recurrence was staged ypT2 ypN0 (0/2) L0 V0 Pn0 R0 > 5 mm cM0 with an infiltration depth of 6 mm. Defect coverage was accomplished using a microvascular vastus lateralis transplant from the patient’s right thigh. In the postoperative interdisciplinary tumour board, it was decided to re-administer the immunotherapeutic agent according to the patient’s will and to additionally perform an adjuvant radio-chemotherapy according to the current German treatment guideline . The following parameters were chosen for quantification and histological assessment of the conducted therapy: 1. CD8 density per mm2 (Cytotoxic T cell marker) 2. TC/TPS %  4. CPS (combined positivity score [(stained tumour cells + stained mononuclear immune cells)/tumour cells)] . Furthermore, one cycle of Pembrolizumab was able to induce significant regressive tumour changes. In detail, approximately 75% of the former tumour bed was represented by large fibrotic and necrotic tumour areas while only 25% was covered by residual vital tumour cells. Pre-treatment biopsies showed no tumour regression or necrosis. Taken together, these results indicate that a single cycle of Pembrolizumab treatment can sufficiently booster the pre-existing antitumoral immune response by increasing the ability of cytotoxic effector T-cells to migrate into the tumour microenvironment and to actively kill tumour cells.To further elucidate immunological processes due to Pembrolizumab treatment we applied PD-L1 (Ventana SP263 assay) and CD8 immunohistochemistry to a baseline tumour sample , a tumour sample derived from the recurrence before administering Pembrolizumab and the final resection specimen after Pembrolizumab treatment (representative images are depicted in  cells)] –23. AlthThe neoadjuvant and adjuvant administration of Pembrolizumab showed no significant side effects. One cycle of Pembrolizumab resulted in significant regressive tumour changes with a large increase in CD8+ immune infiltration, sclerosis, and necrosis of 75% of the tumour mass, as shown by histopathologic assessment. Neither surgical treatment nor adjuvant radiochemotherapy were compromised by the administration of (neo)adjuvant immunotherapy. Although the neoadjuvant use of Pembrolizumab as an individual healing attempt was based on few promising studies and reports in the international literature, in this particular case the patient showed a strong histopathologic response to therapy. At the last follow-up 18 months after salvage surgery, no clinically or radiologically suspicious tumour lesions were detected.Activation of the co-inhibitory PD-1/PD-L1 pathway is among the mechanisms of OSCC tumour cells to evade immune invasion. Binding of PD-1 on the surface of cytotoxic T cells to PD-L1 or PD-L2 expressed on tumour cells and antigen-presenting cells promotes a microenvironment favourable for tumour growth . By thisThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.MW, MK, RL, GF, MH, T-OB, and JG examined and treated the patient and collected the data. ME participated in the pathological diagnosis. MO, MW, and JG discussed the case and data. MO, MW, and JG wrote the manuscript. JR, RL, GF, and T-OB aided with manuscript writing and language prettification. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this study was to estimate the diagnostic performance of synovial fluid polymerase chain reaction (PCR) in periprosthetic hip and knee infection, and whether synovial fluid PCR has greater diagnostic significance than conventional methods.The literature databases PubMed, Scopus, and the Web of Science were searched for English articles describing periprosthetic joint infection (PJI) diagnosis by synovial fluid PCR. Articles were limited to the period between January 1990 and December 2019. Subsequently, conventional methods that were used on at least two occasions were included for further analysis. Data analysis was performed using the Meta-DiSc and Stata software.Eleven studies with 1360 cases were included in the meta-analysis. The pooled sensitivity, specificity, and diagnostic odds ratio (DOR) of synovial fluid PCR were 0.70 (95% CI 0.66–0.74), 0.92 (95% CI 0.90–0.93), and 37.4 (95% CI 17.77–78.74), respectively.Synovial fluid PCR provides an effective tool for rapid diagnosis of PJI, and also in the early stages of culture-negative bacterial infections. Periprosthetic joint infection (PJI) is one of the most severe complication following hip or knee arthroplasty, with high morbidity, mortality and costs –5. HowevJoint aspiration is an invasive diagnostic method and often used as the first step in suspected PJI cases. Currently, various synovial fluid tests have been applied in the clinical diagnosis of PJI , 9. Non-The aim of the present meta-analysis was to assess the diagnostic accuracy of synovial fluid PCR in periprosthetic hip and knee infection. Furthermore, we examined whether synovial fluid PCR has a better diagnostic value compared to conventional methods, especially synovial fluid culture.A systematic literature search of the electronic databases of Web of Science, PubMed, and Scopus was performed for manuscripts of English language between January 1990 and December 2019. The following medical subject headings (MeSH) or text keywords were used: “arthroplasty or joint prosthesis or joint replacement or periprosthetic joint or prosthetic joint”, “infection or infectious or infected”, “synovial fluid”, and “PCR or polymerase chain reaction”.Two authors (LC and LH) independently selected research papers according to the following inclusion criteria: (1) human studies related to synovial fluid PCR in periprosthetic hip or knee infection; (2) clear description of the diagnosis standard of PJI; and (3) values on true-positive (TP), false-positive (FP), true-negative (TN), and false-negative (FN) were provided or could be computed.The combined database was imported to EndNote X7 . All relevant publications on the synovial fluid PCR method in detecting periprosthetic hip or knee joint infection and their reference list were reviewed. Furthermore, included studies from two previous meta-analyses of synovial PCR were reviewed , 16. In Characteristics of the included studies were collected by two reviewers independently and assessed subsequently by a third reviewer. The following information was extracted: first author, year of publication, country, study design, number of total cases, infection site, acquisition time, diagnostic criteria, type of PCR, target gene, antimicrobial use before specimen collection, and diagnostic sensitivity and specificity. The quality of all identified synovial fluid studies was evaluated using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) guidelines. I2 was calculated to evaluate heterogeneity among the studies. If I2 > 50%, the random-effects model was used. Meta-regression analyses were performed to further assess the potential source of heterogeneity, such as type of prosthesis, number of patients, acquisition time, antibiotic use, sample condition, diagnostic standard, and target gene. Deeks’ funnel plot asymmetry test was used to evaluate potential publication bias.To estimate the diagnostic value of synovial fluid PCR for PJI detection, all statistical analyses were performed using Meta-DiSc  and Stata software .Of the identified 145 primary articles, two records were extracted from the reference list of synovial fluid PCR-related studies , 18. FifI2 = 73.9%), specificity (I2 = 86.0%), positive likelihood ratio (PLR) (I2 = 77.4%), negative likelihood ratio (NLR) (I2 = 62.2%), and diagnostic odds ratio (DOR) (I2 = 66.4%). The pooled sensitivity, specificity, PLR, NLR, and DOR estimates for the diagnosis of PJI using synovial fluid PCR were 0.70 (95% CI 0.66–0.74), 0.92 (95% CI 0.90–0.93), 9.09 (95% CI 5.28–15.67), 0.32 (95% CI 0.24–0.42), and 37.4 (95% CI 17.77–78.74), respectively . Intraoperative histology, periprosthetic tissue culture, and sonicate fluid demonstrated better sensitivity and specificity than synovial fluid PCR. Details of the diagnostic values of synovial fluid PCR and other preoperative and intraoperative examination are shown in Table Synovial fluid PCR was shown to have a better sensitivity and specificity than preoperative examination of CRP and erythrocyte sedimentation rate (ESR). Similar sensitivities were observed for conventional synovial fluid culture (70%) and PCR (69%). However, the specificity of synovial fluid PCR was lower than that of synovial fluid culture (91% vsP < 0.001). Compared with the diagnostic standards of MSIS, International Consensus Meeting (ICM), or European Bone and Joint Infection Society (EBJIS), other diagnostic criteria had a higher sensitivity of 0.85 . A small sample size (< 100) was observed to have a higher sensitivity of 0.81 (95% CI 0.71–0.90) compared to studies including more than 100 patients with a sensitivity of 0.71 (95% CI 0.59–0.83) (P = 0.01).Meta-regression analysis was performed in the group of diagnostic standards, acquisition time, number of patients, antibiotic use, and target gene Table . ResultsP = 0.41; Fig. Deeks’ funnel plot analysis did not identify a potential publication bias for synovial fluid PCR , tissue samples , and sonicate fluid  , with moIn recent years, the use of joint fluid for diagnosing PJI was a topic of considerable interest. Multiple synovial fluid tests were applied in clinical practice, with several valuable tests incorporated into the new definition of MSIS . Through. 70%, respectively) and a lower specificity level . Synovial fluid PCR and culture were also observed to have similar results in comparison to the previous meta-analysis of synovial fluid culture, with a sensitivity of 70% and 72%, respectively, and a specificity of 92% and 95%, respectively [Cutibacterium acnes) [vs. two cases), with similar results also reported by Melendez and colleagues [Synovial fluid PCR was compared with preoperative and intraoperative tests from the included studies The PCR test was found to have better sensitivity and specificity than that of serum CRP and ESR. In contrast, lower sensitivity and specificity were observed in comparison to all intraoperative methods. Compared with synovial fluid culture, synovial fluid PCR had an almost identical level of sensitivity with synovial fluid culture (69% vsectively . Althougectively , 23, 25,ectively , 22, 25.lleagues , 21. Duelleagues . The uselleagues  found thAlthough various types of synovial fluid PCR have been tested in the clinical diagnosis of PJI, the diagnostic ability has most likely been disregarded. The most frequently described disadvantage of PCR is FP results, with the potential impacting factors, including the use of different target genes, PCR type, laboratory technician skills, and laboratory conditions , 22, 24.The present study has several limitations. First, the identified studies used different types of PCR; therefore, the overall result may impact the estimates of diagnostic accuracy. Second, meta-regression analysis was not performed in regard to the prosthesis type or sample condition to further explore sources of heterogeneity. The type of prosthesis described in the included studies were the knee or both the hip and knee; however, studies focusing only on the periprosthetic hip were not found. Therefore, further analysis of differences between the hip and knee could not be performed in meta-regression analysis. Regarding the sample condition, frozen specimens were used in the studies; however, there was ambiguity in terms of the use of fresh samples. Third, the diagnostic accuracy of synovial fluid PCR may be affected by the standard definition of PJI . Fourth,The diagnostic capability of synovial fluid PCR is not superior to that of synovial fluid culture. However, in cases of negative synovial fluid culture with highly suspected early-stage infection, synovial fluid PCR can be used as a rapid diagnostic confirmatory tool.


Survival was partially restored with incomplete lymph node resection and CD8 T cell dependent. Treatment with aCTLA4 whilst effective against the primary tumour was ineffective for metastatic lung disease. Conversely, aPD-1/aCD40 treatment was effective in both the primary and metastatic disease settings and restored the detrimental effects of complete dLN resection on survival. In this pre-clinical lung metastatic disease model that closely reflects the clinical setting, we observe decreased frequency of survival after complete lymphadenectomy, which was ameliorated with partial lymph node removal or with early administration of aPD-1/aCD40 therapy. These findings have direct relevance to surgical lymph node resection and adjuvant immunotherapy in lung cancer, and perhaps other cancer, patients.Surgical resection of cancer remains the frontline therapy for millions of patients annually, but post-operative recurrence is common, with a relapse rate of around 45% for non-small cell lung cancer. The tumour draining lymph nodes (dLN) are resected at the time of surgery for staging purposes, and this cannot be a null event for patient survival and future response to immune checkpoint blockade treatment. This project investigates cancer surgery, lymphadenectomy, onset of metastatic disease, and response to immunotherapy in a novel model that closely reflects the clinical setting. In a murine metastatic lung cancer model, primary subcutaneous tumours were resected with associated dLNs remaining intact, completely resected or partially resected. Median survival after surgery was significantly shorter with complete dLN resection at the time of surgery (49 days (95%CI)) compared to when lymph nodes remained intact (> 88 days;  Surgical resection of cancer remains the frontline therapy for some 12 million cancer patients every year , where anot contain tumour cells are usually removed along with any involved ones. Many murine studies by our group [the main site for generation of tumour-specific CTLs is the lymph node draining the tumour compared to any other lymphoid site or blood. Further, there is a decline in anti-tumour immunity over 7–10 days post-tumour resection [During cancer surgery, the dLNs are removed for staging purposes. The extent of nodal dissection in NSCLC resection is still debated, but it does provide accurate staging information for prognostic purposes, determining the need for adjuvant therapy and confirms complete resection. Indeed, the 8th edition TNM recommends that for NSCLC regional LN involvement be assessed by the removal and pathology on a minimum 6 nodes/stations, 3 from the mediastinum, including the subcarinal node (#7) and 3 from N1 zones . In thisur group –8 and byur group , 10 haveesection , and indesection . Howeveresection .In this study, we develop a clinically relevant murine model of surgically resectable, but micrometastatic NSCLC, in which we determine the impact of tumour dLN removal at the time of surgery on the development of metastatic lung disease, and its response to ICPB. More specifically, we identify the tumour dLN for surgical resection by fluorescent IRdye tracking and monitor post-surgery metastatic disease survival. Furthermore, we investigate the efficacy of clinically relevant lung cancer immunotherapies. aCTLA4 and aPD-1, after tumour and dLN resection at the time of surgery.Findings indicate that complete lymphadenectomy at the time of tumour resection is detrimental to survival from lung metastatic disease, and this can be restored with partial lymph node resection or intervention with aPD-1 immunotherapy.Tumour cell lines. The murine mesothelioma cell line AB1 was generated by inoculating crocidolite asbestos intraperitoneally (i.p.) in BALB/c mice [B/c mice . The AB1B/c mice . Line-1 B/c mice . The celB/c mice . Cell liTumour metastatic disease models. Mice received 5 × 105 AB1-HA cells subcutaneously (s.c.) on day 0, tumour growth was monitored with callipers and tumour surgically resected on day 14. On the day of surgery, mice received intravenous (i.v.) injection of AB1-HA_LUC cells. Lung tumour growth was determined by In Vivo Imaging System  after i.p. injection with 100 µl Luciferin . Mice were imaged for peak bioluminescence to 25 min in a Lumina II Imager . Tumour burden was measured as bioluminescence, quantified as photon flux (photons/second), where the limit of sensitivity was considered 0.25 × 107 p/s. Mice were euthanised 88 days post-surgery, or according to clinical signs of severity score sheet, or when tumour burden exceeded 1 × 108 photons/second.6 cells s.c. on day 0, tumour growth was monitored with callipers, and tumour surgically resected at ≥ 140mm2. Lung tumour growth was then determined by IVIS. Mice were euthanised 88 days post-surgery, or according to clinical signs of severity score sheet, or when tumour burden exceeded 1 × 108 photons/second. Upon euthanasia, each mouse was dissected for confirmation of axillary and/or inguinal LN resection.Similarly, for the naturally metastatic Line-1M_LUC cell line, mice received 1 × 10Tumour resection. Analgesic buprenorphine s.c. (0.1 mg/kg) was administered 30 min prior to surgery. Anaesthesia was induced with 4% isoflurane in 100% oxygen and maintained at 2% isoflurane, at a flow rate of 2L/min. Resections were performed via elliptical incision over the subcutaneous tumour, spanning twice the length of the lesion, with ≤ 3 mm lateral margins. Tumour tissue was dissected clear of the adjacent fascia, and wounds were closed with 7-mm wound clips  or uninterrupted sutures . After surgery, mice received 0.05 mg/Kg buprenorphine at 12 and 24 h [and 24 h .For CD8 T cell depletion, mice received 100 µg aCD8 IgG antibodies i.p. at day 4, 8 and 12 post-surgery, and T cell depletion was determined in blood as indicated.Image-guided tumour draining lymph node resection. Shaved mice, bearing s.c. tumours (30–40 mm2) were injected intratumourally with 1.5 nmol of IRDye 800CW PEG  in 25 μl of PBS. Anaesthesia was induced (as for surgery), and mice serially imaged using the Cri Maestro 2 multispectral imaging system .Inguinal lymph nodes adjacent to the tumour were identified and resected from fat and fascia in the tumour resection margin at the time of primary tumour resection. Axillary lymph nodes were approached with a 5-mm mid-axillary oblique incision, and pectoral muscles retracted. Each lymph node was delicately teased away from the axillary vessel, fat and fascia. The axillary lymph node wound was closed with 5/0 Vicryl  uninterrupted sutures. Buprenorphine (0.05 mg/Kg) was administered at 12 and 24 h, or as required. Mice that did not undergo lymph node resection underwent sham surgery.Immunotherapy. On the day of surgery or treatment, mice were randomly assigned to groups. At days 17, 19 and 21 after surgery, mice received immune checkpoint blocking antibodies  by i.p. injection in PBS, 100 μl for monotherapy or 200 μl for combination therapy, at the following dose: 100 μg aCTLA-4 , or 200 μg aPD-1 , and 100 μg aCD40 . Control mice received PBS alone.Histology. At the end of experiment, lung tissue was fixed in 10% formalin , embedded in paraffin, and 750 μm sections prepared for haematoxylin and eosin (H&E) staining. Slides were imaged at 20 × on Aperio ScanScope XT .Preparation of single-cell suspensions. Single-cell suspensions were prepared from lung upon harvest [ harvest . In brieStatistical Analysis. All analysis was performed using GraphPad Prism version 7 software  or SPSS* statistics version 22 .p < 0.05; Fig. Common lung metastatic disease models involve injection of cells intravenously into healthy mice to seed tumour growth in the lung . As suchp = 0.0477). In mice with intact lymph nodes, median survival > 88 days compared to 53 days in the complete LN Rx group , and the onset of lung metastatic disease monitored . Median survival in mice with intact lymph nodes > 88 days compared to median survival of 73.5 days in the complete LN Rx group . Strikingly, the survival frequency from metastatic lung disease following post-surgery CD8 T cell depletion was significantly reduced from 88.9% to 37.5% (p < 0.05). These data indicate an immunological CD8 T cell component is in part responsible for protection from lung metastatic disease after primary tumour resection.We hypothesised that an immune component derived from primary tumour exposure was able to control occult metastatic tumour cell growth in the lung after surgery. In order to determine an immune component to the protective effect of primary tumour exposure on metastatic disease survival, we depleted CD8 T cells after surgery with aCD8 IgG antibodies in mice Fig. b. CD8 T Next, we examined histological sections from early- and late-onset lung metastases and revealed differing morphology. Early-onset metastasis formed a rind in lung tissue, whereas late metastatic disease manifests as nodules Fig. c. We hypThese data highlight the role of the immune system in checking the development of lung metastatic disease.Given the observed immune component to lung metastatic disease development, we determined the efficacy of checkpoint, aCTLA-4 and aPD-1, on survival in both the primary tumour Fig. a.Fig. 4I16. Next, we determined the efficacy of aPD-1 treatment in our subcutaneous primary tumour model (Fig. The efficacy of immunotherapy in primary tumours was first determined. Treatment of primary subcutaneous tumour with aCTLA4 increased survival (Fig. Effective primary tumour treatment strategies, aCTLA4 and aPD-1/CD40, were selected for treatment in the adjuvant setting (schematic, Fig. Notably, mouse lungs harvested after checkpoint blockade, aCTLA4 or aPD-1/aCD40 all grew out AB1-HA_LUC cells, regardless of LN resection.These data indicate that aPD-1/CD40 ICPB therapy after surgery is more effective against lung metastatic disease than aCTLA-4 treatment.Surgery is the gold standard treatment for many cancers; however, long after surgery patients often develop metastatic disease. This is likely due to occult tumours cells remaining at the time of surgery. Cancer surgery generally incorporates lymph node resection, for disease staging; however, this also potentially removes vital anti-tumour immune surveillance mechanisms required for effective control of local and metastatic disease. It is reasonable to consider that LN resection may impact on immunotherapy treatments that aim to modulate systemic anti-tumour immune cell responses. Thus, an understanding of the implications of lymph node resection for metastatic disease and response to immunotherapy is required. This study investigated onset of metastatic disease, the impact of lymph node resection, and efficacy of adjuvant immunotherapy in a novel pre-clinical model of lung metastatic disease that closely reflects the clinical setting.The ability of cancer cells to remain dormant for extended periods of time post-surgery has long been thought due to a state of equilibrium between anti-tumour immune cells and the cancer cells . NotablyWe, and others, have shown that tumour dLNs have been shown to harbour vital tumour-specific T cells , 21, 22.Adjuvant ICPB is under investigation in a number of clinical trials, and it is possible that lymphadenectomy may impact immunotherapy treatment outcomes. Recent studies have indicated that the tumour-specific T cell response in the tumour dLN closely correlates with response to ICPB . SurprisNotably, CTLA-4 is a homologue of CD28 and a negative regulator of T cell function via modulation CD80 and CD86 response . DraininThe success of adjuvant immunotherapy is apparent in findings from clinical trials; however, efficacy for murine primary tumour models has often not translated to patient studies. For example, aCTLA4 is effective in the treatment of solid subcutaneous tumour in mice , 27, butIn summary, we established the model as described to provide a precise scientific method of evaluation because animals, like humans, generate metastases unpredictably. We clearly show a decrease in lung metastatic disease survival following complete lymph node resection, with restoration of survival with conservative lymph node resection in two pre-clinical metastatic disease models. In the AB1-HA metastatic disease model, with controlled systemic introduction of tumour cells at the time of surgery, lymph node retention decreased metastatic disease, implicating immune control of introduced tumour cells. Importantly, notwithstanding the unpredictability of natural metastases in animal models, we did show similar findings of increased survival from metastatic disease were observed in the naturally metastatic, though capricious disease model Line-1 M. In addition, the study highlights the need to test ICPB in relevant clinical models. As such, aCTLA4 therapy was effective in subcutaneous solid tumours but lacked efficacy in the post-surgery metastatic disease setting. Alternately, ICPB treatment with aPD-1/aCD40 was efficacious in both the subcutaneous solid tumour and metastatic disease models of disease. Finally, frequency of survival following aPD-1/aCD40 therapy was similar with or without lymphadenectomy. This study provides valuable information in a relevant pre-clinical model of metastatic disease, demonstrating the impact of lymph node resection, and immune checkpoint blockade response after surgery on survival. Furthermore, the work highlights the importance for suitable pre-clinical modelling to facilitate clinical translation.Finally, there is a paucity of information regarding the impact of lymph node resection in development of metastatic disease and/or response to ICPB, and this reflects the difficulty to address this in patient’ studies. This study is informative to surgeons and addresses many questions relevant to the impact of lymph node resection, recommended by the AJCC Cancer Staging Manual, during primary tumour surgery relevant to patient survival and response to immunotherapy.