Efficacy and Safety of the Melphalan/Hepatic Delivery System in Patients with Unresectable Metastatic Uveal Melanoma: Results from an Open-Label, Single-Arm, Multicenter Phase 3 Study.

BACKGROUND: Uveal melanoma (UM) has a poor prognosis once liver metastases occur. The melphalan/Hepatic Delivery System (melphalan/HDS) is a drug/device combination used for liver-directed treatment of metastatic UM (mUM) patients. The purpose of the FOCUS study was to assess the efficacy and safety of melphalan/HDS in patients with unresectable mUM. METHODS: Eligible patients with mUM received treatment with melphalan (3.0 mg/kg ideal body weight) once every 6 to 8 weeks for a maximum of six cycles. The primary end point was the objective response rate (ORR). The secondary end points included duration of response (DOR), overall survival (OS), and progression-free survival (PFS). RESULTS: The study enrolled 102 patients with mUM. Treatment was attempted in 95 patients, and 91 patients received treatment. In the treated population (n = 91), the ORR was 36.3 % (95 % confidence interval [CI], 26.44-47.01), including 7.7 % of patients with a complete response. Thus, the study met its primary end point because the lower bound of the 95 % CI for ORR exceeded the upper bound (8.3 %) from the benchmark meta-analysis. The median DOR was 14 months, and the median OS was 20.5 months, with an OS of 80 % at 1 year. The median PFS was 9 months, with a PFS of 65 % at 6 months. The most common serious treatment-emergent adverse events were thrombocytopenia (15.8 %) and neutropenia (10.5 %), treated mostly on an outpatient basis with observation. No treatment-related deaths were observed. CONCLUSION: Treatment with melphalan/HDS provides a clinically meaningful response rate and demonstrates a favorable benefit-risk profile in patients with unresectable mUM (study funded by Delcath; ClinicalTrials.gov identifier: NCT02678572; EudraCT no. 2015-000417-44).

The neo-open reading frame peptides that comprise the tumor framome are a rich source of neoantigens for cancer immunotherapy.

Identification of immunogenic cancer neoantigens as targets for therapy is challenging. Here, we integrate cancer whole genome and long-read transcript sequencing to identify the collection of novel open reading frame peptides (NOPs) expressed in tumors, termed the framome. NOPs represent tumor-specific peptides that are different from wild-type proteins and may be strongly immunogenic. We describe an uncharacterized class of hidden NOPs, which derive from structural genomic variants involving an upstream protein coding gene driving expression and translation of non-coding regions of the genome downstream of a rearrangement breakpoint. NOPs represent a vast amount of possible neoantigens particularly in tumors with many (complex) structural genomic variants and a low number of missense mutations. We show that NOPs are immunogenic and epitopes derived from NOPs can bind to MHC class I molecules. Finally, we provide evidence for the presence of memory T-cells specific for hidden NOPs in lung cancer patient peripheral blood.

INHBA is Enriched in HPV-negative Oropharyngeal Squamous Cell Carcinoma and Promotes Cancer Progression.

Patients with oropharyngeal squamous cell carcinoma (OPSCC) caused by human papilloma virus (HPV) exhibit a better prognosis than those with HPV-negative OPSCC. This study investigated the distinct molecular pathways that delineate HPV-negative from HPV-positive OPSCC to identify biologically relevant therapeutic targets. Bulk mRNA from 23 HPV-negative and 39 HPV-positive OPSCC tumors (n = 62) was sequenced to uncover the transcriptomic profiles. Differential expression followed by gene set enrichment analysis was performed to outline the top enriched biological process in the HPV-negative compared with HPV-positive entity. INHBA, the highest overexpressed gene in the HPV-negative tumor, was knocked down. Functional assays (migration, proliferation, cell death, stemness) were conducted to confirm the target's oncogenic role. Correlation analyses to reveal its impact on the tumor microenvironment were performed. We revealed that epithelial-to-mesenchymal transition (EMT) is the most enriched process in HPV-negative compared with HPV-positive OPSCC, with INHBA (inhibin beta A subunit) being the top upregulated gene. INHBA knockdown downregulated the expression of EMT transcription factors and attenuated migration, proliferation, stemness, and cell death resistance of OPSCC cells. We uncovered that INHBA associates with a pro-tumor microenvironment by negatively correlating with antitumor CD8+ T and B cells while positively correlating with pro-tumor M1 macrophages. We identified three miRNAs that are putatively involved in repressing INHBA expression. Our results indicate that the upregulation of INHBA is tumor-promoting. We propose INHBA as an attractive therapeutic target for the treatment of INHBA-enriched tumors in patients with HPV-negative OPSCC to ameliorate prognosis. SIGNIFICANCE: Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC.

Intra-tumoral T cells in pediatric brain tumors display clonal expansion and effector properties.

Brain tumors in children are a devastating disease in a high proportion of patients. Owing to inconsistent results in clinical trials in unstratified patients, the role of immunotherapy remains unclear. We performed an in-depth survey of the single-cell transcriptomes and clonal relationship of intra-tumoral T cells from children with brain tumors. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with the potential to recognize tumor antigens. Such clonally expanded T cells display enrichment of transcripts linked to effector function, tissue residency, immune checkpoints and signatures of neoantigen-specific T cells and immunotherapy response. We identify neoantigens in pediatric brain tumors and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes. Notably, among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and magnitude of T cell response. Our findings suggest that characterization of intra-tumoral T cell responses may enable selection of patients for immunotherapy, an approach that requires prospective validation in clinical trials.

Tumor-Infiltrating CD103+ Tissue-Resident Memory T Cells and CD103-CD8+ T Cells in HNSCC Are Linked to Outcome in Primary but not Metastatic Disease.

PURPOSE: High numbers of tumor-infiltrating lymphocytes (TIL) are linked to better survival in patients with cancer. Tissue-resident memory T cells (TRM; CD8+CD103+) are recognized as a key player of anticancer immune response. To assess TRM cells in primary, metastatic, and recurrent head and neck squamous cell carcinoma (HNSCC), we developed a tissue microarray (TMA) and used multiplex IHC (MxIHC). EXPERIMENTAL DESIGN: Samples from primary tumors of 379 HNSCC cases treated at Southampton Hospitals between 2000 and 2016 were collected and analyzed. Of these, 105 cases had lymph node metastases and 82 recurrences. A TMA was generated with triplicate cores for each sample. MxIHC with a stain-and-strip approach was performed using CD8, CD103, and TIM3. Scanned slides were analyzed (digital image analysis) and quality checked (QC). RESULTS: After QC, 194 primary tumors, 76 lymph node metastases, and 65 recurrences were evaluable. Alcohol consumption was statistically significantly correlated with a reduction of TRM cells in primary tumors (nondrinker vs. heavy drinker: P = 0.0036). The known survival benefit of TRM cell infiltration in primary tumors was not found for lymph node metastasis. In recurrences, a high TRM cell number led to a favorable outcome after 12 months. The checkpoint molecule TIM3, was expressed significantly higher on TRM and non-TRM cells in the lymph node compared with primary tumors (P < 0.0001), which was also seen in recurrences (P = 0.0134 and P = 0.0007, respectively). CONCLUSIONS: We confirm the prognostic impact of TIL in primary tumors and in recurrences. TRM cell density in lymph node metastases was not linked to outcome. The role of TIM3, as a therapeutic target remains to be defined.

Transcriptomes and metabolism define mouse and human MAIT cell populations.

Mucosal-associated invariant T (MAIT) cells are a subset of T lymphocytes that respond to microbial metabolites. We defined MAIT cell populations in different organs and characterized the developmental pathway of mouse and human MAIT cells in the thymus using single-cell RNA sequencing and phenotypic and metabolic analyses. We showed that the predominant mouse subset, which produced IL-17 (MAIT17), and the subset that produced IFN-γ (MAIT1) had not only greatly different transcriptomes but also different metabolic states. MAIT17 cells in different organs exhibited increased lipid uptake, lipid storage, and mitochondrial potential compared with MAIT1 cells. All these properties were similar in the thymus and likely acquired there. Human MAIT cells in lung and blood were more homogeneous but still differed between tissues. Human MAIT cells had increased fatty acid uptake and lipid storage in blood and lung, similar to human CD8 T resident memory cells, but unlike mouse MAIT17 cells, they lacked increased mitochondrial potential. Although mouse and human MAIT cell transcriptomes showed similarities for immature cells in the thymus, they diverged more strikingly in the periphery. Analysis of pet store mice demonstrated decreased lung MAIT17 cells in these so-called "dirty" mice, indicative of an environmental influence on MAIT cell subsets and function.

Advancing Understanding of Non-Small Cell Lung Cancer with Multiplexed Antibody-Based Spatial Imaging Technologies.

Non-small cell lung cancer (NSCLC) remains a cause of significant morbidity and mortality, despite significant advances made in its treatment using immune checkpoint inhibitors (ICIs) over the last decade; while a minority experience prolonged responses with ICIs, benefit is limited for most patients. The development of multiplexed antibody-based (MAB) spatial tissue imaging technologies has revolutionised analysis of the tumour microenvironment (TME), enabling identification of a wide range of cell types and subtypes, and analysis of the spatial relationships and interactions between them. Such study has the potential to translate into a greater understanding of treatment susceptibility and resistance, factors influencing prognosis and recurrence risk, and identification of novel therapeutic approaches and rational treatment combinations to improve patient outcomes in the clinic. Herein we review studies that have leveraged MAB technologies to deliver novel insights into the TME of NSCLC.

JAML immunotherapy targets recently activated tumor-infiltrating CD8+ T cells.

Junctional adhesion molecule-like protein (JAML) serves as a co-stimulatory molecule in γδ T cells. While it has recently been described as a cancer immunotherapy target in mice, its potential to cause toxicity, specific mode of action with regard to its cellular targets, and whether it can be targeted in humans remain unknown. Here, we show that JAML is induced by T cell receptor engagement, reveal that this induction is linked to cis-regulatory interactions between the CD3D and JAML gene loci. When compared with other immunotherapy targets plagued by low target specificity and end-organ toxicity, we find JAML to be mostly restricted to and highly expressed by tissue-resident memory CD8+ T cells in multiple cancer types. By delineating the key cellular targets and functional consequences of agonistic anti-JAML therapy in a murine melanoma model, we show its specific mode of action and the reason for its synergistic effects with anti-PD-1.

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy.

Transcriptional analysis highlights three distinct immune profiles of high-risk oral epithelial dysplasia.

Oral potentially malignant disorders (OPMD) are precursors of oral squamous cell carcinoma (OSCC), and the presence of oral epithelial dysplasia (OED) in OPMD confers an increased risk of malignant transformation. Emerging evidence has indicated a role for the immune system in OPMD disease progression; however, the underlying immune mechanisms remain elusive. In this study, we used immune signatures established from cancer to delineate the immune profiles of moderate and severe OED, which are considered high-risk OPMD. We demonstrated that moderate and severe OEDs exhibit high lymphocyte infiltration and upregulation of genes involved in both immune surveillance (major histocompatibility complex-I, T cells, B cells and cytolytic activity) and immune suppression (immune checkpoints, T regulatory cells, and tumor-associated macrophages). Notably, we identified three distinct subtypes of moderate and severe OED: immune cytotoxic, non-cytotoxic and non-immune reactive. Active immune surveillance is present in the immune cytotoxic subtype, whereas the non-cytotoxic subtype lacks CD8 immune cytotoxic response. The non-immune reactive subtype showed upregulation of genes involved in the stromal microenvironment and cell cycle. The lack of T cell infiltration and activation in the non-immune reactive subtype is due to the dysregulation of CTNNB1, PTEN and JAK2. This work suggests that moderate and severe OED that harbor the non-cytotoxic or non-immune reactive subtype are likely to progress to cancer. Overall, we showed that distinct immune responses are present in high-risk OPMD, and revealed targetable pathways that could lead to potential new approaches for non-surgical management of OED.

Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9+ monocytes associated with COVID-19 hospitalization.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression.

CD33 Expression on Peripheral Blood Monocytes Predicts Efficacy of Anti-PD-1 Immunotherapy Against Non-Small Cell Lung Cancer.

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related deaths globally. Immune checkpoint blockade (ICB) has transformed cancer medicine, with anti-programmed cell death protein 1 (anti-PD-1) therapy now well-utilized for treating NSCLC. Still, not all patients with NSCLC respond positively to anti-PD-1 therapy, and some patients acquire resistance to treatment. There remains an urgent need to find markers predictive of anti-PD-1 responsiveness. To this end, we performed mass cytometry on peripheral blood mononuclear cells from 26 patients with NSCLC during anti-PD-1 treatment. Patients who responded to anti-PD-1 ICB displayed significantly higher levels of antigen-presenting myeloid cells, including CD9+ nonclassical monocytes, and CD33hi classical monocytes. Using matched pre-post treatment samples, we found that the baseline pre-treatment frequencies of CD33hi monocytes predicted patient responsiveness to anti-PD-1 therapy. Moreover, some of these classical and nonclassical monocyte subsets were associated with reduced immunosuppression by T regulatory (CD4+FOXP3+CD25+) cells in the same patients. Our use of machine learning corroborated the association of specific monocyte markers with responsiveness to ICB. Our work provides a high-dimensional profile of monocytes in NSCLC and links CD33 expression on monocytes with anti-PD-1 effectiveness in patients with NSCLC.

Intermittent PI3Kδ inhibition sustains anti-tumour immunity and curbs irAEs.

Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies1-3. Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity4,5, its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (Treg) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on Treg cells. Accordingly, in mouse models, PI3Kδi decreased the number of Treg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 Treg cells, accompanied by expansion of pathogenic T helper 17 (TH17) and type 17 CD8+ T (TC17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity.

Changes in Gene Expression Patterns in the Tumor Microenvironment of Head and Neck Squamous Cell Carcinoma Under Chemoradiotherapy Depend on Response.

Chemoradiotherapy (CRT) is a standard treatment for advanced head and neck squamous cell carcinoma (HNSCC). Unfortunately, not all patients respond to this therapy and require further treatment, either salvage surgery or palliative therapy. The addition of immunotherapy to CRT is currently being investigated and early results describe a mixed response. Therefore, it is important to understand the impact of CRT on the tumor microenvironment (TME) to be able to interpret the results of the clinical trials. Paired biopsies from 30 HNSCC patients were collected before and three months after completion of primary CRT and interrogated for the expression of 1392 immune- and cancer-related genes. There was a relevant difference in the number of differentially expressed genes between the total cohort and patients with residual disease. Genes involved in T cell activation showed significantly reduced expression in these tumors after therapy. Furthermore, gene enrichment for several T cell subsets confirmed this observation. The analysis of tissue resident memory T cells (TRM) did not show a clear association with impaired response to therapy. CRT seems to lead to a loss of T cells in patients with incomplete response that needs to be reversed. It is not clear whether the addition of anti-PD-1 antibodies alone to CRT can prevent treatment failure, as no upregulation of the targets was measurable in the TME.

HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy.

BACKGROUND: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. METHODS: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. RESULTS: We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. CONCLUSION: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Utility of KI-67 as a prognostic biomarker in pulmonary neuroendocrine neoplasms: a systematic review and meta-analysis.

OBJECTIVES: Ki-67, a marker of cellular proliferation, is associated with prognosis across a wide range of tumours, including gastroenteropancreatic neuroendocrine neoplasms (NENs), lymphoma, urothelial tumours and breast carcinomas. Its omission from the classification system of pulmonary NENs is controversial. This systematic review sought to assess whether Ki-67 is a prognostic biomarker in lung NENs and, if feasible, proceed to a meta-analysis. RESEARCH DESIGN AND METHODS: Medline (Ovid), Embase, Scopus and the Cochrane library were searched for studies published prior to 28 February 2019 and investigating the role of Ki-67 in lung NENs. Eligible studies were those that included more than 20 patients and provided details of survival outcomes, namely, HRs with CIs according to Ki-67 percentage. Studies not available as a full text or without an English manuscript were excluded. This study was prospectively registered with PROSPERO. RESULTS: Of 11 814 records identified, seven studies met the inclusion criteria. These retrospective studies provided data for 1268 patients (693 TC, 281 AC, 94 large cell neuroendocrine carcinomas and 190 small cell lung carcinomas) and a meta-analysis was carried out to estimate a pooled effect. Random effects analyses demonstrated an association between a high Ki-67 index and poorer overall survival (HR of 2.02, 95% CI 1.16 to 3.52) and recurrence-free survival (HR 1.42; 95% CI 1.01 to 2.00). CONCLUSION: This meta-analysis provides evidence that high Ki-67 labelling indices are associated with poor clinical outcomes for patients diagnosed with pulmonary NENs. This study is subject to inherent limitations, but it does provide valuable insights regarding the use of the biomarker Ki-67, in a rare tumour. PROSPERO REGISTRATION NUMBER: CRD42018093389.

CD8 T-cell-mediated cerebellitis directed against Purkinje cell antigen after ipilimumab for small cell lung cancer.

We report a rapidly progressive and fatal CD8 T-cell-mediated cerebellitis after ipilimumab (cytotoxic T-lymphocyte-associated protein 4 inhibitor) for small cell lung cancer. Clinical features and histopathology were consistent with an accelerated form of paraneoplastic cerebellar degeneration. A patchy CD8 T-cell infiltrate spatially corresponded to areas of Purkinje cell loss, with occasional CD8 polarisation towards Purkinje cells. CD20-positive B cells were sparse. CD8 T-cell-mediated cerebellitis after immune checkpoint inhibitor treatment may recapitulate the early stages of paraneoplastic cerebellar degeneration.

Analysis of Immune Landscape in Pancreatic and Ileal Neuroendocrine Tumours Demonstrates an Immune Cold Tumour Microenvironment.

INTRODUCTION: Neuroendocrine tumours (NETs) are rare tumours with an increasing incidence. While low- and intermediate-grade pancreatic NET (PanNET) and small intestinal NET (siNET) are slow growing, they have a relatively high rate of metastasizing to the liver, leading to substantially worse outcomes. In many solid tumours, the outcome is determined by the quality of the antitumour immune response. However, the quality and significance of antitumour responses in NETs are incompletely understood. This study provides clinico-pathological analyses of the tumour immune microenvironment in PanNET and siNETs. METHODS: Formalin-fixed paraffin-embedded tissue from consecutive resected PanNETs (61) and siNETs (131) was used to construct tissue microarrays (TMAs); 1-mm cores were taken from the tumour centre, stroma, tumour edge, and adjacent healthy tissue. TMAs were stained with antibodies against CD8, CD4, CD68, FoxP3, CD20, and NCR1. T-cell counts were compared with counts from lung cancers. RESULTS: For PanNET, median counts were CD8+ 35.4 cells/mm2, CD4+ 7.6 cells/mm2, and CD68+ macrophages 117.7 cells/mm2. For siNET, there were CD8+ 39.2 cells/mm2, CD4+ 24.1 cells/mm2, and CD68+ 139.2 cells/mm2. The CD8+ cell density in the tumour and liver metastases were significantly lower than in the adjacent normal tissues, without evidence of a cell-rich area at the tumour edge that might have suggested immune exclusion. T-cell counts in lung cancer were significantly higher than those in PanNET and siNETs: CD8+ 541 cells/mm2 and CD4+ 861 cells/mm2 (p ≤ 0.0001). CONCLUSION: PanNETs and siNETs are immune cold with no evidence of T cell exclusion; the low density of immune infiltrates indicates poor antitumour immune responses.

DNA Vaccines Targeting Novel Cancer-Associated Antigens Frequently Expressed in Head and Neck Cancer Enhance the Efficacy of Checkpoint Inhibitor.

HPV-independent head and neck squamous cell carcinoma (HNSCC) is a common cancer globally. The overall response rate to anti-PD1 checkpoint inhibitors (CPIs) in HNSCC is ~16%. One major factor influencing the effectiveness of CPI is the level of tumor infiltrating T cells (TILs). Converting TILlow tumors to TILhigh tumors is thus critical to improve clinical outcome. Here we describe a novel DNA vaccines to facilitate the T-cell infiltration and control tumor growth. We evaluated the expression of target antigens and their respective immunogenicity in HNSCC patients. The efficacy of DNA vaccines targeting two novel antigens were evaluated with or without CPI using a syngeneic model. Most HNSCC patients (43/44) co-expressed MAGED4B and FJX1 and their respective tetramer-specific T cells were in the range of 0.06-0.12%. In a preclinical model, antigen-specific T cells were induced by DNA vaccines and increased T cell infiltration into the tumor, but not MDSC or regulatory T cells. The vaccines inhibited tumor growth and improved the outcome alone and upon combination with anti-PD1 and resulted in tumor clearance in approximately 75% of mice. Pre-existence of MAGED4B and FJX1-reactive T cells in HNSCC patients suggests that these widely expressed antigens are highly immunogenic and could be further expanded by vaccination. The DNA vaccines targeting these antigens induced robust T cell responses and with the anti-PD1 antibody conferring excellent tumor control. This opens up an opportunity for combination immunotherapy that might benefit a wider population of HNSCC patients in an antigen-specific manner.

COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types.

Common genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in TH2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.