Oncolytic virus treatment differentially affects the CD56dim and CD56bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity.

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where antiviral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, inducing STAT1 and STAT4 signalling in both CD56dim and CD56bright NK cell subsets. Gene expression profiling revealed the dominance of IFN-I responses and identified induction of genes associated with NK cell cytotoxicity and cell cycle progression, with distinct responses in the CD56dim and CD56bright subsets. However, reovirus treatment inhibited IL-15 induced NK cell proliferation in an IFN-I dependent manner and was associated with reduced AKT signalling. In vivo, human CD56dim and CD56bright NK cells responded with similar kinetics to reovirus treatment, but CD56bright NK cells were transiently lost from the peripheral circulation at the peak of the IFN-I response, suggestive of their redistribution to secondary lymphoid tissue. Coupled with the direct, OV-mediated killing of tumour cells, the activation of both CD56dim and CD56bright NK cells by antiviral pathways induces a spectrum of activity that includes the NK cell-mediated killing of tumour cells and modulation of adaptive responses via the trafficking of IFN-γ expressing CD56bright NK cells to lymph nodes.

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

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

Anti-PD-1/anti-CTLA-4 efficacy in melanoma brain metastases depends on extracranial disease and augmentation of CD8+ T cell trafficking.

Inhibition of immune checkpoints programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) on T cells results in durable antitumor activity in melanoma patients. Despite high frequency of melanoma brain metastases (BrM) and associated poor prognosis, the activity and mechanisms of immune checkpoint inhibitors (ICI) in metastatic tumors that develop within the "immune specialized" brain microenvironment, remain elusive. We established a melanoma tumor transplantation model with intracranial plus extracranial (subcutaneous) tumor, mimicking the clinically observed coexistence of metastases inside and outside the brain. Strikingly, intracranial ICI efficacy was observed only when extracranial tumor was present. Extracranial tumor was also required for ICI-induced increase in CD8+ T cells, macrophages, and microglia in brain tumors, and for up-regulation of immune-regulatory genes. Combined PD-1/CTLA-4 blockade had a superior intracranial efficacy over the two monotherapies. Cell depletion studies revealed that NK cells and CD8+ T cells were required for intracranial anti-PD-1/anti-CTLA-4 efficacy. Rather than enhancing CD8+ T cell activation and expansion within intracranial tumors, PD-1/CTLA-4 blockade dramatically (∼14-fold) increased the trafficking of CD8+ T cells to the brain. This was mainly through the peripheral expansion of homing-competent effector CD8+ T cells and potentially further enhanced through up-regulation of T cell entry receptors intercellular adhesion molecule 1 and vascular adhesion molecule 1 on tumor vasculature. Our study indicates that extracranial activation/release of CD8+ T cells from PD-1/CTLA-4 inhibition and potentiation of their recruitment to the brain are paramount to the intracranial anti-PD-1/anti-CTLA-4 activity, suggesting augmentation of these processes as an immune therapy-enhancing strategy in metastatic brain cancer.

The immunological consequences of radiation-induced DNA damage.

Historically, our understanding of the cytotoxicity of radiation has centred on tumour cell-autonomous mechanisms of cell death. Here, tumour cell death occurs when a threshold number of radiation-induced non-reparable double-stranded DNA breaks is exceeded. However, in recent years, the importance of immune mechanisms of cell death has been increasingly recognised, as well as the impact of radiotherapy on non-malignant cellular components of the tumour microenvironment. Conserved antiviral pathways that detect foreign nucleic acid in the cytosol and drive downstream interferon (IFN) responses via the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of IFN genes (cGAS/STING) pathway are key components of the immune response to radiation-induced DNA damage. In preclinical models, acute induction of a type 1 IFN response is important for both direct and abscopal tumour responses to radiation. Inhibitors of the DNA damage response show promise in augmenting this inflammatory IFN response. However, a substantial proportion of tumours show chronic IFN signalling prior to radiotherapy, which paradoxically drives immunosuppression. This chronic IFN signalling leads to treatment resistance, and heterotypic interactions between stromal fibroblasts and tumour cells contribute to an aggressive tumour phenotype. The effect of radiotherapy on myeloid cell populations, particularly tumour-associated macrophages, has an additional impact on the immune tumour microenvironment. It is not yet clear how the above preclinical findings translate into a human context. Human tumours show greater intratumoural genomic heterogeneity and more variable levels of chromosomal instability than experimental murine models. High-quality translational studies of immunological changes occurring during radiotherapy that incorporate intrinsic tumour biology will enable a better understanding of the immunological consequences of radiation-induced DNA damage in patients. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Potentiating Oncolytic Virus-Induced Immune-Mediated Tumor Cell Killing Using Histone Deacetylase Inhibition.

A clinical oncolytic herpes simplex virus (HSV) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), talimogene laherparepvec, causes regression of injected and non-injected melanoma lesions in patients and is now licensed for clinical use in advanced melanoma. To date, limited data are available regarding the mechanisms of human anti-tumor immune priming, an improved understanding of which could inform the development of future combination strategies with improved efficacy. This study addressed direct oncolysis and innate and adaptive human immune-mediated effects of a closely related HSV encoding GM-CSF (HSVGM-CSF) alone and in combination with histone deacetylase inhibition. We found that HSVGM-CSF supported activation of anti-melanoma immunity via monocyte-mediated type I interferon production, which activates NK cells, and viral maturation of immature dendritic cells (iDCs) into potent antigen-presenting cells for cytotoxic T lymphocyte (CTL) priming. Addition of the histone deacetylase inhibitor valproic acid (VPA) to HSVGM-CSF treatment of tumor cells increased viral replication, viral GM-CSF production, and oncolysis and augmented the development of anti-tumor immunity. Mechanistically, VPA increased expression of activating ligands for NK cell recognition and induced expression of tumor-associated antigens, supporting innate NK cell killing and CTL priming. These data support the clinical combination of talimogene laherparepvec with histone deacetylase inhibition to enhance oncolysis and anti-tumor immunity.

Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer.

OBJECTIVE: Oncolytic viruses (OVs) represent promising, proinflammatory cancer treatments. Here, we explored whether OV-induced innate immune responses could simultaneously inhibit HCV while suppressing hepatocellular carcinoma (HCC). Furthermore, we extended this exemplar to other models of virus-associated cancer. DESIGN AND RESULTS: Clinical grade oncolytic orthoreovirus (Reo) elicited innate immune activation within primary human liver tissue in the absence of cytotoxicity and independently of viral genome replication. As well as achieving therapy in preclinical models of HCC through the activation of innate degranulating immune cells, Reo-induced cytokine responses efficiently suppressed HCV replication both in vitro and in vivo. Furthermore, Reo-induced innate responses were also effective against models of HBV-associated HCC, as well as an alternative endogenous model of Epstein-Barr virus-associated lymphoma. Interestingly, Reo appeared superior to the majority of OVs in its ability to elicit innate inflammatory responses from primary liver tissue. CONCLUSIONS: We propose that Reo and other select proinflammatory OV may be used in the treatment of multiple cancers associated with oncogenic virus infections, simultaneously reducing both virus-associated oncogenic drive and tumour burden. In the case of HCV-associated HCC (HCV-HCC), Reo should be considered as an alternative agent to supplement and support current HCV-HCC therapies, particularly in those countries where access to new HCV antiviral treatments may be limited.

Combination Therapy With Reovirus and Anti-PD-1 Blockade Controls Tumor Growth Through Innate and Adaptive Immune Responses.

Oncolytic reovirus can be delivered both systemically and intratumorally, in both preclinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against a variety of tumor types and antitumor activity is directly associated with immune activation by virus replication in tumors. Immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, and the generation of adaptive antitumor immune responses as a result of in vivo priming against tumor-associated antigens. We tested the combination of local oncolytic reovirus therapy with systemic immune checkpoint inhibition. We show that treatment of subcutaneous B16 melanomas with a combination of intravenous (i.v.) anti-PD-1 antibody and intratumoral (i.t.) reovirus significantly enhanced survival of mice compared to i.t. reovirus (P < 0.01) or anti-PD-1 therapy alone. In vitro immune analysis demonstrated that checkpoint inhibition improved the ability of NK cells to kill reovirus-infected tumor cells, reduced T(reg) activity, and increased the adaptive CD8(+) T-cell-dependent antitumor T-cell response. PD-1 blockade also enhanced the antiviral immune response but through effector mechanisms which overlapped with but also differed from those affecting the antitumor response. Therefore, combination with checkpoint inhibition represents a readily translatable next step in the clinical development of reovirus viroimmunotherapy.

Anti-cancer effects of oncolytic viral therapy combined with photodynamic therapy in human pancreatic cancer cell lines.

BACKGROUND: Oncolytic viral therapy and photodynamic therapy are potential therapies for inoperable or advanced pancreatic cancer. Our aim was to investigate the anti-cancer killing effects of reovirus therapy combined with protoporphyrin IX (PpIX)-mediated photodynamic therapy on a variety of human pancreatic cancer cell lines. METHODS: Pancreatic cancer cell lines (PsPC-1 and BXPC-3) and a non-cancer control cell line (HEK293) were infected with reovirus serotype 3 strain Dearing (T3D) at 0, 0·1, 1, and 10 plaque-forming units (PFU) per cell for 48 h. Cells were incubated with PpIX pro-drug 5-aminolevulinic acid (5-ALA) at 0, 1, 2, 3, and 4 mM for 4 h. Then, cells were photo-irradiated for 15 min with visible red light-emitting diodes with a light-fluence of 0·54 J/cm(2) of 653 nm (PpIX optimal excitation wavelength). The killing effects of reovirus combined with PpIX-mediated photodynamic therapy were analysed in methylthiazoltetrazolium (MTT) and trypan blue assays. The effect of adding reovirus after photodynamic therapy was also assessed. The statistical significance of the difference between groups was assessed with the two-tailed Student's t test. p<0·05 was considered statistically significant. FINDINGS: Reovirus monotherapy induced cell death in the two pancreatic lines (mean 57% [SE 10·2] at 10 PFU per cell). PpIX-mediated PDT monotherapy induced cell death in a dose-dependent manner (mean 10% [SE 2·2], 30 [6·4], 50 [8·2], and 70 [13·2] after 1, 2, 3, and 4 mM 5-ALA, respectively). Reovirus with PpIX-mediated photodynamic therapy resulted in a significantly increased cytotoxic effect compared with reovirus monotherapy and photodynamic therapy (p=0·042) with 100% cell death observed across pancreatic cell lines with 10 PFU per cell combined with 1 and 2 mM 5-ALA. There was no difference in cytotoxicity observed between added reovirus before or after photodynamic therapy. INTERPRETATION: To our knowledge, this is the first in-vitro study to combine reovirus oncolytic viral therapy with PpIX-mediated photodynamic therapy to treat pancreatic cancer. These results show a significant additive effect in cell killing and they provide initial evidence for a novel combined therapeutic intervention. FUNDING: National Institute for Health Research.

A phase-I trial of pre-operative, margin intensive, stereotactic body radiation therapy for pancreatic cancer: the 'SPARC' trial protocol.

BACKGROUND: Standard therapy for borderline-resectable pancreatic cancer in the UK is surgery with adjuvant chemotherapy, but rates of resection with clear margins are unsatisfactory and overall survival remains poor. Meta-analysis of single-arm studies shows the potential of neo-adjuvant chemo-radiotherapy but the relative radio-resistance of pancreatic cancer means the efficacy of conventional dose schedules is limited. Stereotactic radiotherapy achieves sufficient accuracy and precision to enable pre-operative margin-intensive dose escalation with the goal of increasing rates of clear resection margins and local disease control. METHODS/DESIGN: SPARC is a "rolling-six" design single-arm study to establish the maximum tolerated dose for margin-intensive stereotactic radiotherapy before resection of pancreatic cancer at high risk of positive resection margins. Eligible patients will have histologically or cytologically proven pancreatic cancer defined as borderline-resectable per National Comprehensive Cancer Network criteria or operable tumour in contact with vessels increasing the risk of positive margin. Up to 24 patients will be recruited from up to 5 treating centres and a 'rolling-six' design is utilised to minimise delays and facilitate ongoing recruitment during dose-escalation. Radiotherapy will be delivered in 5 daily fractions and surgery, if appropriate, will take place 5-6 weeks after radiotherapy. The margin-intense radiotherapy concept includes a systematic method to define the target volume for a simultaneous integrated boost in the region of tumour-vessel infiltration, and up to 4 radiotherapy dose levels will be investigated. Maximum tolerated dose is defined as the highest dose at which no more than 1 of 6 patients or 0 of 3 patients experience a dose limiting toxicity. Secondary endpoints include resection rate, resection margin status, response rate, overall survival and progression free survival at 12 and 24 months. Translational work will involve exploratory analyses of the cytological and humoral immunological responses to stereotactic radiotherapy in pancreatic cancer. Radiotherapy quality assurance of target definition and radiotherapy planning is enforced with pre-trial test cases and on-trial review. Recruitment began in April 2015. DISCUSSION: This prospective multi-centre study aims to establish the maximum tolerated dose of pre-operative margin-intensified stereotactic radiotherapy in pancreatic cancer at high risk of positive resection margins with a view to subsequent definitive comparison with other neoadjuvant treatment options. TRIAL REGISTRATION: ISRCTN14138956 . Funded by CRUK.

Mutated BRAF Emerges as a Major Effector of Recurrence in a Murine Melanoma Model After Treatment With Immunomodulatory Agents.

We used a VSV-cDNA library to treat recurrent melanoma, identifying immunogenic antigens, allowing us to target recurrences with immunotherapy or chemotherapy. Primary B16 melanoma tumors were induced to regress by frontline therapy. Mice with recurrent tumors were treated with VSV-cDNA immunotherapy. A Th17 recall response was used to screen the VSV-cDNA library for individual viruses encoding rejection antigens, subsequently targeted using immunotherapy or chemotherapy. Recurrent tumors were effectively treated with a VSV-cDNA library using cDNA from recurrent B16 tumors. Recurrence-associated rejection antigens identified included Topoisomerase-IIα, YB-1, cdc7 kinase, and BRAF. Fourteen out of 16 recurrent tumors carried BRAF mutations (595-605 region) following frontline therapy, even though the parental B16 tumors were BRAF wild type. The emergence of mutated BRAF-containing recurrences served as an excellent target for BRAF-specific immune-(VSV-BRAF), or chemo-(PLX-4720) therapies. Successful PLX-4720 therapy of recurrent tumors was associated with the development of a broad spectrum of T-cell responses. VSV-cDNA technology can be used to identify recurrence specific antigens. Emergence of mutated BRAF may be a major effector of melanoma recurrence which could serve as a target for chemo or immune therapy. This study suggests a rationale for offering patients with initially wild-type BRAF melanomas an additional biopsy to screen for mutant BRAF upon recurrence.

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

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

Progress in clinical oncolytic virus-based therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) carries a dismal prognosis, with advanced disease being resistant to both radiotherapy and conventional cytotoxic drugs, whilst anti-angiogenic drugs are marginally efficacious. Oncolytic viruses (OVs) offer the promise of selective cancer therapy through direct and immune-mediated mechanisms. The premise of OVs lies in their preferential genomic replication, protein expression and productive infection of malignant cells. Numerous OVs are being tested in preclinical models of HCC, with good evidence of direct and immune-mediated anti-tumour efficacy. Efforts to enhance the performance of these agents have concentrated on engineering OV cellular specificity, immune evasion, enhancing anti-tumour potency and improving delivery. The lead agent in HCC clinical trials, JX-594, a recombinant Wyeth strain vaccinia virus, has demonstrated evidence for significant benefit and earned orphan drug status. Thus, JX-594 appears to be transcending the barrier between novel laboratory science and credible clinical therapy. Relatively few other OVs have entered clinical testing, a hurdle that must be overcome if significant progress is to be made in this field. This review summarizes the preclinical and clinical experience of OV therapy in the difficult-to-treat area of HCC.

Cytokine conditioning enhances systemic delivery and therapy of an oncolytic virus.

Optimum clinical protocols require systemic delivery of oncolytic viruses in the presence of an intact immune system. We show that preconditioning with immune modulators, or loading virus onto carrier cells ex vivo, enhances virus-mediated antitumor activity. Our early trials of systemic reovirus delivery showed that after infusion reovirus could be recovered from blood cells--but not from plasma--suggesting that rapid association with blood cells may protect virus from neutralizing antibody. We therefore postulated that stimulation of potential carrier cells directly in vivo before intravenous viral delivery would enhance delivery of cell-associated virus to tumor. We show that mobilization of the CD11b(+) cell compartment by granulocyte macrophage-colony stimulating factor immediately before intravenous reovirus, eliminated detectable tumor in mice with small B16 melanomas, and achieved highly significant therapy in mice bearing well-established tumors. Unexpectedly, cytokine conditioning therapy was most effective in the presence of preexisting neutralizing antibody. Consistent with this, reovirus bound by neutralizing antibody effectively accessed monocytes/macrophages and was handed off to tumor cells. Thus, preconditioning with cytokine stimulated recipient cells in vivo for enhanced viral delivery to tumors. Moreover, preexisting neutralizing antibody to an oncolytic virus may, therefore, even be exploited for systemic delivery to tumors in the clinic.

The profile of tumor antigens which can be targeted by immunotherapy depends upon the tumor's anatomical site.

Previously, we showed that vesicular stomatitis virus (VSV) engineered to express a cDNA library from human melanoma cells (ASMEL, Altered Self Melanoma Epitope Library) was an effective systemic therapy to treat subcutaneous (s.c.) murine B16 melanomas. Here, we show that intravenous treatment with the same ASMEL VSV-cDNA library was an effective treatment for established intra-cranial (i.c.) melanoma brain tumors. The optimal combination of antigens identified from the ASMEL which treated s.c. B16 tumors (VSV-N-RAS+VSV-CYTC-C+VSV-TYRP-1) was ineffective against i.c. B16 brain tumors. In contrast, combination of VSV-expressed antigens-VSV-HIF-2α+VSV-SOX-10+VSV-C-MYC+VSV-TYRP1-from ASMEL which was highly effective against i.c. B16 brain tumors, had no efficacy against the same tumors growing subcutaneously. Correspondingly, i.c. B16 tumors expressed a HIF-2α(Hi), SOX-10(Hi), c-myc(Hi), TYRP1, N-RAS(lo)Cytc(lo) antigen profile, which differed significantly from the HIF-2α(lo), SOX-10(lo), c-myc(lo), TYRP1, N-RAS(Hi)Cytc(Hi) phenotype of s.c. B16 tumors, and was imposed upon the tumor cells by CD11b(+) cells within the local brain tumor microenvironment. Combining T-cell costimulation with systemic VSV-cDNA treatment, long-term cures of mice with established i.c. tumors were achieved in about 75% of mice. Our data show that the anatomical location of a tumor profoundly affects the profile of antigens that it expresses.

The efficacy versus toxicity profile of combination virotherapy and TLR immunotherapy highlights the danger of administering TLR agonists to oncolytic virus-treated mice.

Injection of oncolytic vesicular stomatitis virus (VSV) into established B16ova melanomas results in tumor regression, in large part by inducing innate immune reactivity against the viral infection, mediated by MyD88- and type III interferon (IFN)-, but not TLR-4-, signaling. We show here that intratumoral (IT) treatment with lipopolysaccharide (LPS), a TLR-4 agonist, significantly enhanced the local therapy induced by VSV by combining activation of different innate immune pathways. Therapy was further enhanced by co-recruiting a potent antitumor, adaptive T-cell response by using a VSV engineered to express the ovalbumin tumor-associated antigen ova, in combination with LPS. However, the combination of IT LPS with systemically delivered VSV resulted in rapid morbidity and mortality in the majority of mice. Decreasing the intravenous (IV) dose of VSV to levels at which toxicity was ameliorated did not enhance therapy compared with IT LPS alone. Toxicity of the systemic VSV + IT LPS regimen was associated with rapidly elevated levels of serum tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, which neither systemic VSV, nor IT LPS, alone induced. These data show that therapy associated with direct IT injections of oncolytic viruses can be significantly enhanced by combination with agonists of innate immune activation pathways, which are not themselves activated by the virus alone. Importantly, they also highlight possible, unforeseen dangers of combination therapies in which an immunotherapy, even delivered locally at the tumor site, may systemically sensitize the patient to a cytokine shock-like response triggered by IV delivery of oncolytic virus.

Functional cloning of recurrence-specific antigens identifies molecular targets to treat tumor relapse.

Aggressive regrowth of recurrent tumors following treatment-induced dormancy represents a major clinical challenge for treatment of malignant disease. We reported previously that recurrent prostate tumors, which underwent complete macroscopic regression followed by aggressive regrowth, could be cured with a vesicular stomatitis virus (VSV)-expressed cDNA library derived from recurrent tumor cells. By screening the protective, recurrence-derived VSV-cDNA library, here we identify topoisomerase-IIα (TOPO-IIα) as a recurrence-specific tumor antigen against which tolerance can be broken. Tumor recurrences, in two different types of tumor (prostate and melanoma), which had evaded two different frontline treatments (immunotherapy or chemotherapy), significantly overexpressed TOPO-IIα compared with their primary tumor counterparts, which conferred a novel sensitivity to doxorubicin (DOX) chemotherapy upon the recurrent tumors. This was exploited in vivo using combination therapies to cure mice, which would otherwise have relapsed, after suboptimal primary therapy in both models. Our data show that recurrent tumors-across histologies and primary treatments-express distinct antigens compared with the primary tumor which can be identified using the VSV-cDNA library technology. These results suggest that it may be possible to design a few common second-line therapies against a variety of tumor recurrences, in some cases using agents with no obvious activity against the primary tumor.

DNA-PKcs is required for cGAS/STING-dependent viral DNA sensing in human cells.

To mount an efficient interferon response to virus infection, intracellular pattern recognition receptors (PRRs) sense viral nucleic acids and activate anti-viral gene transcription. The mechanisms by which intracellular DNA and DNA viruses are sensed are relevant not only to anti-viral innate immunity, but also to autoinflammation and anti-tumour immunity through the initiation of sterile inflammation by self-DNA recognition. The PRRs that directly sense and respond to viral or damaged self-DNA function by signaling to activate interferon regulatory factor (IRF)-dependent type one interferon (IFN-I) transcription. We and others have previously defined DNA-dependent protein kinase (DNA-PK) as an essential component of the DNA-dependent anti-viral innate immune system. Here, we show that DNA-PK is essential for cyclic GMP-AMP synthase (cGAS)- and stimulator of interferon genes (STING)-dependent IFN-I responses in human cells during stimulation with exogenous DNA and infection with DNA viruses.

Intratumoral therapies in head and neck squamous cell carcinoma: A systematic review and future perspectives.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) presents an ideal scenario for intratumoral therapies (IT), due to its local recurrence pattern and frequent superficial extension. IT therapies aim to effect tumor regression by directly injecting antineoplastic agents into lesions. However, there is a lack of updated evidence regarding IT therapies in HNSCC. PATIENTS AND METHODS: A systematic literature search (CRD42023462291) was conducted using WebOfScience, ClinicalTrials.gov, and conference abstracts from ESMO and ASCO, identifying for IT clinical trials in patients with HNSCC, from database creation to September 12th, 2023. Efficacy as well as safety (grade ≥ 3 treatment-related adverse events[trAEs]) were reported. RESULTS: After evaluation of 1180 articles identified by the systematic search, 31 studies treating 948 patients were included. IT injectables were categorized as chemotherapies with or without electroporation (k = 4, N = 268), oncolytic viruses, plasmids, and bacteria-based (k = 16, N = 446), immunotherapies and EGFR-based therapies (k = 5, N = 160), radioenhancer particles (k = 2, N = 68), and calcium electroporation (k = 1, n = 6). EGFR-antisense plasmids, NBTXR3 radioenhancer and immune innate agonists show best overall response rates, at 83 %, 81 % and 44 % respectively. Eleven (35 %) studies added systemic therapy or radiotherapy to the IT injections. No study used predictive biomarkers to guide patient selection. 97 % studies were phase I-II. Safety-wise, electroporation and epinephrine-based injectable trials had significant local symptoms such as necrosis, fistula formation and post-injection dysphagia. Treatment-related tumor haemorrhages of various grades were described in several trials. Grade ≥ 3 trAEs attributable to the other therapies mainly comprised general symptoms such as fatigue. There were 3 injectable-related deaths across the systematic review. CONCLUSION: This is the first review to summarize all available evidence of IT in HNSCC. As of today, IT therapies lack sufficient evidence to recommend their use in clinical practice. Continuing research on potential molecules, patient selection, safe administration of injections and controlled randomized trials are needed to assess their added benefit.

Longitudinal Assessment of Tumor-Infiltrating Lymphocytes in Primary Breast Cancer Following Neoadjuvant Radiation Therapy.

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have prognostic significance in several cancers, including breast cancer. Despite interest in combining radiation therapy with immunotherapy, little is known about the effect of radiation therapy itself on the tumor-immune microenvironment, including TILs. Here, we interrogated longitudinal dynamics of TILs and systemic lymphocytes in patient samples taken before, during, and after neoadjuvant radiation therapy (NART) from PRADA and Neo-RT breast clinical trials. METHODS AND MATERIALS: We manually scored stromal TILs (sTILs) from longitudinal tumor samples using standardized guidelines as well as deep learning-based scores at cell-level (cTIL) and cell- and tissue-level combination analyses (SuperTIL). In parallel, we interrogated absolute lymphocyte counts from routine blood tests at corresponding time points during treatment. Exploratory analyses studied the relationship between TILs and pathologic complete response (pCR) and long-term outcomes. RESULTS: Patients receiving NART experienced a significant and uniform decrease in sTILs that did not recover at the time of surgery (P < .0001). This lymphodepletive effect was also mirrored in peripheral blood. Our SuperTIL deep learning score showed good concordance with manual sTILs and importantly performed comparably to manual scores in predicting pCR from diagnostic biopsies. The analysis suggested an association between baseline sTILs and pCR, as well as sTILs at surgery and relapse, in patients receiving NART. CONCLUSIONS: This study provides novel insights into TIL dynamics in the context of NART in breast cancer and demonstrates the potential for artificial intelligence to assist routine pathology. We have identified trends that warrant further interrogation and have a bearing on future radioimmunotherapy trials.