Jacob Ricca

Heterogeneous Tumor-Immune Microenvironments among Differentially Growing Metastases in an Ovarian Cancer Patient

Summary We present an exceptional case of a patient with high-grade serous ovarian cancer, treated with multiple chemotherapy regimens, who exhibited regression of some metastatic lesions with concomitant progression of other lesions during a treatment-free period. Using immunogenomic approaches, we found that progressing metastases were characterized by immune cell exclusion, whereas regressing and stable metastases were infiltrated by CD8+ and CD4+ T cells and exhibited oligoclonal expansion of specific T cell subsets. We also detected CD8+ T cell reactivity against predicted neoepitopes after isolation of cells from a blood sample taken almost 3 years after the tumors were resected. These findings suggest that multiple distinct tumor immune microenvironments co-exist within a single individual and may explain in part the heterogeneous fates of metastatic lesions often observed in the clinic post-therapy. Video Abstract

Intratumoral modulation of the inducible co-stimulator ICOS by recombinant oncolytic virus promotes systemic anti-tumour immunity

Emerging data suggest that locoregional cancer therapeutic approaches with oncolytic viruses can lead to systemic anti-tumour immunity, although the appropriate targets for intratumoral immunomodulation using this strategy are not known. Here we find that intratumoral therapy with Newcastle disease virus (NDV), in addition to the activation of innate immunity, upregulates the expression of T-cell co-stimulatory receptors, with the inducible co-stimulator (ICOS) being most notable. To explore ICOS as a direct target in the tumour, we engineered a recombinant NDV-expressing ICOS ligand (NDV-ICOSL). In the bilateral flank tumour models, intratumoral administration of NDV-ICOSL results in enhanced infiltration with activated T cells in both virus-injected and distant tumours, and leads to effective rejection of both tumours when used in combination with systemic CTLA-4 blockade. These findings highlight that intratumoral immunomodulation with an oncolytic virus expressing a rationally selected ligand can be an effective strategy to drive systemic efficacy of immune checkpoint blockade.

Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic antitumor immunity via STING and Batf3-dependent dendritic cells.

Inactivated vaccinia virus induces antitumor immunity that can overcome resistance to checkpoint inhibitors. A viral STING operation for tumors The immune response to viruses can be harnessed to attack tumors. Here, Dai et al. demonstrate that inactivated modified vaccinia virus Ankara (iMVA) can induce antitumor responses in two different models of cancer in mice. This effect relied on both Batf3-dependent dendritic cells and the cGAS-STING cytosolic DNA-sensing pathway. Moreover, intratumor injection of iMVA overcame tumor resistance to checkpoint inhibitors. Viral-induced innate immune responses may therefore tip the scale to successful cancer immunotherapy. Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immunosuppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as immunotherapeutic agents. We show that infection of conventional dendritic cells (DCs) with heat- or ultraviolet-inactivated MVA leads to higher levels of interferon induction than MVA alone through the cGAS (cyclic guanosine monophosphate–adenosine monophosphate synthase)–STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA) was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than in wild-type mice, indicating that both cytosolic DNA sensing and Batf3-dependent CD103+/CD8α+ DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral tumor implantation models as well as in a unilateral large established tumor model. Our results suggest that inactivated vaccinia virus could be used as an immunotherapeutic agent for human cancers.

Immune-Active Microenvironment in Small Cell Carcinoma of the Ovary, Hypercalcemic Type: Rationale for Immune Checkpoint Blockade

Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a highly aggressive monogenic cancer driven by SMARCA4 mutations. Here, we report responses to anti-PD1 immunotherapy in four patients and characterize the immune landscape of SCCOHT tumors using quantitative immunofluorescence and gene expression profiling. Unexpectedly for a low mutation burden cancer, the majority of the tumors (eight of 11 cases) demonstrated PD-L1 expression with strong associated T-cell infiltration (R2 = 0.60–0.95). PD-L1 expression was detected in both tumor and stromal cells, with macrophages being the most abundant PD-L1-positive cells in some tumors (three of 11 cases). Transcriptional profiling revealed increased expression of genes related to Th1 and cytotoxic cell function in PD-L1-high tumors, suggesting that PD-L1 acts as a pathway of adaptive immune resistance in SCCOHT. These findings suggest that although SCCOHT are low–mutational burden tumors, their immunogenic microenvironment resembles the landscape of tumors that respond well to treatment with PD-1/PD-L1 blockade.

Pre-Existing Immunity to Oncolytic Virus Potentiates Its Immunotherapeutic Efficacy

Anti-viral immunity presents a major hurdle for systemically administered oncolytic viruses (OV). Intratumoral OV therapy has a potential to overcome this problem through activation of anti-tumor immune response, with local and abscopal effects. However, the effects of anti-viral immunity in such a setting are still not well defined. Using Newcastle Disease Virus (NDV) as a model, we explore the effects of pre-existing anti-viral immunity on therapeutic efficacy in syngeneic mouse tumor models. Unexpectedly, we find that while pre-existing immunity to NDV limits its replication in tumors, tumor clearance, abscopal anti-tumor immune effects, and survival are not compromised and, on the contrary, are superior in NDV-immunized mice. These findings demonstrate that pre-existing immunity to NDV may increase its therapeutic efficacy through potentiation of systemic anti-tumor immunity, which provides clinical rationale for repeated therapeutic dosing and prompts investigation of such effects with other OVs.

Validation of Anti-Mouse PDL-1 Goat Polyclonal Antibody Staining with Mouse PDL-1 In Situ Hybridization on Adjacent Sections of Cell Pellets and Mouse Tumors

Finding a valid antibody to detect mouse programmed death ligand 1 (PDL-1) by immunohistochemistry or immunofluorescence staining has been notoriously difficult. Successful validation of an antibody requires the use of multiple detection methods with the ability to compare appropriate positive and negative controls. Here, we describe in detail the protocols used to validate a mouse-specific PDL-1 antibody used in immunohistochemistry staining with an mRNA in situ hybridization on adjacent sections of mouse B16 tumor. This validation is supported by immunohistochemistry staining of PDL-1 on B16 cell pellets either treated or not treated with IFN-gamma.

Lysis-independent potentiation of immune checkpoint blockade by oncolytic virus

Intratumoral therapy with oncolytic viruses is increasingly being explored as a strategy to potentiate an immune response against cancer, but it remains unknown whether such therapy should be restricted to cancers sensitive to virus-mediated lysis. Using Newcastle Disease Virus (NDV) as a model, we explore immunogenic potential of an oncolytic virus in bladder cancer, where existing immunotherapy with PD-1 and PD-L1-targeting antibodies to date has shown suboptimal response rates. Infection of human and mouse bladder cancer cells with NDV resulted in immunogenic cell death, activation of innate immune pathways, and upregulation of MHC and PD-L1 in all tested cell lines, including the cell lines completely resistant to NDV-mediated lysis. In a bilateral flank NDV-lysis-resistant syngeneic murine bladder cancer model, intratumoral therapy with NDV led to an increase of immune infiltration in both treated and distant tumors and a shift from an inhibitory to effector T cell phenotype. Consequently, combination of intratumoral NDV with systemic PD-1 or CTLA-4 blockade led to improved local and abscopal tumor control and overall survival. These findings encourage future clinical trials combining intratumoral NDV therapy with systemic immunomodulatory agents and underscore the rationale for such treatments irrespective of tumor cell sensitivity to NDV-mediated lysis.

Abstract B09: Heterogeneous fates of metastatic lesions linked to immune escape in an ovarian cancer patient

The effect of chemotherapy on tumor heterogeneity and activation of the immune system in cancer is poorly understood. We present an unusual case of a patient with high-grade serous ovarian cancer, treated with multiple chemotherapy regimens, who exhibited spontaneous regression of some metastatic lesions with concomitant progression of other lesions during a treatment-free period. The primary tumor was resected before chemotherapy treatment started. After 5 years of chemotherapy without response, treatment was stopped, the patient transitioned to best supportive care and was followed clinically with regular CA125 biomarker evaluation. During the course of 2 years of clinical follow up, CA125 decreased and computerized tomography (CT) scans revealed one regressing (right upper quadrant), one stable (liver), and two progressing (spleen and vaginal cuff) metastases. Due to abdominal discomfort, metastases were simultaneously resected. We performed: a) CT scan-based radiomics to anlayze tumor heterogeneity, b) exome sequencing to identify somatic mutations, predict neoepitopes, and estimate neoepitope depletion, and c) immunofluorescence staining to analyze immune cell infiltration on the primary and four metastatic tumors. We found that chemotherapy increases tumor heterogeneity, and metastases that developed after multi-line chemotherapy have a higher somatic mutation rate, more neoepitopes, and mutations in immune recognition molecules compared to metastases that appeared earlier. The right upper quadrant regressing and the liver stable metastases were infiltrated by CD4+ and CD8+ T cells. The vaginal cuff progressing metastasis was characterized by immune cell exclusion, while the spleen progressing metastasis was infiltrated only by CD8+ T cells. Finally, we detected neoepitope depletion only in the right upper quadrant regressing metastasis, potentially indicating an active immunoediting process. These findings indicate that multiple distinct tumor immune microenvironments can co-exist within a single individual, evidencing potential clinical challenges for the appropriate application of immunotherapy and chemotherapy combinations to overcome tumor heterogeneity at the genetic and tumor-microenvironment levels. Citation Format: Alejandro Jimenez-Sanchez, Harini Veeraraghavan, Yanyun Li, Hebert Alberto Vargas, Michael B. Gill, Kay J. Park, Oliver Zivanovic, Jason Konner, Jacob Ricca, Dmitriy Zamarin, Carol Aghajanian, Jedd D. Wolchok, Taha Merghoub, Evis Sala, Alexandra Snyder, Martin L. Miller. Heterogeneous fates of metastatic lesions linked to immune escape in an ovarian cancer patient. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B09.