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Purpose: The successful implementation of immune checkpoint inhibitors (ICI) in the clinical management of various solid tumors has raised considerable expectations for patients with epithelial ovarian carcinoma (EOC). However, EOC is poorly responsive to ICIs due to immunologic features including limited tumor mutational burden (TMB) and poor lymphocytic infiltration. An autologous dendritic cell (DC)-based vaccine (DCVAC) has recently been shown to be safe and to significantly improve progression-free survival (PFS) in a randomized phase II clinical trial enrolling patients with EOC (SOV01, NCT02107937). Patients and Methods: We harnessed sequencing, flow cytometry, multispectral immunofluorescence microscopy, and IHC to analyze (pretreatment) tumor and (pretreatment and posttreatment) peripheral blood samples from 82 patients enrolled in SOV01, with the aim of identifying immunologic biomarkers that would improve the clinical management of patients with EOC treated with DCVAC. Results: Although higher-than-median TMB and abundant CD8+ T-cell infiltration were associated with superior clinical benefits in patients with EOC receiving standard-of-care chemotherapy, the same did not hold true in women receiving DCVAC. Conversely, superior clinical responses to DCVAC were observed in patients with lower-than-median TMB and scarce CD8+ T-cell infiltration. Such responses were accompanied by signs of improved effector functions and tumor-specific cytotoxicity in the peripheral blood. Conclusions: Our findings suggest that while patients with highly infiltrated, “hot” EOCs benefit from chemotherapy, women with “cold” EOCs may instead require DC-based vaccination to jumpstart clinically relevant anticancer immune responses.

At odds with other solid tumors, epithelial ovarian cancer (EOC) is poorly sensitive to immune checkpoint inhibitors (ICIs), largely reflecting active immunosuppression despite CD8

Purpose:The successful implementation of immune checkpoint inhibitors (ICI) in the clinical management of various solid tumors has raised considerable expectations for patients with epithelial ovarian carcinoma (EOC). However, EOC is poorly responsive to ICIs due to immunologic features including limited tumor mutational burden (TMB) and poor lymphocytic infiltration. An autologous dendritic cell (DC)-based vaccine (DCVAC) has recently been shown to be safe and to significantly improve progression-free survival (PFS) in a randomized phase II clinical trial enrolling patients with EOC (SOV01, NCT02107937).Patients and Methods:We harnessed sequencing, flow cytometry, multispectral immunofluorescence microscopy, and IHC to analyze (pretreatment) tumor and (pretreatment and posttreatment) peripheral blood samples from 82 patients enrolled in SOV01, with the aim of identifying immunologic biomarkers that would improve the clinical management of patients with EOC treated with DCVAC.Results:Although higher-than-median TMB and abundant CD8+ T-cell infiltration were associated with superior clinical benefits in patients with EOC receiving standard-of-care chemotherapy, the same did not hold true in women receiving DCVAC. Conversely, superior clinical responses to DCVAC were observed in patients with lower-than-median TMB and scarce CD8+ T-cell infiltration. Such responses were accompanied by signs of improved effector functions and tumor-specific cytotoxicity in the peripheral blood.Conclusions:Our findings suggest that while patients with highly infiltrated, “hot” EOCs benefit from chemotherapy, women with “cold” EOCs may instead require DC-based vaccination to jumpstart clinically relevant anticancer immune responses.

Supplementary Figure from An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors

Supplementary Data from An Autologous Dendritic Cell Vaccine Promotes Anticancer Immunity in Patients with Ovarian Cancer with Low Mutational Burden and Cold Tumors

Purpose:In multiple oncological settings, expression of the coinhibitory ligand PD-L1 by malignant cells and tumor infiltration by immune cells expressing coinhibitory receptors such as PD-1, CTLA4, LAG-3, or TIM-3 conveys prognostic or predictive information. Conversely, the impact of these features of the tumor microenvironment on disease outcome among high-grade serous carcinoma (HGSC) patients remains controversial.Experimental Design:We harnessed a retrospective cohort of 80 chemotherapy-naïve HGSC patients to investigate PD-L1 expression and tumor infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ dendritic cells as well as by PD-1+, CTLA4+, LAG-3+, and TIM-3+ cells in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on a second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 308 HGSC samples was used as a confirmatory approach.Results:High levels of PD-L1 and high densities of PD-1+ cells in the microenvironment of HGSCs were strongly associated with an immune contexture characterized by a robust TH1 polarization and cytotoxic orientation that enabled superior clinical benefits. Moreover, PD-1+TIM-3+CD8+ T cells presented all features of functional exhaustion and correlated with poor disease outcome. However, although PD-L1 levels and tumor infiltration by TIM-3+ cells improved patient stratification based on the intratumoral abundance of CD8+ T cells, the amount of PD-1+ cells failed to do so.Conclusions:Our data indicate that PD-L1 and TIM-3 constitute prognostically relevant biomarkers of active and suppressed immune responses against HGSC, respectively.

While type I interferon (IFN) is best known for its key role against viral infection, accumulating preclinical and clinical data indicate that robust type I IFN production in the tumor microenvironment promotes cancer immunosurveillance and contributes to the efficacy of various antineoplastic agents, notably immunogenic cell death inducers. Here, we report that malignant blasts from patients with acute myeloid leukemia (AML) release type I IFN via a Toll-like receptor 3 (TLR3)-dependent mechanism that is not driven by treatment. While in these patients the ability of type I IFN to stimulate anticancer immune responses was abolished by immunosuppressive mechanisms elicited by malignant blasts, type I IFN turned out to exert direct cytostatic, cytotoxic and chemosensitizing activity in primary AML blasts, leukemic stem cells from AML patients and AML xenograft models. Finally, a genetic signature of type I IFN signaling was found to have independent prognostic value on relapse-free survival and overall survival in a cohort of 132 AML patients. These findings delineate a clinically relevant, therapeutically actionable and prognostically informative mechanism through which type I IFN mediates beneficial effects in patients with AML.

Epithelial ovarian cancer (EOC) is among the top five causes of cancer-related death in women, largely reflecting early, prediagnosis dissemination of malignant cells to the peritoneum. Despite improvements in medical therapies, particularly with the implementation of novel drugs targeting homologous recombination deficiency, the survival rates of patients with EOC remain low. Unlike other neoplasms, EOC remains relatively insensitive to immune checkpoint inhibitors, which is correlated with a tumor microenvironment (TME) characterized by poor infiltration by immune cells and active immunosuppression dominated by immune components with tumor-promoting properties, especially tumor-associated macrophages (TAMs). In recent years, TAMs have attracted interest as potential therapeutic targets by seeking to reverse the immunosuppression in the TME and enhance the clinical efficacy of immunotherapy. Here, we review the key biological features of TAMs that affect tumor progression and their relevance as potential targets for treating EOC. We especially focus on the therapies that might modulate the recruitment, polarization, survival, and functional properties of TAMs in the TME of EOC that can be harnessed to develop superior combinatorial regimens with immunotherapy for the clinical care of patients with EOC.

BackgroundMost patients with epithelial ovarian cancer (EOC) relapse despite primary debulking surgery and chemotherapy (CT). Autologous dendritic cell immunotherapy (DCVAC) can present tumor antigens to elicit a durable immune response. We hypothesized that adding parallel or sequential DCVAC to CT stimulates antitumor immunity and improves clinical outcomes in patients with EOC. Based on the interim results of sequential DCVAC/OvCa administration and to accommodate the increased interest in maintenance treatment in EOC, the trial was amended by adding Part 2.MethodsPatients with International Federation of Gynecology and Obstetrics stage III EOC (serous, endometrioid, or mucinous), who underwent cytoreductive surgery up to 3 weeks prior to randomization and were scheduled for first-line platinum-based CT were eligible. Patients, stratified by tumor residuum (0 or + monocytes, pulsed with two allogenic OvCa cell lines (SK-OV-3, OV-90), and matured in the presence of polyinosinic:polycytidylic acid. We report the safety outcomes (safety analysis set, Parts 1 and 2 combined) along with the primary (progression-free survival (PFS)) and secondary (overall survival (OS)) efficacy endpoints. Efficacy endpoints were assessed in the modified intention-to-treat (mITT) analysis set in Part 1.ResultsBetween November 2013 and March 2016, 99 patients were randomized. The mITT (Part 1) comprised 31, 29, and 30 patients in Groups A, B, and C, respectively. Baseline characteristics and DCVAC/OvCa exposure were comparable across the treatment arms. DCVAC/OvCa showed a good safety profile with treatment-emergent adverse events related to DCVAC/OvCa in 2 of 34 patients (5.9%) in Group A and 2 of 53 patients (3.8%) in Group B. Median PFS was 20.3, not reached, and 21.4 months in Groups A, B, and C, respectively. The HR (95% CI) for Group A versus Group C was 0.98 (0.48 to 2.00; p=0.9483) and the HR for Group B versus Group C was 0.39 (0.16 to 0.96; p=0.0336). This was accompanied by a non-significant trend of improved OS in Groups A and B. Median OS was not reached in any group after a median follow-up of 66 months (34% of events).ConclusionsDCVAC/OvCa and leukapheresis was not associated with significant safety concerns in this trial. DCVAC/OvCa sequential to CT was associated with a statistically significant improvement in PFS in patients undergoing first-line treatment of EOC.Trial registration numberNCT02107937, EudraCT2010-021462-30.

Epithelial ovarian carcinoma (EOC) is a relatively rare malignancy but is the fifth-leading cause of cancer-related death in women, largely reflecting early, prediagnosis dissemination of malignant disease to the peritoneum. At odds with other neoplasms, EOC is virtually insensitive to immune checkpoint inhibitors, correlating with a tumor microenvironment that exhibits poor infiltration by immune cells and active immunosuppression. Here, we comparatively summarize the humoral and cellular features of primary and metastatic EOC, comparatively analyze their impact on disease outcome, and propose measures to alter them in support of treatment sensitivity and superior patient survival. ispartof: JOURNAL FOR IMMUNOTHERAPY OF CANCER vol:9 issue:10 ispartof: location:England status: published

Objective DCVAC/OvCa is an active cellular immunotherapy designed to stimulate an immune response against ovarian cancer. We explored the safety and efficacy of DCVAC/OvCa plus carboplatin and gemcitabine in platinum-sensitive ovarian cancer. Methods In this open-label, parallel-group, phase 2 trial ( ClinicalTrials.gov number NCT02107950 ), patients with platinum-sensitive ovarian cancer relapsing after first-line chemotherapy were randomized to DCVAC/OvCa and chemotherapy or chemotherapy alone. DCVAC/OvCa was administered every 3–6 weeks (10 doses). Endpoints included safety, progression-free survival (PFS; primary efficacy endpoint) and overall survival (OS; secondary efficacy endpoint). Results Between November 2013 and May 2015, 71 patients were randomized to chemotherapy in combination with DCVAC/OvCa or to chemotherapy alone. Treatment-emergent adverse events related to DCVAC/OvCa, leukapheresis and chemotherapy occurred in six (16.2%), two (5.4%), and 35 (94.6%) patients in the DCVAC/OvCa group. Chemotherapy-related events occurred in all patients in the chemotherapy group. Seven patients in the DCVAC/OvCa group were excluded from primary efficacy analyses due to failure to receive ≥1 dose of DCVAC/OvCa. PFS was not improved (hazard ratio [HR] 0.73, 95% confidence interval [CI] 0.42–1.28, P = 0.274, data maturity 78.1%). Median OS was significantly prolonged (by 13.4 months) in the DCVAC/OvCa group (HR 0.38, 95% CI 0.20–0.74, P = 0.003; data maturity 56.3%). A signal for enhanced surrogate antigen-specific T-cell activity was seen with DCVAC/OvCa. Conclusions DCVAC/OvCa combined with chemotherapy had a favorable safety profile in patients with platinum-sensitive ovarian cancer. DCVAC/OvCa did not improve PFS, but the exploratory analyses revealed OS prolongation and enhanced surrogate antigen-specific T-cell activity.

In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns. Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). Calreticulin exposure not only correlated with enhanced T-cell-dependent antitumor immunity in this setting but also affected the number of circulating natural killer (NK) cells upon restoration of normal hematopoiesis. Here, we report that calreticulin exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of calreticulin to stimulate NK-cells relies on CD11c+CD14high cells that, upon exposure to CRT, express higher levels of IL-15Rα, maturation markers (CD86 and HLA-DR) and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferon. This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL-15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed calreticulin favors NK-cell activation in AML patients.

Purpose: In multiple oncological settings, expression of the coinhibitory ligand PD-L1 by malignant cells and tumor infiltration by immune cells expressing coinhibitory receptors such as PD-1, CTLA4, LAG-3, or TIM-3 conveys prognostic or predictive information. Conversely, the impact of these features of the tumor microenvironment on disease outcome among high-grade serous carcinoma (HGSC) patients remains controversial. Experimental Design: We harnessed a retrospective cohort of 80 chemotherapy-naïve HGSC patients to investigate PD-L1 expression and tumor infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ dendritic cells as well as by PD-1+, CTLA4+, LAG-3+, and TIM-3+ cells in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on a second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 308 HGSC samples was used as a confirmatory approach. Results: High levels of PD-L1 and high densities of PD-1+ cells in the microenvironment of HGSCs were strongly associated with an immune contexture characterized by a robust TH1 polarization and cytotoxic orientation that enabled superior clinical benefits. Moreover, PD-1+TIM-3+CD8+ T cells presented all features of functional exhaustion and correlated with poor disease outcome. However, although PD-L1 levels and tumor infiltration by TIM-3+ cells improved patient stratification based on the intratumoral abundance of CD8+ T cells, the amount of PD-1+ cells failed to do so. Conclusions: Our data indicate that PD-L1 and TIM-3 constitute prognostically relevant biomarkers of active and suppressed immune responses against HGSC, respectively.

Accumulating evidence indicates that immune checkpoint inhibitors (ICIs) can restore CD8+ cytotoxic T lymphocyte (CTL) functions in preclinical models of acute myeloid leukemia (AML). However, ICIs targeting programmed cell death 1 (PDCD1, best known as PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) have limited clinical efficacy in patients with AML. Natural killer (NK) cells are central players in AML-targeting immune responses. However, little is known on the relationship between co-inhibitory receptors expressed by NK cells and the ability of the latter to control AML. Here, we show that hepatitis A virus cellular receptor 2 (HAVCR2, best known as TIM-3) is highly expressed by NK cells from AML patients, correlating with improved functional licensing and superior effector functions. Altogether, our data indicate that NK cell frequency as well as TIM-3 expression levels constitute prognostically relevant biomarkers of active immunity against AML.

LTX-315 is a nonameric oncolytic peptide in early clinical development for the treatment of solid malignancies. Preclinical and clinical evidence indicates that the anticancer properties of LTX-315 originate not only from its ability to selectively kill cancer cells, but also from its capacity to promote tumor-targeting immune responses. Here, we investigated the therapeutic activity and immunological correlates of intratumoral LTX-315 administration in three syngeneic mouse models of breast carcinoma, with a focus on the identification of possible combinatorial partners. We found that breast cancer control by LTX-315 is accompanied by a reconfiguration of the immunological tumor microenvironment that supports the activation of anticancer immunity and can be boosted by radiation therapy. Mechanistically, depletion of natural killer (NK) cells compromised the capacity of LTX-315 to limit local and systemic disease progression in a mouse model of triple-negative breast cancer, and to extend the survival of mice bearing hormone-accelerated, carcinogen-driven endogenous mammary carcinomas. Altogether, our data suggest that LTX-315 controls breast cancer progression by engaging NK cell-dependent immunity.

Drug repurposing is a promising strategy for identifying new applications for approved drugs. Here, we describe a polymer biomaterial composed of the antiretroviral drug ritonavir derivative (5-methyl-4-oxohexanoic acid ritonavir ester; RD), covalently bound to HPMA copolymer carrier via a pH-sensitive hydrazone bond (P-RD). Apart from being more potent inhibitor of P-glycoprotein in comparison to ritonavir, we found RD to have considerable cytostatic activity in six mice (IC50 ~ 2.3–17.4 μM) and six human (IC50 ~ 4.3–8.7 μM) cancer cell lines, and that RD inhibits the migration and invasiveness of cancer cells in vitro. Importantly, RD inhibits STAT3 phosphorylation in CT26 cells in vitro and in vivo, and expression of the NF-κB p65 subunit, Bcl-2 and Mcl-1 in vitro. RD also dampens chymotrypsin-like and trypsin-like proteasome activity and induces ER stress as documented by induction of PERK phosphorylation and expression of ATF4 and CHOP. P-RD nanomedicine showed powerful antitumor activity in CT26 and B16F10 tumor-bearing mice, which, moreover, synergized with IL-2-based immunotherapy. P-RD proved very promising therapeutic activity also in human FaDu xenografts and negligible toxicity predetermining these nanomedicines as side-effect free nanosystem. The therapeutic potential could be highly increased using the fine-tuned combination with other drugs, i.e. doxorubicin, attached to the same polymer system. Finally, we summarize that described polymer nanomedicines fulfilled all the requirements as potential candidates for deep preclinical investigation.

In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns. Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). Calreticulin exposure not only correlated with enhanced T-cell-dependent antitumor immunity in this setting but also affected the number of circulating natural killer (NK) cells upon restoration of normal hematopoiesis. Here, we report that calreticulin exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of calreticulin to stimulate NK-cells relies on CD11c+CD14high cells that, upon exposure to CRT, express higher levels of IL-15Rα, maturation markers (CD86 and HLA- DR) and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferon. This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL- 15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed calreticulin favors NK-cell activation in AML patients. Disclosure Information I. Truxova: None. L. Kasikova: None. C. Salek: None. M. Hensler: None. D. Lysak: None. P. Holicek: None. P. Bilkova: None. M. Holubova: None. X. Chen: None. R. Mikyskova: None. M. Reinis: None. M. Kovar: None. B. Tomalova: None. J.P. Kline: None. L. Galluzzi: None. R. Spisek: None. J. Fucikova: None.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BackgroundThe immunological microenvironment of primary high-grade serous carcinomas (HGSCs) has a major impact on disease outcome. Conversely, little is known on the microenvironment of metastatic HGSCs and its potential influence on patient survival. Here, we explore the clinical relevance of the immunological configuration of HGSC metastases.MethodsRNA sequencing was employed on 24 paired primary tumor microenvironment (P-TME) and metastatic tumor microenvironment (M-TME) chemotherapy-naive HGSC samples. Immunohistochemistry was used to evaluate infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ (lysosomal-associated membrane protein 3) dendritic cells (DCs), NKp46+ (natural killer) cells and CD68+CD163+ M2-like tumor-associated macrophages (TAMs), abundance of PD-1+ (programmed cell death 1), LAG-3+ (lymphocyte-activating gene 3) cells, and PD-L1 (programmed death ligand 1) expression in 80 samples. Flow cytometry was used for functional assessments on freshly resected HGSC samples.Results1468 genes were differentially expressed in the P-TME versus M-TME of HGSCs, the latter displaying signatures of extracellular matrix remodeling and immune infiltration. M-TME infiltration by immune effector cells had little impact on patient survival. Accordingly, M-TME-infiltrating T cells were functionally impaired, but not upon checkpoint activation. Conversely, cytokine signaling in favor of M2-like TAMs activity appeared to underlie inhibited immunity in the M-TME and poor disease outcome.ConclusionsImmunosuppressive M2-like TAM infiltrating metastatic sites limit clinically relevant immune responses against HGSCs.

Calreticulin (CALR) is an endoplasmic reticulum (ER)-resident protein involved in a spectrum of cellular processes. In healthy cells, CALR operates as a chaperone and Ca(2+) buffer to assist correct protein folding within the ER. Besides favoring the maintenance of cellular proteostasis, these cell-intrinsic CALR functions support Ca(2+)-dependent processes, such as adhesion and integrin signaling, and ensure normal antigen presentation on MHC Class I molecules. Moreover, cancer cells succumbing to immunogenic cell death (ICD) expose CALR on their surface, which promotes the uptake of cell corpses by professional phagocytes and ultimately supports the initiation of anticancer immunity. Thus, loss-of-function CALR mutations promote oncogenesis not only as they impair cellular homeostasis in healthy cells, but also as they compromise natural and therapy-driven immunosurveillance. However, the prognostic impact of total or membrane-exposed CALR levels appears to vary considerably with cancer type. For instance, while genetic CALR defects promote pre-neoplastic myeloproliferation, patients with myeloproliferative neoplasms bearing CALR mutations often experience improved overall survival as compared to patients bearing wild-type CALR. Here, we discuss the context-dependent impact of CALR on malignant transformation, tumor progression and response to cancer therapy.

Immunogenic cell death (ICD), a functionally peculiar type of apoptosis, represents a unique way to deliver danger-associated molecular patterns (DAMPs) to the tumor microenvironment. Once emitted by dying cancer cells, DAMPs orchestrate antigen-specific immune responses by acting on both innate and adaptive components of the immune system. Accumulating preclinical and clinical evidence indicates that one of these DAMPs, calreticulin (CALR) represents a novel powerful prognostic biomarker, reflecting the activation of a clinically relevant anticancer immune response in different cancer malignancies. Therefore, the assessment of CALR emission can provide a therapeutic tool for the stratification of cancer patients and the identification of individuals that are intrinsically capable to respond to a particular treatment. Here we describe methods for the quantification of CALR exposure in the tumor microenvironment of cancer patients by flow cytometry and immunohistochemistry.

The term 'immunogenic cell death' (ICD) denotes an immunologically unique type of regulated cell death that enables, rather than suppresses, T cell-driven immune responses that are specific for antigens derived from the dying cells. The ability of ICD to elicit adaptive immunity heavily relies on the immunogenicity of dying cells, implying that such cells must encode and present antigens not covered by central tolerance (antigenicity), and deliver immunostimulatory molecules such as damage-associated molecular patterns and cytokines (adjuvanticity). Moreover, the host immune system must be equipped to detect the antigenicity and adjuvanticity of dying cells. As cancer (but not normal) cells express several antigens not covered by central tolerance, they can be driven into ICD by some therapeutic agents, including (but not limited to) chemotherapeutics of the anthracycline family, oxaliplatin and bortezomib, as well as radiation therapy. In this Trial Watch, we describe current trends in the preclinical and clinical development of ICD-eliciting chemotherapy as partner for immunotherapy, with a focus on trials assessing efficacy in the context of immunomonitoring. ispartof: ONCOIMMUNOLOGY vol:9 issue:1 ispartof: location:United States status: published

Immunogenic cell death (ICD), a functionally peculiar type of apoptosis, represents a unique way to deliver danger-associated molecular patterns (DAMPs) to the tumor microenvironment. Once emitted by dying cancer cells, DAMPs orchestrate antigen-specific immune responses by acting on both innate and adaptive components of the immune system. Accumulating preclinical and clinical evidence indicates that one of these DAMPs, calreticulin (CALR) represents a novel powerful prognostic biomarker, reflecting the activation of a clinically relevant anticancer immune response in different cancer malignancies. Therefore, the assessment of CALR emission can provide a therapeutic tool for the stratification of cancer patients and the identification of individuals that are intrinsically capable to respond to a particular treatment. Here we describe methods for the quantification of CALR exposure in the tumor microenvironment of cancer patients by flow cytometry and immunohistochemistry.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation. ispartof: JOURNAL FOR IMMUNOTHERAPY OF CANCER vol:8 issue:1 ispartof: location:England status: published

Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.

Dendritic cells have been widely investigated in cancer immunotherapy clinical trials for the last two decades mainly due to their robust ability to elicit an adaptive anticancer immune response of the cellular and humoral types. Immature DCs can be easily loaded with desired antigens. However, to become efficient antigen-presenting cells, DCs must first undergo a process of maturation. Protocols for the generation of DCs for use in cancer immunotherapy, including the generation of a large number of immature DCs for antigen pulsing and the selection of a well-defined immunostimulatory agent to achieve complete and reproducible maturation, which is a crucial step for further stimulation of T cell activation, must carefully consider the characteristics of DC physiology. In this report, we provided a detailed protocol for DC generation, pulsation and activation with the subsequent induction of T cell-specific immune responses.

Antibodies targeting the co-inhibitory receptor programmed cell death 1 (PDCD1, best known as PD-1) or its main ligand CD274 (best known as PD-L1) have shown some activity in patients with metastatic triple-negative breast cancer (TNBC), especially in a recent Phase III clinical trial combining PD-L1 blockade with taxane-based chemotherapy. Despite these encouraging findings, however, most patients with TNBC fail to derive significant benefits from PD-L1 blockade, calling for the identification of novel therapeutic approaches. Here, we used the 4T1 murine mammary cancer model of metastatic and immune-resistant TNBC to test whether focal radiation therapy (RT), a powerful inducer of immunogenic cell death, in combination with various immunotherapeutic strategies can overcome resistance to immune checkpoint blockade. Our results suggest that focal RT enhances the therapeutic effects of PD-1 blockade against primary 4T1 tumors and their metastases. Similarly, the efficacy of an antibody specific for V-set immunoregulatory receptor (VSIR, another co-inhibitory receptor best known as VISTA) was enhanced by focal RT. Administration of cyclophosphamide plus RT and dual PD-1/VISTA blockade had superior therapeutic effects, which were associated with activation of tumor-infiltrating CD8+ T cells and depletion of intratumoral granulocytic myeloid-derived suppressor cells (MDSCs). Overall, these results demonstrate that RT can sensitize immunorefractory tumors to VISTA or PD-1 blockade, that this effect is enhanced by the addition of cyclophosphamide and suggest that a multipronged immunotherapeutic approach may also be required to increase the incidence of durable responses in patients with TNBC.

Caspase 3 (CASP3) has a key role in the execution of apoptosis, and many cancer cells are believed to disable CASP3 as a mechanism of resistance to cytotoxic therapeutics. Alongside, CASP3 regulates stress-responsive immunomodulatory pathways, including secretion of type I interferon (IFN). Here, we report that mouse mammary carcinoma TSA cells lacking Casp3 or subjected to chemical caspase inhibition were as sensitive to the cytostatic and cytotoxic effects of radiation therapy (RT) in vitro as their control counterparts, yet secreted increased levels of type I IFN. This effect originated from the accrued accumulation of irradiated cells with cytosolic DNA, likely reflecting the delayed breakdown of cells experiencing mitochondrial permeabilization in the absence of CASP3. Casp3-/- TSA cells growing in immunocompetent syngeneic mice were more sensitive to RT than their CASP3-proficient counterparts, and superior at generating bona fide abscopal responses in the presence of an immune checkpoint blocker. Finally, multiple genetic signatures of apoptotic proficiency were unexpectedly found to have robust negative (rather than positive) prognostic significance in a public cohort of breast cancer patients. However, these latter findings were not consistent with genetic signatures of defective type I IFN signaling, which were rather associated with improved prognosis. Differential gene expression analysis on patient subgroups with divergent prognosis (as stratified by independent signatures of apoptotic proficiency) identified SLC7A2 as a new biomarker with independent prognostic value in breast cancer patients. With the caveats associated with the retrospective investigation of heterogeneous, public databases, our data suggest that apoptotic caspases may influence the survival of breast cancer patients (or at least some subsets thereof) via mechanisms not necessarily related to type I IFN signaling as they identify a novel independent prognostic biomarker that awaits prospective validation.

BackgroundOncolytic peptides are attractive tools for the development of novel anticancer regimens [1]. LTX-315 is a synthetic peptide with a marked capacity to elicit tumor-targeting immunity in preclinical cancer models [2]. Indeed, LTX-315 has been shown to elicit immunogenic cell death (ICD) in malignant cells [3, 4] and to deplete immunosuppressive cells such as CD4+CD25+FOXP3+ TREG cells and myeloid-derived suppressor cells (MDSCs) from the tumor microenvironment (TME) [5]. Accordingly, LTX-315 synergized with immunogenic chemotherapeutics or immune checkpoint inhibitors (ICIs) in preclinical tumor models [5, 6]. Moreover, recent findings from a Phase I clinical trial in patients with advanced solid tumors (NCT01986426) indicate that intratumoral LTX-315 is safe, clinically active, and elicits alterations in the TME that support the initiation of anticancer immunity [7, 8]. However, the dependency of LTX-315 therapeutic effects on the immune system in preclinical models of breast cancer has not been mechanistically investigated.MethodsWe harnessed three distinct mouse models of ICI-resistant breast cancer, namely hormone receptor (HR)-positive TS/A established and triple-negative breast cancer (TNBC) 4T1 cells established in immunocompetent syngeneic BALB/c mice, as well as medroxyprogesterone acetate (MPA, M)-initiated, 7,12-dimethylbenz[a]anthracene (DMBA, D)-driven mammary carcinomas evolving in C57BL/6 mice to assess the immunotherapeutic effects of LTX-315 optionally combined with radiation therapy (RT), based on the primary tumor growth, metastatic dissemination and overall survival (depending on model). Multilesion models, rechallenge assays, antibody-mediated depletion experiments as well as experiments in Rag1-/- mice were employed to elucidate the mechanistic involvement of the immune system.ResultsIn the multilesion TS/A models, intratumoral LTX-315 to one lesion combined with hypofractionated RT to another lesion resulted in superior systemic disease control as manifested by eradication of a 3rd untreated lesion in up to 50% of mice, which were protected from a subsequent rechallenge with living TS/A cells. In the single lesion 4T1 model, LTX-315 mediated enable robust local and metastatic disease control, which could be enhanced (only locally) with RT and dependent on natural killer (NK) cells, but less so on T lymphocytes (as determined with anti-asialo GM1 antibodies and Rag1-/- hosts). In the M/D-driven model, LTX-315 considerably controlled the growth of primary tumors and delayed relapse, an effect that depended on NK cells (as demonstrated with anti-NK1.1 antibodies).ConclusionsLTX-315, alone and combined with RT, mediates robust immunotherapeutic effects in multiple models of ICI-resistant breast cancer. Intriguingly, NK cells appear to be required for such effects, potentially linked to the emergence of immunological memory.AcknowledgementsWe are indebted to Dr. Fred Miller (Karmanos Cancer Center, Detroit, MI) for the kind gift of 4T1 cells, as well as to Dr. Karsten A. Pilones (Weill Cornell Medicine, New York, NY) and Maria E. Rodriguez-Ruiz (University of Navarra, Pamplona, Spain) for help with clonogenic assays. This work has been sponsored by a research grant by Lytix Biopharma (Oslo, Norway) to S.D. and L.G.ReferencesKepp, O. et al. (2020) Oncolysis without viruses - inducing systemic anticancer immune responses with local therapies. Nat Rev Clin Oncol 17 (1), 49–64.Vitale, I. et al. (2021) Targeting Cancer Heterogeneity with Immune Responses Driven by Oncolytic Peptides. Trends Cancer 7 (6), 557–572.Eike, L.M. et al. (2015) The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells. Oncotarget 6 (33), 34910–23.Zhou, H. et al. (2016) The oncolytic peptide LTX-315 triggers immunogenic cell death. Cell Death Dis 7 (3), e2134.Yamazaki, T. et al. (2016) The oncolytic peptide LTX-315 overcomes resistance of cancers to immunotherapy with CTLA4 checkpoint blockade. Cell Death Differ 23 (6), 1004–15.Camilio, K.A. et al. (2019) Combining the oncolytic peptide LTX-315 with doxorubicin demonstrates therapeutic potential in a triple-negative breast cancer model. Breast Cancer Res 21 (1), 9.Jebsen, N.L. et al. (2019) Enhanced T-lymphocyte infiltration in a desmoid tumor of the thoracic wall in a young woman treated with intratumoral injections of the oncolytic peptide LTX-315: a case report. J Med Case Rep 13 (1), 177.Spicer, J. et al. (2021) Safety, Antitumor Activity, and T-cell Responses in a Dose-Ranging Phase I Trial of the Oncolytic Peptide LTX-315 in Patients with Solid Tumors. Clin Cancer Res 27 (10), 2755–2763.Ethics ApprovalThis study was approved by Weill Cornell Medical College’s Ethics Board; approval number 2015-0028, 2018-0002.

5521 Background: Most patients with EOC relapse despite PDS and CMT. Autologous DCVAC can present tumor antigens to elicit a durable immune response. We hypothesized that adding DCVAC to CMT stimulates antitumor immunity and improves clinical outcomes. Methods: Key eligibility criteria were FIGO stage III EOC (serous, endometrioid, or mucinous), post-PDS with < 1 cm maximal residuum, no prior systemic therapy, and ECOG 0-2. In part 1, patients were randomized up to 6 weeks after PDS, 1:1:1, into arm A (A; DCVAC concomitant with CMT), arm B (B; DCVAC sequential after CMT), and arm C (C; CMT). Patients were stratified by tumor residuum (0 or < 1 cm). CMT consisted of 6 cycles of carboplatin (AUC 5-7) and paclitaxel (175 mg/m2). Patients in A and B received up to 10 doses of DCVAC (1×107 DCs/dose). The primary endpoint was radiologically assessed progression-free survival (PFS). The key secondary endpoint was overall survival (OS). Results presented refer to a protocol-defined modified intention-to-treat population (mITT) including patients who received ≥1 CMT dose in C or ≥1 DCVAC dose in A and B. Results: Between November 2013 and March 2016, 99 patients were randomized. At the final analysis, the mITT included 31 patients in A, 29 patients in B, and 30 patients in C. Key baseline characteristics and DCVAC exposure were comparable across treatment arms. Median PFS was 20.3 months in A, not reached in B, and 21.4 months in C, with corresponding HRs (95% CI) compared to C of 0.98 (0.48-2.00) in A and 0.39 (0.16-0.96) in B. The PFS benefit in B was statistically significant (p = 0.034) This was supported by a non-significant trend in OS in A and B. Median OS was not reached in any arm at the time of median follow-up of 66 months (34% of events). Patients with low CD8+ T-cell counts (CD8Lo) in tumor samples in A and B had significantly improved clinical outcomes compared to patients in C with CD8Lo: median PFS gain of 6 months (19 vs 13 months) and a more robust OS gain (median not reached vs 31 months), with minimal difference between A and B. This effect could not be attributed to statistical differences in high CD8+ T-cell counts (CD8Hi) density patients. These findings indicated the best clinical outcome in DCVAC patients with immunologically “cold” tumors in both DCVAC arms. DCVAC showed a good safety profile with only 8 DCVAC-related adverse events (Grade 1-2). Conclusions: DCVAC improved PFS and OS outcomes in patients with newly diagnosed EOC, predominantly in patients with immunologically “cold” tumors, thus representing a promising treatment option in this patient population. Clinical trial information: NCT02107937.

Immunotherapy of cancer has the potential to be effective mostly in patients with a low tumour burden. Rising PSA (prostate-specific antigen) levels in patients with prostate cancer represents such a situation. We performed the present clinical study with dendritic cell (DC)-based immunotherapy in this patient population. The single-arm phase I/II trial registered as EudraCT 2009-017259-91 involved 27 patients with rising PSA levels. The study medication consisted of autologous DCs pulsed with the killed LNCaP cell line (DCVAC/PCa). Twelve patients with a favourable PSA response continued with the second cycle of immunotherapy. The primary and secondary objectives of the study were to assess the safety and determine the PSA doubling time (PSADT), respectively. No significant side effects were recorded. The median PSADT in all treated patients increased from 5.67 months prior to immunotherapy to 18.85 months after 12 doses (p

Dendritic cells (DCs) are key regulators of immune responses that operate at the interface between innate and adaptive immunity, and defects in DC functions contribute to the pathogenesis of a variety of disorders. For instance, cancer evolves in the context of limited DC activity, and some autoimmune diseases are initiated by DC-dependent antigen presentation. Thus, correcting aberrant DC functions stands out as a promising therapeutic paradigm for a variety of diseases, as demonstrated by an abundant preclinical and clinical literature accumulating over the past two decades. However, the therapeutic potential of DC-targeting approaches remains to be fully exploited in the clinic. Here, we discuss the unique features of DCs that underlie the high therapeutic potential of DC-targeting strategies and critically analyze the obstacles that have prevented the full realization of this promising paradigm.

Calreticulin (CALR) exposed on the surface of cancer cells succumbing to therapy delivers robust phagocytic signals that support the activation of adaptive anticancer immune responses. Recent data from our group demonstrate that spontaneous CARL exposure on leukemic blasts also supports innate anticancer immunity by natural killer (NK) cells via an indirect mechanism relying on myeloid CD11c+CD14+ cells.

Caspases are known for their ability to precipitate apoptosis. Our findings indicate that accelerating the terminal inactivation of cells dying in response to radiation therapy limit their immunogenicity as a consequence of reduced type I interferon secretion. Thus, caspase inhibitors stand out as promising combinatorial partners to improve the immunogenicity of radiation therapy in the clinic.

A high density of tumor-infiltrating CD8+ T cells and CD20+ B cells correlates with prolonged survival in patients with a wide variety of human cancers, including high-grade serous ovarian carcinoma (HGSC). However, the potential impact of mature dendritic cells (DCs) in shaping the immune contexture of HGSC, their role in the establishment of T cell-dependent antitumor immunity, and their potential prognostic value for HGSC patients remain unclear. We harnessed immunohistochemical tests and biomolecular analyses to demonstrate that a high density of tumor-infiltrating DC-LAMP+ DCs is robustly associated with an immune contexture characterized by TH1 polarization and cytotoxic activity. We showed that both mature DCs and CD20+ B cells play a critical role in the generation of a clinically-favorable cytotoxic immune response in HGSC microenvironment. In line with this notion, robust tumor infiltration by both DC-LAMP+ DCs and CD20+ B cells was associated with most favorable overall survival in two independent cohorts of chemotherapy-naïve HGSC patients. Our findings suggest that the presence of mature, DC-LAMP+ DCs in the tumor microenvironment may represent a novel, powerful prognostic biomarker for HGSC patients that reflects the activation of clinically-relevant anticancer immunity. Electronic supplementary material The online version of this article (10.1186/s40425-018-0446-3) contains supplementary material, which is available to authorized users.

Toll-like receptor (TLR) agonists demonstrate therapeutic promise as immunological adjuvants for anticancer immunotherapy. To date, three TLR agonists have been approved by US regulatory agencies for use in cancer patients. Additionally, the potential of hitherto experimental TLR ligands to mediate clinically useful immunostimulatory effects has been extensively investigated over the past few years. Here, we summarize recent preclinical and clinical advances in the development of TLR agonists for cancer therapy.

Photodynamic therapy (PDT) has attracted great attention in cancer treatment. Porphyrin derivative with ethylene glycol chains linked to meta position of meso-tetraphenylporphyrin, mTPP(EG)4, is one of assumed compounds that can be used for treatment of cancer in photodynamic therapy (PDT). The aim of the study is to demonstrate the photodynamic efficacy in vitro and in vivo as well as to analyze its therapeutic mechanism. The cell viability, cell surface exposure of immunogenic molecules, maturation and phagocytic capacity of PDT-treated tumor cells were monitored by flow cytometry. The kinetics of key components of ER stress-mediated apoptotic pathway was analyzed by Western blotting. Our first results indicate a new and attractive possibility of immunogenic cell death induction. Initiation of immunogenic cell death by PDT was successfully demonstrated in vitro on human and mouse tumor cell lines. The effects of mTPP(EG)4 were compared with the effects of mTPP(EG)4 without subsequent photoactivation, hypericin-mediated PDT, and high hydrostatic pressure. In vivo assays evaluated immunogenicity of mTPP(EG)4 in mice and assessed the ability of PDT treated cells to induce effective immunity against transplanted syngeneic tumors (mouse TRAMP-C2, TC-1 and B16). The results obtained in this study will provide a basis for possible further preclinical experiments with mTPP(EG)4 or other derivatives, testing their potential in PDT and development of new antitumor therapies. Citation Format: Sarka Vosahlikova, Irena Kusova Moserova, Jarmila Kralova, Milan Reinis, Romana Mikyskova, Jitka Palich Fucikova. Studies on photo-sensitivity of a glycol porphyrin derivative and its anti-tumor efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B35.

A high density of tumor-infiltrating CD8+ T cells and CD20+ B cells correlates with prolonged survival in patients with a wide variety of human cancers, including high-grade serous ovarian carcinoma (HGSC). However, the potential impact of mature dendritic cells (DCs) in shaping the immune contexture of HGSC, their role in the establishment of T cell-dependent antitumor immunity, and their potential prognostic value for HGSC patients remain unclear. We harnessed immunohistochemical tests and biomolecular analyses to demonstrate that a high density of tumor-infiltrating DC-LAMP+ DCs is robustly associated with an immune contexture characterized by TH1 polarization and cytotoxic activity. We showed that both mature DCs and CD20+ B cells play a critical role in the generation of a clinically favorable cytotoxic immune response in HGSC microenvironment. In line with this notion, robust tumor infiltration by both DC-LAMP+ DCs and CD20+ B cells was associated with most favorable overall survival in two independent cohorts of chemotherapy-naive HGSC patients. Our findings suggest that the presence of mature, DC-LAMP+ DCs in the tumor microenvironment may represent a novel, powerful prognostic biomarker for HGSC patients that reflects the activation of clinically relevant anticancer immunity. Citation Format: Iva Truxova, Lenka Kasikova, Michal Hensler, Petr Skapa, Jan Laco, Ladislav Pecen, Lucie Belicova, Ivan Praznovec, Michael J. Halaska, Tomas Brtnicky, Eva Salkova, Lukas Rob, Roman Kodet, Jeremy Goc, Catherine Sautes-Fridman, Wolf Herman Fridman, Ales Ryska, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Mature dendritic cells correlate with favorable immune infiltrate and improved prognosis in ovarian carcinoma patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B76.

In some settings, cancer cells responding to treatment undergo an immunogenic form of cell death that is associated with the abundant emission of danger signals in the form of damage-associated molecular patterns (DAMPs). Accumulating preclinical and clinical evidence indicates that danger signals play a crucial role in the (re-)activation of antitumor immune responses in vivo, thus having a major impact on patient prognosis. We have previously demonstrated that the presence of calreticulin (CRT) on the surface of malignant blasts is a positive prognostic biomarker for patients with acute myeloid leukemia (AML). CRT exposure not only correlated with enhanced T cell-dependent antitumor immunity in this setting but also affected the number of circulating NK cells upon restoration of normal hematopoiesis. Here, we report that CRT exposure on malignant blasts is associated with enhanced NK cell cytotoxic and secretory functions, both in AML patients and in vivo in mice. The ability of CRT to stimulate NK cells relies on CD11c+CD14high cells that, upon exposure to CRT, display higher levels of IL-15Rα, maturation markers (CD86 and HLA-DR), and CCR7. CRT exposure on malignant blasts also correlates with the upregulation of genes coding for type I interferons (IFNs). This suggests that CD11c+CD14high cells have increased capacity to migrate to secondary lymphoid organs, where can efficiently deliver stimulatory signals (IL-15Rα/IL-15) to NK cells. These findings delineate a multipronged, clinically relevant mechanism whereby surface-exposed CRT favors NK cell activation in AML patients. Citation Format: Iva Truxova, Lenka Kasikova, Cyril Salek, Michal Hensler, Daniel Lysak, Peter Holicek, Pavla Bilkova, Monika Holubova, Xiufen Chen, Romana Mikyskova, Milan Reinis, Marek Kovar, Barbora Tomalova, Justin Kline, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Calreticulin exposure on malignant blasts correlates with improved NK cell-mediated cytotoxicity in AML patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B96.

A high density of tumor-infiltrating CD8+ T lymphocytes (CTLs), key mediator of anticancer immunity, and CD20+ B cells correlates with positive prognostic value and prolonged survival in patients with a various solid tumors including high-grade serous ovarian carcinoma (HGSC). The majority of HGSCs containing high frequencies of CD8+ CTLs are also robustly infiltrated by CD20+ B cells, and patients whose tumors exhibit abundant coinfiltration have higher survival rates than patients with tumors that only contain high amounts of CD8+ CTLs. However, the cellular mechanism, the potential impact of mature dendritic cells (DCs) in shaping the immune contexture of HGSC, their role in the establishment of T cell-dependent antitumor immunity, and their potential prognostic value for HGSC patients remain unclear. Three independent cohorts of patients with resectable HGSC who did not receive neoadjuvant chemotherapy were involved in the study. Lysosomal-associated membrane protein 3 (DC-LAMP) and its colocalization with CD20+B cells in samples was analyzed by immunohistochemistry (IHC) and cell quantification. Prognostic impact of DC-LAMP+ cells in patient cohort was stratified based on median density of DC-LAMP+ cells in the tumor stroma and nest, followed by retrospective RFS and OS analysis. Comparison of DC-LAMPHi and DC-LAMPLo patients and impact of mature DCs on the composition of the immune contexture characterized by TH1 polarization and cytotoxic activity was performed by biomolecular analyses (flow cytometry, NGS analysis). Patients with high density of DC-LAMP+ cells in the tumor stroma exhibited significantly longer RFS and OS than DC-LAMP- patients. Compared to DC-LAMPLo, DC-LAMPHi tumors exhibited an over-representation of gene sets specifically clustering to the following immunologic functions: T cells, CD8 cytotoxicity, TH1 polarization, T cell activation, NK cells, and plasma cells. Also, a significantly higher percentage of CD3+CD45+ and CD3+CD8+ T cells among live mononuclear cells and higher density of NK cells in the tumor stroma in DC-LAMPHi were observed. We found that both mature DCs and CD20+ B cells play a critical role in the generation of a clinically favorable cytotoxic immune response in HGSC microenvironment. Robust tumor infiltration by both DC-LAMP+ DCs and CD20+ B cells was associated with most favorable overall survival in independent cohorts of chemotherapy-naive HGSC patients. Our findings suggest that the presence of mature, DC-LAMP+ DCs in the tumor microenvironment may represent a novel, powerful prognostic biomarker for HGSC patients that reflects the activation of clinically relevant anticancer immunity. Citation Format: Iva Truxova, Lenka Kasikova, Michal Hensler, Petr Skapa, Jan Laco, Ladislav Pecen, Lucie Belicova, Sarka Vosahlikova, Ivan Praznovec, Michael Halaska, Tomas Brtnicky, Eva Salkova, Lukas Rob, Roman Kodet, Jeremy Goc, Catherine Sautes-Fridman, Wolf Herve Fridman, Ales Ryska, Lorenzo Galluzzi, Radek Spisek, Jitka Fucikova. Mature dendritic cells correlate with favorable immune infiltrate and improved prognosis in ovarian carcinoma patients [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A24.

M2-polarized macrophages have been shown to adapt their 3D migration mode to physical properties of surrounding extracellular matrix. They migrate in the integrin-mediated adhesion and proteolytic activity-dependent “mesenchymal” mode in stiff matrices and in the integrin and protease-independent “amoeboid” mode in low density, porous environments. To find out what impact the switching between the migration modes has on expression of both protein-coding and non-coding genes we employed RNA sequencing of total RNA depleted of ribosomal RNA isolated from macrophages migrating in either mode in 3D collagens. Differentially expressed genes from both categories have been detected and the changes in expression of selected genes were further validated with RT-qPCR. The acquired data will facilitate better understanding of how mechanical properties of tissue microenvironment reflect in macrophage immune function and how the transitions between mesenchymal and amoeboid migratory modes are regulated at the gene expression level.

Peptide-based anticancer vaccination aims at stimulating an immune response against one or multiple tumor-associated antigens (TAAs) following immunization with purified, recombinant or synthetically engineered epitopes. Despite high expectations, the peptide-based vaccines that have been explored in the clinic so far had limited therapeutic activity, largely due to cancer cell-intrinsic alterations that minimize antigenicity and/or changes in the tumor microenvironment that foster immunosuppression. Several strategies have been developed to overcome such limitations, including the use of immunostimulatory adjuvants, the co-treatment with cytotoxic anticancer therapies that enable the coordinated release of damage-associated molecular patterns, and the concomitant blockade of immune checkpoints. Personalized peptide-based vaccines are also being explored for therapeutic activity in the clinic. Here, we review recent preclinical and clinical progress in the use of peptide-based vaccines as anticancer therapeutics.Abbreviations: CMP: carbohydrate-mimetic peptide; CMV: cytomegalovirus; DC: dendritic cell; FDA: Food and Drug Administration; HPV: human papillomavirus; MDS: myelodysplastic syndrome; MHP: melanoma helper vaccine; NSCLC: non-small cell lung carcinoma; ODD: orphan drug designation; PPV: personalized peptide vaccination; SLP: synthetic long peptide; TAA: tumor-associated antigen; TNA: tumor neoantigen

International audience; Oncolytic viruses selectively target and kill cancer cells in an immunogenic fashion, thus supporting the establishment of therapeutically relevant tumor-specific immune responses. In 2015, the US Food and Drug Administration (FDA) approved the oncolytic herpes simplex virus T-VEC for use in advanced melanoma patients. Since then, a plethora of trials has been initiated to assess the safety and efficacy of multiple oncolytic viruses in patients affected with various malignancies. Here, we summarize recent preclinical and clinical progress in the field of oncolytic virotherapy.

International audience; Cytokines regulate virtually aspects of innate and adaptive immunity, including the initiation, execution and extinction of tumor-targeting immune responses. Over the past three decades, the possibility of using recombinant cytokines as a means to elicit or boost clinically relevant anticancer immune responses has attracted considerable attention. However, only three cytokines have been approved so far by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, namely, recombinant interleukin (IL)-2 and two variants of recombinant interferon alpha 2 (IFN-α2a and IFN-α2b). Moreover, the use of these cytokines in the clinics is steadily decreasing, mostly as a consequence of: (1) the elevated pleiotropism of IL-2, IFN-α2a and IFN-α2b, resulting in multiple unwarranted effects; and (2) the development of highly effective immunostimulatory therapeutics, such as immune checkpoint blockers. Despite this and other obstacles, research in the field continues as alternative cytokines with restricted effects on specific cell populations are being evaluated. Here, we summarize research preclinical and clinical developments on the use of recombinant cytokines for immunostimulation in cancer patients.

Immune checkpoint blockers (ICBs) are literally revolutionizing the clinical management of an ever more diversified panel of oncological indications. Although considerable attention persists around the inhibition of cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1) signaling, several other co-inhibitory T-cell receptors are being evaluated as potential targets for the development of novel ICBs. Moreover, substantial efforts are being devoted to the identification of biomarkers that reliably predict the likelihood of each patient to obtain clinical benefits from ICBs in the absence of severe toxicity. Tailoring the delivery of specific ICBs or combinations thereof to selected patient populations in the context of precision medicine programs constitutes indeed a major objective of the future of ICB-based immunotherapy. Here, we discuss recent preclinical and clinical advances on the development of ICBs for oncological indications.

The death of cancer cells can be categorized as either immunogenic (ICD) or nonimmunogenic, depending on the initiating stimulus. The immunogenic processes of immunogenic cell death are mainly mediated by damage-associated molecular patterns (DAMPs), which include surface exposure of calreticulin (CRT), secretion of adenosine triphosphate (ATP), release of non-histone chromatin protein high-mobility group box 1 (HMGB1) and the production of type I interferons (IFNs). DAMPs are recognized by various receptors that are expressed by antigen-presenting cells (APCs) and potentiate the presentation of tumor antigens to T lymphocytes. Accumulating evidence indicates that CRT exposure constitutes one of the major checkpoints, that determines the immunogenicity of cell death both in vitro and in vivo in mouse models. Moreover, recent studies have identified CRT expression on tumor cells not only as a marker of ICD and active anti-tumor immune reactions but also as a major predictor of a better prognosis in various cancers. Here, we discuss the recent information on the CRT capacity to activate anticancer immune response as well as its prognostic and predictive role for the clinical outcome in cancer patients.

Dendritic cell (DC)-based vaccines pulsed with high hydrostatic pressure (HHP)-inactivated tumor cells have recently been shown to be a promising tool for prostate cancer chemoimmunotherapy. In this study, DC-based vaccines, both pulsed and unpulsed, were as effective as docetaxel (DTX) in reducing prostate tumors in the orthotopic transgenic adenocarcinoma of the mouse prostate (TRAMP) model. However, we did not observe any additive or synergic effects of chemoimmunotherapy on the tumor growth, while only the combination of DTX and pulsed dendritic cells resulted in significantly lower proliferation detected by Ki67 staining in histological samples. The DC-based vaccine pulsed with HHP-treated tumor cells was also combined with another type of cytostatic, cyclophosphamide, with similar results. In another clinically relevant setting, minimal residual tumor disease after surgery, administration of DC-based vaccines after the surgery of poorly immunogenic transplanted TRAMP-C2, as well as in immunogenic TC-1 tumors, reduced the growth of tumor recurrences. To identify the effector cell populations after DC vaccine application, mice were twice immunized with both pulsed and unpulsed DC vaccine, and the cytotoxicity of the spleen cells populations was tested. The effector cell subpopulations were defined as CD4+ and NK1.1+, which suggests rather unspecific therapeutic effects of the DC-based vaccines in our settings. Taken together, our data demonstrate that DC-based vaccines represent a rational tool for the treatment of human prostate cancer.

Owing to their central role in the initiation and regulation of antitumor immunity, dendritic cells (DCs) have been widely tested for use in cancer immunotherapy. Despite several encouraging clinical applications, existing DC-based immunotherapy efforts have yielded inconsistent results. Recent work has identified strategies that may allow for more potent DC-based vaccines, such as the combination with antitumor agents that have the potential to synergistically enhance DC functions. Selected cytotoxic agents may stimulate DCs either by directly promoting their maturation or through the induction of immunogenic tumor cell death. Moreover, they may support DC-induced adaptive immune responses by disrupting tumor-induced immunosuppressive mechanisms via selective depletion or inhibition of regulatory subsets, such as myeloid-derived suppressor cells and/or regulatory T cells (Tregs). Here, we summarize our current knowledge on the capacity of anticancer chemotherapeutics to modulate DC phenotype and functions and the results of ongoing clinical trials evaluating the use of DC-based immunotherapy in combination with chemotherapy in cancer patients.

Recent studies have identified molecular events characteristic of immunogenic cell death (ICD), including surface exposure of calreticulin (CRT), the heat shock proteins HSP70 and HSP90, the release of high-mobility group box protein 1 (HMGB1) and the release of ATP from dying cells. We investigated the potential of high hydrostatic pressure (HHP) to induce ICD in human tumor cells. HHP induced the rapid expression of HSP70, HSP90 and CRT on the cell surface. HHP also induced the release of HMGB1 and ATP. The interaction of dendritic cells (DCs) with HHP-treated tumor cells led to a more rapid rate of DC phagocytosis, upregulation of CD83, CD86 and HLA-DR and the release of interleukin IL-6, IL-12p70 and TNF-α. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor-specific T cells. DCs pulsed with HHP-treated tumor cells also induced the lowest number of regulatory T cells. In addition, we found that the key features of the endoplasmic reticulum stress-mediated apoptotic pathway, such as reactive oxygen species production, phosphorylation of the translation initiation factor eIF2α and activation of caspase-8, were activated by HHP treatment. Therefore, HHP acts as a reliable and potent inducer of ICD in human tumor cells.