Search

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.

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.