Calreticulin exposure on malignant blasts correlates with improved natural killer cell-mediated cytotoxicity in acute myeloid leukemia patients.

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.

Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients.

Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called "damage-associated molecular patterns," DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response.

Tertiary lymphoid structures and B cells determine clinically relevant T cell phenotypes in ovarian cancer.

Intratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8+ effector T (TEFF) cells and TIM3+PD1+, hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8+ T cells. Conversely, CD8+ T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1+ PD1+ T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8+ T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that - at odds with NSCLC - HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1+PD1+ CD8+ T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC.

Type I interferon signaling in malignant blasts contributes to treatment efficacy in AML patients.

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.

Immunological control of ovarian carcinoma by chemotherapy and targeted anticancer agents.

At odds with other solid tumors, epithelial ovarian cancer (EOC) is poorly sensitive to immune checkpoint inhibitors (ICIs), largely reflecting active immunosuppression despite CD8+ T cell infiltration at baseline. Accumulating evidence indicates that both conventional chemotherapeutics and targeted anticancer agents commonly used in the clinical management of EOC not only mediate a cytostatic and cytotoxic activity against malignant cells, but also drive therapeutically relevant immunostimulatory or immunosuppressive effects. Here, we discuss such an immunomodulatory activity, with a specific focus on molecular and cellular pathways that can be harnessed to develop superior combinatorial regimens for clinical EOC care.

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

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.

Peripheral gene signatures reveal distinct cancer patient immunotypes with therapeutic implications for autologous DC-based vaccines.

Dendritic cells (DCs) have received considerable attention as potential targets for the development of novel cancer immunotherapies. However, the clinical efficacy of DC-based vaccines remains suboptimal, largely reflecting local and systemic immunosuppression at baseline. An autologous DC-based vaccine (DCVAC) has recently been shown to improve progression-free survival and overall survival in randomized clinical trials enrolling patients with lung cancer (SLU01, NCT02470468) or ovarian carcinoma (SOV01, NCT02107937), but not metastatic castration-resistant prostate cancer (SP005, NCT02111577), despite a good safety profile across all cohorts. We performed biomolecular and cytofluorometric analyses on peripheral blood samples collected prior to immunotherapy from 1000 patients enrolled in these trials, with the objective of identifying immunological biomarkers that may improve the clinical management of DCVAC-treated patients. Gene signatures reflecting adaptive immunity and T cell activation were associated with favorable disease outcomes and responses to DCVAC in patients with prostate and lung cancer, but not ovarian carcinoma. By contrast, the clinical benefits of DCVAC were more pronounced among patients with ovarian carcinoma exhibiting reduced expression of T cell-associated genes, especially those linked to TH2-like signature and immunosuppressive regulatory T (TREG) cells. Clinical responses to DCVAC were accompanied by signs of antitumor immunity in the peripheral blood. Our findings suggest that circulating signatures of antitumor immunity may provide a useful tool for monitoring the potency of autologous DC-based immunotherapy.

TIM-3 levels correlate with enhanced NK cell cytotoxicity and improved clinical outcome in AML patients.

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.

Detection of immunogenic cell death and its relevance for cancer therapy.

Chemotherapy, radiation therapy, as well as targeted anticancer agents can induce clinically relevant tumor-targeting immune responses, which critically rely on the antigenicity of malignant cells and their capacity to generate adjuvant signals. In particular, immunogenic cell death (ICD) is accompanied by the exposure and release of numerous damage-associated molecular patterns (DAMPs), which altogether confer a robust adjuvanticity to dying cancer cells, as they favor the recruitment and activation of antigen-presenting cells. ICD-associated DAMPs include surface-exposed calreticulin (CALR) as well as secreted ATP, annexin A1 (ANXA1), type I interferon, and high-mobility group box 1 (HMGB1). Additional hallmarks of ICD encompass the phosphorylation of eukaryotic translation initiation factor 2 subunit-α (EIF2S1, better known as eIF2α), the activation of autophagy, and a global arrest in transcription and translation. Here, we outline methodological approaches for measuring ICD markers in vitro and ex vivo for the discovery of next-generation antineoplastic agents, the development of personalized anticancer regimens, and the identification of optimal therapeutic combinations for the clinical management of cancer.

Side-by-side comparison of flow cytometry and immunohistochemistry for detection of calreticulin exposure in the course of immunogenic cell death.

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.

Assessment of NK cell-mediated cytotoxicity by flow cytometry after rapid, high-yield isolation from peripheral blood.

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.

M2-like macrophages dictate clinically relevant immunosuppression in metastatic ovarian cancer.

BACKGROUND: The 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. METHODS: RNA 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. RESULTS: 1468 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. CONCLUSIONS: Immunosuppressive M2-like TAM infiltrating metastatic sites limit clinically relevant immune responses against HGSCs.

Genomic Structure of Hstx2 Modifier of Prdm9-Dependent Hybrid Male Sterility in Mice.

F1 hybrids between mouse inbred strains PWD and C57BL/6 represent the most thoroughly genetically defined model of hybrid sterility in vertebrates. Hybrid male sterility can be fully reconstituted from three components of this model, the Prdm9 gene, intersubspecific homeology of Mus musculus musculus and Mus musculus domesticus autosomes, and the X-linked Hstx2 locus. Hstx2 modulates the extent of Prdm9-dependent meiotic arrest and harbors two additional factors responsible for intersubspecific introgression-induced oligospermia (Hstx1) and meiotic recombination rate (Meir1). To facilitate positional cloning and to overcome the recombination suppression within the 4.3 Mb encompassing the Hstx2 locus, we designed Hstx2-CRISPR and SPO11/Cas9 transgenes aimed to induce DNA double-strand breaks specifically within the Hstx2 locus. The resulting recombinant reduced the Hstx2 locus to 2.70 Mb (chromosome X: 66.51-69.21 Mb). The newly defined Hstx2 locus still operates as the major X-linked factor of the F1 hybrid sterility, and controls meiotic chromosome synapsis and meiotic recombination rate. Despite extensive further crosses, the 2.70 Mb Hstx2 interval behaved as a recombination cold spot with reduced PRDM9-mediated H3K4me3 hotspots and absence of DMC1-defined DNA double-strand-break hotspots. To search for structural anomalies as a possible cause of recombination suppression, we used optical mapping and observed high incidence of subspecies-specific structural variants along the X chromosome, with a striking copy number polymorphism of the microRNA Mir465 cluster. This observation together with the absence of a strong sterility phenotype in Fmr1 neighbor (Fmr1nb) null mutants support the role of microRNA as a likely candidate for Hstx2.

Calreticulin exposure correlates with robust adaptive antitumor immunity and favorable prognosis in ovarian carcinoma patients.

BACKGROUND: Adjuvanticity, which is the ability of neoplastic cells to deliver danger signals, is critical for the host immune system to mount spontaneous and therapy-driven anticancer immune responses. One of such signals, i.e., the exposure of calreticulin (CALR) on the membrane of malignant cells experiencing endoplasmic reticulum (ER) stress, is well known for its role in the activation of immune responses to dying cancer cells. However, the potential impact of CALR on the immune contexture of primary and metastatic high-grade serous carcinomas (HGSCs) and its prognostic value for patients with HGSC remains unclear. METHOD: We harnessed a retrospective cohort of primary (no = 152) and metastatic (no = 74) tumor samples from HGSC patients to investigate the CALR expression in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 302 HGSC samples was used as a confirmatory approach. RESULTS: We demonstrate that CALR exposure on the surface of primary and metastatic HGSC cells is driven by a chemotherapy-independent ER stress response and culminates with the establishment of a local immune contexture characterized by TH1 polarization and cytotoxic activity that enables superior clinical benefits. CONCLUSIONS: Our data indicate that CALR levels in primary and metastatic HGSC samples have robust prognostic value linked to the activation of clinically-relevant innate and adaptive anticancer immune responses.

TIM-3 Dictates Functional Orientation of the Immune Infiltrate in Ovarian Cancer.

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.

Relevance of the chaperone-like protein calreticulin for the biological behavior and clinical outcome of cancer.

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.

Mature dendritic cells correlate with favorable immune infiltrate and improved prognosis in ovarian carcinoma patients.

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.