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.

Phase I/II trial of dendritic cell-based active cellular immunotherapy with DCVAC/PCa in patients with rising PSA after primary prostatectomy or salvage radiotherapy for the treatment of prostate cancer.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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 < 0.0018). Twelve patients who continued immunotherapy with the second cycle had a median PSADT of 58 months that remained stable after the second cycle. In the peripheral blood, specific PSA-reacting T lymphocytes were increased significantly already after the fourth dose, and a stable frequency was detected throughout the remainder of DCVAC/PCa treatment. Long-term immunotherapy of prostate cancer patients experiencing early signs of PSA recurrence using DCVAC/PCa was safe, induced an immune response and led to the significant prolongation of PSADT. Long-term follow-up may show whether the changes in PSADT might improve the clinical outcome in patients with biochemical recurrence of the prostate cancer.

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.

Omega-3 phospholipids from fish suppress hepatic steatosis by integrated inhibition of biosynthetic pathways in dietary obese mice.

Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Antiinflammatory, hypolipidemic, and insulin-sensitizing effects ofDHA/EPA depend on their lipid form, with marine phospholipids showing better efficacy than fish oils. We characterized the mechanisms underlying beneficial effects of DHA/EPA phospholipids, alone or combined with an antidiabetic drug, on hepatosteatosis. C57BL/6N mice were fed for 7 weeks an obesogenic high-fat diet (cHF) or cHF-based interventions: (i) cHF supplemented with phosphatidylcholine-rich concentrate from herring (replacing 10% of dietary lipids; PC), (ii) cHF containing rosiglitazone (10 mg/kg diet; R), or (iii) PC + R. Metabolic analyses, hepatic gene expression and lipidome profiling were performed. Results showed that PC and PC + R prevented cHlF-induced weight gain and glucose intolerance, while all interventions reduced abdominal fat and plasma triacylglycerols. PC and PC + R also lowered hepatic and plasma cholesterol and reduced hepatosteatosis. Microarray analysis revealed integrated downregulation of hepatic lipogenic and cholesterol biosynthesis pathways by PC, while R-induced lipogenesis was fully counteracted in PC + R Gene expression changes in PC and PC + R were associated with preferential enrichment of hepatic phosphatidylcholine and phosphatidylethanolamine fractions by DHA/EPA. The complex downregulation of hepatic lipogenic and cholesterol biosynthesis genes and the antisteatotic effects were unique to DHA/EPA-containing phospholipids, since they were absent in mice fed soy-derived phosphatidylcholine. Thus, inhibition of lipid and cholesterol biosynthesis associated with potent antisteatotic effects in the liver in response to DHA/EPA-containing phospholipids support their use in NAFLD prevention and treatment.

Tissue contexture determines the pattern and density of tumor-infiltrating immune cells in HPV-associated squamous cell carcinomas of oropharynx and uterine cervix.

The profile of the antitumor immune response is an important factor determining patient clinical outcome. However, the influence of the tissue contexture on the composition of the tumor microenvironments of virally induced tumors is not clearly understood. Therefore, we analyzed the immune landscape of two HPV-associated malignancies: oropharyngeal squamous cell carcinoma (OPSCC) and squamous cell carcinoma of uterine cervix (CESC). We employed multiplex immunohistochemistry and immunofluorescence to evaluate the density and spatial distribution of immune cells in retrospective cohorts of OPSCC and CESC patients. This approach was complemented by transcriptomic analysis of purified primary tumor cells and in silico analysis of publicly available RNA sequencing data. Transcriptomic analysis showed similar immune profiles in OPSCC and CESC samples. Interestingly, immunostaining of OPSCC tissues revealed high densities of immune cells in both tumor stroma and tumor epithelium, whereas CESC samples were mainly characterized by the lack of immune cells in the tumor epithelium. However, in contrast to other immune cell populations, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were abundant in both segments of CESC samples and CESC-derived tumor cells expressed markedly higher levels of the PMN-MDSC chemoattractants CXCL1, CXCL5, and CXCL6 than OPSCC tumor cells. Taken together, despite their having the same etiologic agent, the immune infiltration pattern significantly differs between OPSCC and CESC, with a noticeable shift toward prominent MDSC infiltration in the latter. Our data thus present a rationale for a diverse approach to targeted therapy in patients with HPV-associated tumors of different tissue origins.

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.

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.

The inhibition of fat cell proliferation by n-3 fatty acids in dietary obese mice.

BACKGROUND: Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) of marine origin exert multiple beneficial effects on health. Our previous study in mice showed that reduction of adiposity by LC n-3 PUFA was associated with both, a shift in adipose tissue metabolism and a decrease in tissue cellularity. The aim of this study was to further characterize the effects of LC n-3 PUFA on fat cell proliferation and differentiation in obese mice. METHODS: A model of inducible and reversible lipoatrophy (aP2-Cre-ERT2 PPARγL2/L2 mice) was used, in which the death of mature adipocytes could be achieved by a selective ablation of peroxisome proliferator-activated receptor γ in response to i.p. injection of tamoxifen. Before the injection, obesity was induced in male mice by 8-week-feeding a corn oil-based high-fat diet (cHF) and, subsequently, mice were randomly assigned (day 0) to one of the following groups: (i) mice injected by corn-oil-vehicle only, i.e."control" mice, and fed cHF; (ii) mice injected by tamoxifen in corn oil, i.e. "mutant" mice, fed cHF; (iii) control mice fed cHF diet with15% of dietary lipids replaced by LC n-3 PUFA concentrate (cHF+F); and (iv) mutant mice fed cHF+F. Blood and tissue samples were collected at days 14 and 42. RESULTS: Mutant mice achieved a maximum weight loss within 10 days post-injection, followed by a compensatory body weight gain, which was significantly faster in the cHF as compared with the cHF+F mutant mice. Also in control mice, body weight gain was depressed in response to dietary LC n-3 PUFA. At day 42, body weights in all groups stabilized, with no significant differences in adipocyte size between the groups, although body weight and adiposity was lower in the cHF+F as compared with the cHF mice, with a stronger effect in the mutant than in control mice. Gene expression analysis documented depression of adipocyte maturation during the reconstitution of adipose tissue in the cHF+F mutant mice. CONCLUSION: Dietary LC n-3 PUFA could reduce both hypertrophy and hyperplasia of fat cells in vivo. Results are in agreement with the involvement of fat cell turnover in control of adiposity.

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.

LTX-315-enabled, radiotherapy-boosted immunotherapeutic control of breast cancer by NK cells.

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.

Converging focal radiation and immunotherapy in a preclinical model of triple negative breast cancer: contribution of VISTA blockade.

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.

Additive Effects of Omega-3 Fatty Acids and Thiazolidinediones in Mice Fed a High-Fat Diet: Triacylglycerol/Fatty Acid Cycling in Adipose Tissue.

Long-chain n-3 polyunsaturated fatty acids (Omega-3) and anti-diabetic drugs thiazolidinediones (TZDs) exhibit additive effects in counteraction of dietary obesity and associated metabolic dysfunctions in mice. The underlying mechanisms need to be clarified. Here, we aimed to learn whether the futile cycle based on the hydrolysis of triacylglycerol and re-esterification of fatty acids (TAG/FA cycling) in white adipose tissue (WAT) could be involved. We compared Omega-3 (30 mg/g diet) and two different TZDs-pioglitazone (50 mg/g diet) and a second-generation TZD, MSDC-0602K (330 mg/g diet)-regarding their effects in C57BL/6N mice fed an obesogenic high-fat (HF) diet for 8 weeks. The diet was supplemented or not by the tested compound alone or with the two TZDs combined individually with Omega-3. Activity of TAG/FA cycle in WAT was suppressed by the obesogenic HF diet. Additive effects in partial rescue of TAG/FA cycling in WAT were observed with both combined interventions, with a stronger effect of Omega-3 and MSDC-0602K. Our results (i) supported the role of TAG/FA cycling in WAT in the beneficial additive effects of Omega-3 and TZDs on metabolism of diet-induced obese mice, and (ii) showed differential modulation of WAT gene expression and metabolism by the two TZDs, depending also on Omega-3.

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.

Publisher Correction: Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer.

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

Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer.

Hormone receptor (HR)+ breast cancer (BC) causes most BC-related deaths, calling for improved therapeutic approaches. Despite expectations, immune checkpoint blockers (ICBs) are poorly active in patients with HR+ BC, in part reflecting the lack of preclinical models that recapitulate disease progression in immunocompetent hosts. We demonstrate that mammary tumors driven by medroxyprogesterone acetate (M) and 7,12-dimethylbenz[a]anthracene (D) recapitulate several key features of human luminal B HR+HER2- BC, including limited immune infiltration and poor sensitivity to ICBs. M/D-driven oncogenesis is accelerated by immune defects, demonstrating that M/D-driven tumors are under immunosurveillance. Safe nutritional measures including nicotinamide (NAM) supplementation efficiently delay M/D-driven oncogenesis by reactivating immunosurveillance. NAM also mediates immunotherapeutic effects against established M/D-driven and transplantable BC, largely reflecting increased type I interferon secretion by malignant cells and direct stimulation of immune effector cells. Our findings identify NAM as a potential strategy for the prevention and treatment of HR+ BC.

Apoptotic caspases inhibit abscopal responses to radiation and identify a new prognostic biomarker for breast cancer patients.

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.

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.