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Radiotherapy and immunotherapy can be concomitantly or sequentially combined seeking synergistic effects in terms of control of irradiated tumors and abscopal effects on nonirradiated lesions. Clinical-trial testing of such combinations faces several obstacles to demonstrate efficacy and needs improvements in trial design, patient selection, evaluation of results and biomarker discovery. See related article by Foster et al., p. 5510 ., (©2021 American Association for Cancer Research.)

One of the most important mechanisms by which cancer fosters its own development is the generation of an immune microenvironment that inhibits or impairs antitumor immune responses. A cancer permissive immune microenvironment is present in a large proportion of the patients with cancer who do not respond to immunotherapy approaches intended to trigger preexisting antitumor immune responses, for instance, immune checkpoint blockade. High circulating levels of IL8 in patients with cancer quite accurately predict those who will not benefit from checkpoint-based immunotherapy. IL8 has been reported to favor cancer progression and metastases via different mechanisms, including proangiogenesis and the maintenance of cancer stem cells, but its ability to attract and functionally modulate neutrophils and macrophages is arguably one of the most important factors. IL8 does not only recruit neutrophils to tumor lesions, but also triggers the extrusion of neutrophil extracellular traps (NET). The relevance and mechanisms underlying the contribution of both neutrophils and NETs to cancer development and progression are starting to be uncovered and include both direct effects on cancer cells and changes in the tumor microenvironment, such as facilitating metastasis, awakening micrometastases from dormancy, and facilitating escape from cytotoxic immune cells. Blockade of IL8 or its receptors (CXCR1 and CXCR2) is being pursued in drug development, and clinical trials alone or in combination with anti-PD-L1 checkpoint inhibitors are already ongoing., (©2020 American Association for Cancer Research.)

Immunomodulatory agents blocking the PD-1/PD-L1 pathway have shown a new way to treat cancer. The explanation underlying the success of these agents may be the selective expression of PD-L1 with dominant immune-suppressive activities in the tumor microenvironment (TME), supporting a more favorable tumor response-to-toxicity ratio. However, despite the big success of these drugs, most patients with cancer show primary or acquired resistance, calling for the identification of new immune modulators in the TME. Using a genome-scale T-cell activity array in combination with bioinformatic analysis of human cancer databases, we identified Siglec-15 as a critical immune suppressor with broad upregulation on various cancer types and a potential target for cancer immunotherapy. Siglec-15 has unique molecular features compared with many other known checkpoint inhibitory ligands. It shows prominent expression on macrophages and cancer cells and a mutually exclusive expression with PD-L1, suggesting that it may be a critical immune evasion mechanism in PD-L1-negative patients. Interestingly, Siglec-15 has also been identified as a key regulator for osteoclast differentiation and may have potential implications in bone disorders not limited to osteoporosis. Here, we provide an overview of Siglec-15 biology, its role in cancer immune regulation, the preliminary and encouraging clinical data related to the first-in-class Siglec-15 targeting mAb, as well as many unsolved questions in this pathway. As a new player in the cancer immunotherapeutic arena, Siglec-15 may represent a novel class of immune inhibitors with tumor-associated expression and divergent mechanisms of action to PD-L1, with potential implications in anti-PD-1/PD-L1-resistant patients., (©2020 American Association for Cancer Research.)

It has been reported that a group of patients with advanced non-small cell lung cancer showed circulating T cells with a senescent phenotype, and an abundance of such cells is associated with worse clinical response to immune checkpoint inhibitors. This study encourages further analysis of the role of senescent T cells in resistance to lung cancer immunotherapy. See related article by Ferrara et al., p. 492 ., (©2020 American Association for Cancer Research.)

Immune checkpoint inhibitors (ICI) have revolutionized the management of advanced non-small cell lung cancer (NSCLC). However, most pivotal phase III trials systematically excluded patients with active brain metastases, precluding the generalization of the results. Although theoretically restricted from crossing the blood-brain barrier, the novel pharmacokinetic/pharmacodynamic profiles of anti-PD-1/PD-L1 drugs have prompted studies to evaluate their activity in patients with NSCLC with active central nervous system (CNS) involvement. Encouraging results have suggested that ICI could be active in the CNS in selected patients with driver-negative advanced NSCLC with high PD-L1 expression and low CNS disease burden. Single-agent CNS response rates around 30% have been reported. Beyond this particular setting, anti-PD-1/PD-L1 antibodies have been evaluated in patients receiving local therapy for brain metastases (BM), addressing concerns about potential neurologic toxicity risks associated with radiotherapy, more specifically, radionecrosis (RN). Accordingly, a variety of clinical and imaging strategies are being appropriately developed to evaluate tumor response and to rule out pseudoprogression or radionecrosis. Our purpose is to critically summarize the advances regarding the role of systemic anti-PD-1/PD-L1 antibodies for the treatment of NSCLC BM. Data were collected from the PubMed database, reference lists, and abstracts from the latest scientific meetings. Recent reports suggest anti-PD-1/PD-L1 agents are active in a subset of patients with NSCLC with BM showing acceptable toxicity. These advances are expected to change soon the management of these patients but additional research is required to address concerns regarding radionecrosis and the appropriate sequencing of local and systemic therapy combinations., (©2020 American Association for Cancer Research.)

Purpose: To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non-small cell lung cancer (NSCLC)., Experimental Design: Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment., Results: PD-1, LAG-3, and TIM-3 were detected in tumor-infiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progression-free survival., Conclusions: PD-1, LAG-3, and TIM-3 have distinct tissue/cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways., (©2019 American Association for Cancer Research.)

Immune-checkpoint inhibitors (ICI), particularly inhibitors of the PD-1 axis, have altered the management of non-small cell lung cancer (NSCLC) over the last 10 years. First demonstrated to improve outcomes in second-line or later therapy of advanced disease, ICIs were shown to improve overall survival compared with chemotherapy in first-line therapy for patients whose tumors express PD-L1 on at least 50% of cells. More recently, combining ICIs with chemotherapy has been shown to improve survival in patients with both squamous and nonsquamous NSCLC, regardless of PD-L1 expression. However, PD-L1 and, more recently, tumor mutational burden have not proven to be straightforward indicative biomarkers. We describe the advances to date in utilizing these biomarkers, as well as novel markers of tumor inflammation, to ascertain which patients are most likely to benefit from ICIs. Ongoing translational work promises to improve the proportion of patients who benefit from these agents., (©2019 American Association for Cancer Research.)

Purpose: To determine the expression level, associations, and biological role of PD-L1, IDO-1, and B7-H4 in non-small cell lung cancer (NSCLC)., Experimental Design: Using multiplexed quantitative immunofluorescence (QIF), we measured the levels of PD-L1, IDO-1, B7-H4, and different tumor-infiltrating lymphoycte (TIL) subsets in 552 stages I-IV lung carcinomas from two independent populations. Associations between the marker levels, TILs, and major clinicopathologic variables were determined. Validation of findings was performed using mRNA expression data from The Cancer Genome Atlas (TCGA) and in vitro stimulation of lung adenocarcinoma A549 cells with IFNγ and IL10., Results: PD-L1 was detected in 16.9% and 21.8% of cases in each population. IDO-1 was expressed in 42.6% and 49.8%; and B7-H4 in 12.8% and 22.6% of cases, respectively. Elevated PD-L1 and IDO-1 were consistently associated with prominent B- and T-cell infiltrates, but B7-H4 was not. Coexpression of the three protein markers was infrequent, and comparable results were seen in the lung cancer TCGA dataset. Levels of PD-L1 and IDO-1 (but not B7-H4) were increased by IFNγ stimulation in A549 cells. Treatment with IL10 upregulated B7-H4 but did not affect PD-L1 and IDO-1 levels., Conclusions: PD-L1, IDO-1, and B7-H4 are differentially expressed in human lung carcinomas and show limited co-expression. While PD-L1 and IDO-1 are associated with increased TILs and IFNγ stimulation, B7-H4 is not. The preferential expression of discrete immune evasion pathways in lung cancer could participate in therapeutic resistance and support design of optimal clinical trials. Clin Cancer Res; 23(2); 370-8. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: Myeloid-derived suppressor cells (MDSC) are considered an important T-cell immunosuppressive component in cancer-bearing hosts. The factors that attract these cells to the tumor microenvironment are poorly understood. IL8 (CXCL8) is a potent chemotactic factor for neutrophils and monocytes., Experimental Design: MDSC were characterized and sorted by multicolor flow cytometry on ficoll-gradient isolated blood leucokytes from healthy volunteers (n = 10) and advanced cancer patients (n = 28). In chemotaxis assays, sorted granulocytic and monocytic MDSC were tested in response to recombinant IL8, IL8 derived from cancer cell lines, and patient sera. Neutrophil extracellular traps (NETs) formation was assessed by confocal microscopy, fluorimetry, and time-lapse fluorescence confocal microscopy on short-term MDSC cultures., Results: IL8 chemoattracts both granulocytic (GrMDSC) and monocytic (MoMDSC) human MDSC. Monocytic but not granulocytic MDSC exerted a suppressor activity on the proliferation of autologous T cells isolated from the circulation of cancer patients. IL8 did not modify the T-cell suppressor activity of human MDSC. However, IL8 induced the formation of NETs in the GrMDSC subset., Conclusions: IL8 derived from tumors contributes to the chemotactic recruitment of MDSC and to their functional control. Clin Cancer Res; 22(15); 3924-36. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: Interleukin-8 (IL8) is a chemokine produced by malignant cells of multiple cancer types. It exerts various functions in shaping protumoral vascularization and inflammation/immunity. We evaluated sequential levels of serum IL8 in preclinical tumor models and in patients to assess its ability to estimate tumor burden., Experimental Design: IL8 levels were monitored by sandwich ELISAs in cultured tumor cells supernatants, tumor-xenografted mice serum, and in samples from 126 patients with cancer. We correlated IL8 serum levels with baseline tumor burden and with treatment-induced changes in tumor burden, as well as with prognosis., Results: IL8 concentrations correlated with the number of IL8-producing tumor cells in culture. In xenografted neoplasms, IL8 serum levels rapidly dropped after surgical excision, indicating an accurate correlation with tumor burden. In patients with melanoma (n = 16), renal cell carcinoma (RCC; n = 23), non-small cell lung cancer (NSCLC; n = 21), or hepatocellular carcinoma (HCC; n = 30), serum IL8 concentrations correlated with tumor burden and stage, survival (melanoma, n = 16; RCC, n = 23; HCC, n = 33), and objective responses to therapy, including those to BRAF inhibitors (melanoma, n = 16) and immunomodulatory monoclonal antibodies (melanoma, n = 8). IL8 concentrations in urine (n = 18) were mainly elevated in tumors with direct contact with the urinary tract., Conclusions: IL8 levels correlate with tumor burden in preclinical models and in patients with cancer. IL8 is a potentially useful biomarker to monitor changes in tumor burden following anticancer therapy, and has prognostic significance., (©2014 American Association for Cancer Research.)

Purpose: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen., Experimental Design: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays., Results: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8(+) and CD4(+) T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy., Conclusion: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151-62. ©2013 AACR.

Therapy for cancer can be achieved by artificially stimulating antitumor T and natural killer (NK) lymphocytes with agonist monoclonal antibodies (mAb). T and NK cells express several members of the TNF receptor (TNFR) family specialized in delivering a costimulatory signal on their surface. Engagement of these receptors is typically associated with proliferation, elevated effector functions, resistance to apoptosis, and differentiation into memory cells. These receptors lack any intrinsic enzymatic activity and their signal transduction relies on associations with TNFR-associated factor (TRAF) adaptor proteins. Stimulation of CD137 (4-1BB), CD134 (OX40), and glucocorticoid-induced TNFR (GITR; CD357) promotes impressive tumor-rejecting immunity in a variety of murine tumor models. The mechanisms of action depend on a complex interplay of CTL, T-helper cells, regulatory T cells, dendritic cells, and vascular endothelium in tumors. Agonist mAbs specific for CD137 have shown signs of objective clinical activity in patients with metastatic melanoma, whereas anti-OX40 and anti-GITR mAbs have entered clinical trials. Preclinical evidence suggests that engaging TNFR members would be particularly active with conventional cancer therapies and additional immunotherapeutic approaches. Indeed, T-cell responses elicited to tumor antigens by means of immunogenic tumor cell death are amplified by these immunostimulatory agonist mAbs. Furthermore, anti-CD137 mAbs have been shown to enhance NK-mediated cytotoxicity elicited by rituximab and trastuzumab. Combinations with other immunomodulatory mAb that block T-cell checkpoint blockade receptors such as CTLA-4 and PD-1 are also promising., (©2013 AACR.)

Type I interferons (IFN-I) are well-known inducers of tumor cell apoptosis and antiangiogenesis via signaling through a common receptor interferon alpha receptor (IFNAR). IFNAR induces the Janus activated kinase-signal transducer and activation of transcription (JAK-STAT) pathway in most cells, along with other biochemical pathways that may differentially operate, depending on the responding cell subset, and jointly control a large collection of genes. IFNs-I were found to systemically activate natural killer (NK) cell activity. Recently, mouse experiments have shown that IFNs-I directly activate other cells of the immune system, such as antigen-presenting dendritic cells (DC) and CD4 and CD8 T cells. Signaling through the IFNAR in T cells is critical for the acquisition of effector functions. Cross-talk between IFNAR and the pathways turned on by other surface lymphocyte receptors has been described. Importantly, IFNs-I also increase antigen presentation of the tumor cells to be recognized by T lymphocytes. These IFN-driven immunostimulatory pathways offer opportunities to devise combinatorial immunotherapy strategies., (©2011 AACR.)