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Supplementary Figure Legends 1-5 from Immune Infiltration of Spontaneous Mouse Astrocytomas Is Dominated by Immunosuppressive Cells from Early Stages of Tumor Development

Supplementary Figures 1-3 from Preferential Expression of Very Late Antigen-4 on Type 1 CTL Cells Plays a Critical Role in Trafficking into Central Nervous System Tumors

Immune infiltration of advanced human gliomas has been shown, but it is doubtful whether these immune cells affect tumor progression. It could be hypothesized that this infiltrate reflects recently recruited immune cells that are immediately overwhelmed by a high tumor burden. Alternatively, if there is earlier immune detection and infiltration of the tumor, the question arises as to when antitumor competency is lost. To address these issues, we analyzed a transgenic mouse model of spontaneous astrocytoma (GFAP-V12HA-ras mice), which allows the study of immune interactions with developing glioma, even at early asymptomatic stages. T cells, including a significant proportion of Tregs, are already present in the brain before symptoms develop, followed later by macrophages, natural killer cells, and dendritic cells. The effector potential of CD8 T-cells is defective, with the absence of granzyme B expression and low expression of IFN-γ, tumor necrosis factor, and interleukin 2. Overall, our results show an early defective endogenous immune response to gliomas, and local accumulation of immunosuppressive cells at the tumor site. Thus, the antiglioma response is not simply overwhelmed at advanced stages of tumor growth, but is counterbalanced by an inhibitory microenvironment from the outset. Nevertheless, we determined that effector molecule expression (granzyme B, IFN-γ) by brain-infiltrating CD8 T-cells could be enhanced, despite this unfavorable milieu, by strong immune stimuli. This potential to modulate the strong imbalance in local antiglioma immunity is encouraging for the development and optimization of future glioma immunotherapies. Cancer Res; 70(12); 4829–39. ©2010 AACR.

Vaccines that can coordinately induce multi-epitope T cell–mediated immunity, T helper functions, and immunologic memory may offer effective tools for cancer immunotherapy. Here, we report the development of a new class of recombinant protein cancer vaccines that deliver different CD8+ and CD4+ T-cell epitopes presented by MHC class I and class II alleles, respectively. In these vaccines, the recombinant protein is fused with Z12, a novel cell-penetrating peptide that promotes efficient protein loading into the antigen-processing machinery of dendritic cells. Z12 elicited an integrated and multi-epitopic immune response with persistent effector T cells. Therapy with Z12-formulated vaccines prolonged survival in three robust tumor models, with the longest survival in an orthotopic model of aggressive brain cancer. Analysis of the tumor sites showed antigen-specific T-cell accumulation with favorable modulation of the balance of the immune infiltrate. Taken together, the results offered a preclinical proof of concept for the use of Z12-formulated vaccines as a versatile platform for the development of effective cancer vaccines. Cancer Res; 75(15); 3020–31. ©2015 AACR.

Legend for Supplementary Figures S1-S7.

Supplementary Figure 5 from Immune Infiltration of Spontaneous Mouse Astrocytomas Is Dominated by Immunosuppressive Cells from Early Stages of Tumor Development

We have previously shown preferential tumor-homing and therapeutic efficacy of adoptively transferred type 1 CTL (Tc1) when compared with type 2 CTL (Tc2) in mice bearing intracranial ovalbumin-transfected melanoma (M05). Further characterizing the expression of a panel of homing receptors on Tc1 and Tc2 cells, we found that very late antigen (VLA)-4 (a heterodimer of CD49d and CD29), but none of other receptors evaluated, was expressed at significantly higher levels on Tc1 cells than on Tc2 cells. Although CD49d (α4 integrin) can form heterodimers with both β1 (CD29) and β7 integrins, α4β7 complexes were not expressed by either Tc1 or Tc2 cells, suggesting that CD49d is solely expressed in VLA-4 complexes. VLA-4 expression on Tc2 cells was down-regulated in an interleukin (IL)-4 dose-dependent manner but not by other type 2 cytokines, such as IL-10 and IL-13, suggesting that IL-4 uniquely down-regulates VLA-4 expression on these cells. In accordance with the differential expression of VLA-4 on Tc1 versus Tc2 cells, Tc1 cells alone were competent to adhere to plate-bound VCAM-1-Ig fusion protein. Finally, the efficient trafficking of Tc1 cells into intracranial M05 lesions in vivo was efficiently blocked by administration of monoclonal antibodies against CD49d or VCAM-1 or small interfering RNA–mediated silencing of CD49d on Tc1 cells. Collectively, these data support the critical role of VLA-4 in the effective intracranial tumor homing of adoptive-transferred, antigen-specific Tc1 cells and suggest that more effective vaccine and/or ex vivo T-cell activation regimens may be developed by promoting the generation of VLA-4+ antitumor Tc1 cells. [Cancer Res 2007;67(13):6451–8]

Peptides and proteins used in the study, with all the minimal epitopes including 4 flanking residues (S1); Purified proteins were analyzed by Coomassie blue and Western Blot (S2); Z12 transports efficiently its cargo into the cytoplasm of DC (S3); Choice of adjuvant influences proportions of effector and memory phenotype antigen-specific CD8+ T cells induced after Z12-OVALP vaccination (S4); Persistance of antigen-specific cytotoxic T cells after vaccination (S5); Balance of brain tumor infiltrating lymphocytes is altered by Z12-OVALP vaccination (S6); Effector cytokine expression by brain tumor infiltrating CD8+ T cells is augmented by Z13-MultiE2 vaccination (S7).

Supplementary Figure 3 from Immune Infiltration of Spontaneous Mouse Astrocytomas Is Dominated by Immunosuppressive Cells from Early Stages of Tumor Development

Supplementary Figure 2 from Immune Infiltration of Spontaneous Mouse Astrocytomas Is Dominated by Immunosuppressive Cells from Early Stages of Tumor Development

Supplementary Figure 4 from Immune Infiltration of Spontaneous Mouse Astrocytomas Is Dominated by Immunosuppressive Cells from Early Stages of Tumor Development

Tissue programmed death ligand-1 (PD-L1) protein expression is predictive of immune checkpoint inhibitor (ICI) benefit. However, tissue PD-L1 can be fraught with tissue acquisition and heterogeneity limitations. Plasma testing can overcome these limitations. However, the overall survival (OS) predictive benefit of plasma PD-L1 assays have not been well characterized. Patients with stage IV non-small cell lung cancer (NSCLC) and plasma cfRNA PD-L1 by PCR expression were identified and assessed for OS. 16 patients treated with front-line ICI-based regimens were assessed and represented a real-world patient population with over half with a performance status of 2 or greater. 10 contemporaneous patients at the same institution treated with chemotherapy alone were also identified and assessed. With a median follow-up of 33 months, median OS was 13 months with a 30% 3-year OS for the ICI treated patients compared to a median OS of 3 months and a 10% 3-year OS for those treated with chemotherapy alone. Comparative log-rank test p-value = 0.014 and a hazard ratio 0.376 (95%-CI 0.134-1.057). Plasma cfRNA PD-L1 was associated with a statistically significant survival benefit from ICI-based treatment compared to chemotherapy in the first line treatment of a real-world patient population of advanced NSCLC.

Immune checkpoint inhibitors (ICIs) are a breakthrough in therapy for many solid malignancies. ICIs suppress the inhibitory signaling of several immune cell subtypes by the blocking the ligation of...

The original version of this article unfortunately contained a mistake. The second sentence of the section "irAEs and ICI efficacy" should read as.

Background Glioblastoma (GBM) is refractory to immune checkpoint inhibitor (ICI) therapy. We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed. Methods We used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally ICI-refractory (GBM and sarcoma) or ICI-responsive (renal cell carcinoma), as well as mouse models of GBM that are ICI-responsive (GL261) or ICI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations. Results ICI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells (DCs), and fewer myeloid cells, in particular PD-L1+ tumor-associated macrophages. The SB28 mouse model of GBM responded to ICI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying ICI resistance in GBM. The response to ICI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded DCs, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation. Conclusions Our data suggest that a major obstacle for effective immunotherapy of GBM is poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified DCs and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo.

Cancer vaccines based on synthetic peptides are a safe, well-tolerated immunotherapy able to specifically stimulate tumor-reactive T cells. However, their clinical efficacy does not approach that achieved with other immunotherapies such as immune checkpoint blockade. Nevertheless, major advances have been made in selecting tumor antigens to target, identifying epitopes binding to classical and non-classical HLA molecules, and incorporating these into optimal sized peptides for formulation into a vaccine. Limited potency of currently used adjuvants and the immunosuppressive tumor microenvironment are now understood to be major impediments to vaccine efficacy that need to be overcome. Rationally designed combination therapies are now being tested and should ultimately enable peptide vaccination to be added to immuno-oncology treatment options.

Background Advances in early diagnosis and curative treatment have reduced high mortality rates associated with non‐small cell lung cancer. However, racial disparity in survival persists partly because Black patients receive less curative treatment than White patients. Methods We performed a 5‐year pragmatic, trial at five cancer centers using a system‐based intervention. Patients diagnosed with early stage lung cancer, aged 18‐85 were eligible. Intervention components included: (1) a real‐time warning system derived from electronic health records, (2) race‐specific feedback to clinical teams on treatment completion rates, and (3) a nurse navigator. Consented patients were compared to retrospective and concurrent controls. The primary outcome was receipt of curative treatment. Results There were 2841 early stage lung cancer patients (16% Black) in the retrospective group and 360 (32% Black) in the intervention group. For the retrospective baseline, crude treatment rates were 78% for White patients vs 69% for Black patients (P

e21050 Background: Programmed death ligand-1 (PD-L1) expression is predictive of immunotherapy benefit. However, tissue PD-L1 protein immunohistochemical testing can be fraught with tissue acquisition and heterogeneity limitations. PD-L1 expression by RNA sequencing can be performed by both tissue and plasma with tissue PD-L1 protein correlations. What has not been well characterized is the correlation of plasma cell free circulating tumor RNA (cfRNA) PD-L1 and clinical outcomes with immunotherapy. Plasma cfRNA PD-L1 expression was evaluated and correlated with immunotherapy benefit in advanced non-small cell lung cancers (NSCLC). Methods: Patients with advanced NSCLC undergoing plasma next-generation sequencing including plasma cfRNA.PD-L1 testing in a Clinical Laboratory Improvement Amendments (CLIA) and College of American pathologists (CAP) accredited laboratory were retrospectively identified and evaluated at a single institution. Plasma PD-L1 positive patients underwent a de-identified chart abstraction to identify those patients with advanced NSCLC treated with front line immunotherapy regimens and those who received cytotoxic chemotherapy alone. Results: Sixteen patients with plasma PD-L1 expression treated with front-line immunotherapy regimens including single-agent immune checkpoint inhibitors, and combinatorial chemo-immune or chemo-immune-bevacizumab regimens were assessed for overall survival (OS). Eleven patients with plasma PD-L1 expression who received chemotherapy were used as a non-immunotherapy OS comparison. Median OS for the immunotherapy treated patients was thirteen months with a thirty percent three year landmark OS versus four months median OS and a ten percent three-year landmark OS for those treated with chemotherapy alone. Comparative log-rank test p-value 0.0091 and a hazard ratio of 0.36 (95%-CI 0.13-0.99). Conclusions: Plasma cfRNA PD-L1 expression is predictive of a statistically significant survival benefit from immunotherapy treatment compared to chemotherapy in the first line treatment of advanced NSCLC. The three year landmark OS of thirty percent parallels tissue PD-L1 directed immunotherapy-based treatment outcomes. The clinical utility of plasma cfRNA PD-L1 to overcome tissue acquisition and PD-L1 protein heterogeneity limitations and to study the dynamic nature of PD-L1 expression with non-immune cancer treatments and potential immunotherapy response monitoring are undergoing ongoing research.

Extracellular vesicle (EV) secretion is a ubiquitous cellular process with both physiologic and pathologic consequences. EVs are small lipid bilayer vesicles that encompass both microvesicles and exosomes and which are secreted by virtually all cells including cancer cells. In this review, we will focus on the roles of EVs in mediating the crosstalk between glioblastoma (GBM) cells and innate and adaptive immune cells and the potential impact on glioma progression. Glioma-derived EVs contain many bioactive cargoes that can broaden and amplify glioma cell mediated immunosuppressive functions and thereby contribute to shaping the tumor microenvironment. We will discuss evidence demonstrating that the low oxygen (hypoxia) in the GBM microenvironment, in addition to cell-intrinsic effects, can affect intercellular communication through EV release, raising the possibility that properties of the tumor core can more widely impact the tumor microenvironment. Recent advances in glioma-derived EV research have shown their importance not only as message carriers, but also as mediators of immune escape, with the capacity to reprogram tumor infiltrating immune cells. Exploring EV function in cancer-immune crosstalk is therefore becoming an important research area, opening up opportunities to develop EV monitoring for mechanistic studies as well as novel diagnostic glioma biomarker applications. However, robust and reproducible EV analysis is not always routinely established, whether in research or in clinical settings. Taking into account the current state of the art in EV studies, we will discuss the challenges and opportunities for extending the many exciting findings in basic research to a better interpretation of glioma and its response to current and future immunotherapies.

High-grade glioma is one of the most aggressive types of cancer with a low survival rate ranging from 12 to 15 months after the first diagnosis. Though being the most common strategy for glioma therapy, conventional chemotherapy suffers providing the therapeutic dosage of common therapeutics mostly because of limited permeability of blood–brain barrier (BBB), and blood–brain tumor barrier (BBTB) to anticancer agents. Among various nanoformulations, liposomes are considered as the most popular carriers aimed for glioma therapy. However, non-targeted liposomes which passively accumulate in most of the cancer tissues mainly through the enhanced permeation and retention effect (EPR), may not be applicable for glioma therapy due to BBB tight junctions. In the recent decade, the surface modification of liposomes with different active targeting ligands has shown promising results by getting different chemotherapeutics across the BBB and BBTB and leading them into the glioma cells. The present review discusses the major barriers for drug delivery systems to glioma, elaborates the existing mechanisms for liposomes to traverse across the BBB, and explores the main strategies for incorporation of targeting ligands onto the liposomes. It subsequently investigates the most recent and relevant studies of actively targeted liposomes modified with antibodies, aptamers, monosaccharides, polysaccharides, proteins, and peptides applied for effective glioma therapy, and highlights the common challenges facing this area. Finally, the actively targeted liposomes undergoing preclinical and clinical studies for delivery of different anticancer agents to glioma cells will be reviewed.

Purpose/Objective(s) Immune checkpoint inhibitors (ICIs) have been widely adapted as part of the treatment of locally advance and metastatic lung cancer. ICIs can lead to severe immunogenic related cardiotoxicities, however, the overall cardiotoxic profile of these agents when combined with thoracic radiation therapy is not well defined. Here, we assessed the risk of cardiotoxicity in ICI-treated lung cancer patients with or without cardiac radiation from thoracic radiotherapy. Materials/Methods Retrospective data was collected on Stage III-IV small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) patients who received ICIs between 2015 and 2018 at a large academic tertiary care hospital. All cardiotoxicities associated with ICI were assessed by a cardiologist and in correlation with history of thoracic radiotherapy (RT), the timing of RT in relation to ICI, and the mean RT heart dose. Multivariate analysis was performed on multiple logistic regression including the Framingham risk score and steroid use during the ICI therapy. Results Of 194 ICI-treated patients evaluated, 66 (34.02%) and 128 (65.98%) had Stage III and IV SCLC or NSCLC, respectively, the median age was 64 years (range, 36-88), and 42.47% were female. Within the total cohort, 55.2% (n = 107/194) patients had received thoracic RT at a median dose of 60.4 Gy (range, 15-75). Cardiotoxicities such as non-ST elevated myocardial infarction and new onset supraventricular tachycardias were observed in 13 (12.2%) of those who had thoracic RT versus 9 (10.3%) who did not (P = 0.87). There were 38 patients who received RT concurrently with ICI and did not develop any cardiotoxicity whereas cardiotoxicity was observed in 14.1% (n = 22/156) of those who did not receive concurrent RT (univariate, P = 0.030; multivariate, P = 0.055). There were no significant differences in the mean heart RT dose, Framingham risk score, and steroid treatment between patients that received concurrent RT with ICI versus non-concurrent RT/ICI. Conclusion ICI-related cardiotoxicities were not significantly associated with patients who received concurrent thoracic radiotherapy. Univariate analysis suggests a potential cardioprotective effect of thoracic RT with ICI's, which may be due to modulation of the immune response through reduction of lymphocytes involved in autoimmune processes. A larger cohort study will be conducted to further evaluate this potential cardioprotective effect.

Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and β1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles’ heel for a significant and unique subset of GBM patients., Seguin et al demonstrated in glioblastoma patient-derived stem cells that a subset of glioblastoma tumours is dependent on macropinocytosis mediated survival through a Galectin-3/RAB10/beta 1 integrin axis. They used both genetic and pharmacologic inhibition of Galectin-3 in vivo and in vitro to identify underlying mechanisms and define a Galectin-3/macropinocytosis molecular signature, which could inform the development of anti-tumour therapeutic strategies.

BackgroundGlioblastoma (GBM) is refractory to checkpoint blockade immunotherapy (CPI). We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed.MethodsWe used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally CPI-refractory (GBM and sarcoma) or CPI-responsive (renal cell carcinoma), as well as mouse models of GBM that are CPI-responsive (GL261) or CPI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations.ResultsCPI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells, and fewer myeloid cells, in particular PD-L1+ tumor associated macrophages. The SB28 mouse model of GBM responded to CPI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying CPI resistance in GBM. The response to CPI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded dendritic cells, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation.ConclusionsOur data suggest that a major obstacle for effective immunotherapy of GBM is the low antigenicity of the tumor cells coupled with poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified dendritic cells and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo.Graphical AbstractIn BriefIn mice and humans, tumors that were sensitive to checkpoint blockade had consistent immunological features. A mouse model of glioma that is refractory to checkpoint blockade was sensitized by increasing antigen presentation through a variety of approaches.

Tumor-associated macrophage (TAM) phagocytic activity is emerging as a new mechanism to harness for cancer treatment. Currently, many approaches are investigated at the preclinical level and some modalities have now reached clinical trials, including the targeting of the phagocytosis inhibitor CD47. The rationale for increasing TAM phagocytic activity is to improve innate anticancer immunity, and to promote T-cell mediated adaptive immune responses. In this context, a clear understanding of the impact of TAM phagocytosis on both innate and adaptive immunity is critical. Indeed, uncertainties persist regarding the capacity of TAM to present tumor antigens to CD8 T cells by cross-presentation. This process is critical for an optimal cytotoxic T-cell immune response and can be mediated by dendritic cells but also potentially by macrophages. In addition, the engulfment of cancer cells affects TAM functionality, as apoptotic cell uptake (a process termed efferocytosis) promotes macrophage anti-inflammatory functions. Because of the abundance of TAM in most solid tumors and the common use of apoptosis inducers such as radiotherapy to treat patients with cancer, efferocytosis potentially affects the overall immune balance within the tumor microenvironment (TME). In this review, we will discuss how cancer cell phagocytosis by TAM impacts antitumor immunity. First, we will focus on the potential of the phagocytic activity of TAM per se to control tumor progression. Second, we will examine the potential of TAM to act as antigen presenting cells for tumor specific CD8 T cells, considering the different characteristics of this process in the tumor tissue and at the molecular level. Finally, we will see how phagocytosis and efferocytosis affect TAM functionality and how these mechanisms impact on antitumor immunity. A better understanding of these aspects will enable us to better predict and interpret the consequences of cancer therapies on the immune status of the TME. Future cancer treatment regimens can thereby be designed to not only impact directly on cancer cells, but also to favorably modulate TAM phagocytic activity to benefit from the potential of this central immune player to achieve more potent therapeutic efficacy.

Introduction: The development of immune-related adverse events (irAEs) has been associated with improved efficacy of immune checkpoint inhibitors in patients with urothelial cancer, melanoma, and NSCLC. Whether this association exists in patients with SCLC is currently unknown. Methods: We conducted a multicenter retrospective study to evaluate the relationship between irAEs and immunotherapy efficacy in SCLC. To account for the lead-time bias resulting from the time-dependent nature of irAEs, the development of irAEs was considered as a time-varying covariate in univariate and multivariate Cox proportional hazard models. Results: Of the 183 patients treated with immunotherapy, 73 (39.9%) experienced at least one irAE. A total of 42 patients (22.9%) had grade 1 to 2 irAEs, whereas 31 patients (16.9%) had grade 3 to 4 irAEs. The median time of onset to the first irAE was 24 days (interquartile range: 14–55). The baseline clinicopathologic features were well-balanced between patients with and without irAEs. At a median follow-up of 24 months (95% confidence interval [CI]: 17.0–31.6), the median progression-free survival was significantly longer in the irAE group than the non-irAE group (3.8 versus 1.3 mo, p < 0.0001). The median overall survival was also significantly longer among patients with irAEs than patients without irAEs (13.8 versus 2.9 mo, p < 0.0001). When analyzed as a time-varying covariate, the development of irAEs was associated with a significant improvement in progression-free survival (hazard ratio: 0.44 [95% CI: 0.29–0.66], p < 0.001) and overall survival (hazard ratio: 0.47 [95% CI: 0.32–0.71], p < 0.001) in multivariate models. Conclusions: The development of irAEs is associated with improved clinical outcomes for immunotherapy in patients with advanced SCLC.

Background CRP is an acute-phase protein produced primarily in response to interleukin IL-6 via transcriptional activation of the STAT3. Recent data have provided mechanistic insights into the immune suppressive role of elevated CRP by elucidating its influence on effector T-cell function and antigen presentation.1 Furthermore, melanoma patients in Checkmate-064 and 067 with high baseline and on-treatment CRP were seen to have a lower response rate and shorter survival to immune checkpoint inhibitors (ICIs).2 Given these observations, we sought to evaluate the role of CRP as a prognostic biomarker in advanced NSCLC treated with ICIs from a multi-center international cohort. Methods Between 2015–2019, 420 adult patients with advanced NSCLC treated with ICIs alone or with concurrent chemotherapy (Chemo-ICI) were identified at four (1 US and 3 European) academic centers. CRP level in peripheral blood samples collected up to 2 weeks before starting ICI based treatments was considered as baseline. Based on previously validated data, a CRP cutoff of 10 mg/l was used to define CRP-normal (CRP-N) and CRP-high (CRP-H). Association of baseline CRP with median progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method and multivariate proportional hazards regression adjusted for multiple variables. Results Baseline CRP value was available in 75.5% of patients, with 66% having CRP-H. The median CRP was 21.0 mg/l. Single-agent nivolumab (44%) and Chemo-ICI (33.3%) were the two most common therapies. CRP-H showed a trend for stronger association with squamous histology (73.7% vs 63.3%; p= 0.063) and female sex (70.8 vs 60.0%; p=0.062) but did not show an association with PD-L1 status (0%, 1–49%, or ≥50%). Patients with CRP-H had a lower objective response rate compared with patients with CRP-N (26.9% vs. 47.6% PR; p=0.029). Compared to those with CRP-N (figure 1), patients with CRP-H had a significantly shorter median PFS [3.9 vs. 6.6 months, HR 1.41 95% CI: (1.07–1.86); p=0.0138] and OS (8.6 vs. 14.8 months, HR 1.55 95% CI [1.13- 2.14]; p=0.0060). In Cox regression analysis, CRP-H was again found to be independently associated with shorter median PFS and OS. Conclusions This is the largest international real-world dataset demonstrating significantly inferior outcomes associated with CRP > 10 mg/l in NSCLC patients treated with ICI based therapies. The potential influence of the immune suppressive effects of elevated CRP and IL-6 on the anti-tumor efficacy of ICIs needs prospective evaluation and could potentially be exploited as a therapeutic avenue in NSCLC. Acknowledgements Susan Eubanks and Sue-Ann Joyner at the ECU IRB for their help and support. Ethics Approval The primary IRB approval for this study was conducted under an ECU (P-MAIT- UMCIRB-15-001400). Individual approval was also obtained from the respective IRB of each participating institution. References Yoshida T, Ichikawa J, Giuroiu I, et al. C reactive protein impairs adaptive immunity in immune cells of patients with melanoma. Journal for ImmunoTherapy of Cancer 2020. Weber, et al. Journal of Clinical Oncology37, no. 15_suppl (May 20, 2019) 100–100

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, characterized by a high degree of intertumoral heterogeneity. However, a common feature of the GBM microenvironment is hypoxia, which can promote radio- and chemotherapy resistance, immunosuppression, angiogenesis, and stemness. We experimentally defined common GBM adaptations to physiologically relevant oxygen gradients, and we assessed their modulation by the metabolic drug metformin. We directly exposed human GBM cell lines to hypoxia (1% O2) and to physioxia (5% O2). We then performed transcriptional profiling and compared our in vitro findings to predicted hypoxic areas in vivo using in silico analyses. We observed a heterogenous hypoxia response, but also a common gene signature that was induced by a physiologically relevant change in oxygenation from 5% O2 to 1% O2. In silico analyses showed that this hypoxia signature was highly correlated with a perinecrotic localization in GBM tumors, expression of certain glycolytic and immune-related genes, and poor prognosis of GBM patients. Metformin treatment of GBM cell lines under hypoxia and physioxia reduced viable cell number, oxygen consumption rate, and partially reversed the hypoxia gene signature, supporting further exploration of targeting tumor metabolism as a treatment component for hypoxic GBM.

Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might affect the tumor microenvironment and sensitize cancer cells to immunotherapy. We used 2 glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti-programmed cell death 1 and anti-cytotoxic T lymphocyte-associated protein 4; anti-PD-1 and anti-CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade-resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T cell-independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti-PD-1 and anti-CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.

The original version of this article unfortunately contained a mistake. The second sentence of the section "irAEs and ICI efficacy" should read as.

Background Besides modeling/simulation-based analysis, no post-approval studies have evaluated optimal administration frequency of pembrolizumab in non-small cell lung cancer (NSCLC). Patients and Methods We performed a multicenter retrospective cohort study to evaluate association between survival outcomes and treatment extensions/delays of pembrolizumab-based regimens in advanced NSCLC patients. Those who had received at least four cycles in routine practice were divided into two groups: non-standard (Non-Std: ≥2 cycles at intervals >3weeks +3days) and standard (Std: all cycles every 3weeks or 1 cycle >3weeks +3days). Results Among 150 patients, 92 (61%) were eligible for the study (Non-Std:27, Std:65). Reasons for patients with extensions/delays in the Non-Std group included: immune-related adverse events (irAEs,33%), non-irAE-related medical issues (26%), and patient-physician preference (41%). Non-Std group was more likely to have higher PD-L1 tumor proportion score, higher number of treatment cycles and pembrolizumab monotherapy. Univariate and 6-month landmark analyses showed longer median overall survival (OS) and progression-free survival (PFS) in Non-Std group compared to the Std group. After multivariable adjustment for confounding factors, there was no significant difference in OS [HR 1.2 (95%C.I.: 0.3–4.8), p=0.824] or PFS [HR 2.6 (95%C.I.: 0.7–9.6), p=0.157] between the two groups. Conclusion Our study shows that a significant proportion of advanced NSCLC patients receive pembrolizumab-based regimens with extended intervals or delays in routine clinical practice and with similar outcomes to those receiving treatment at label-specified 3-week intervals. Given the durability of benefit seen and the potential for cost reduction and decreased infusion frequency in these patients, this requires validation in prospective trials., Highlights • The most cost-effective dosing frequency of pembrolizumab in advanced lung cancer is unknown. • We found delays/extensions of pembrolizumab-based treatment regimens in routine practice. • These delays/extensions were due to immune-related adverse events, medical issues or preferences. • Treatment delays/extensions were not associated with worse outcomes in advanced NSCLC. • Prospective trials are needed to evaluate extended pembrolizumab dosing regimens., The most cost-effective administration frequency of pembrolizumab in advanced non-small cell lung cancer (NSCLC) is unknown. We found that a significant proportion of these patients receive pembrolizumab-based regimens with extended intervals or delays in routine practice, with similar outcomes to those on label-specified 3-week interval treatments. Prospective evaluation of alternative dosing strategies is warranted to develop a more fiscally viable and patient-centered model.

As one of the deadliest diseases, cancer frequently resists existing therapeutics because they do not target all cells within a progressing tumor, for example both tumor stem and proliferating cells. This frequently results in enrichment of invasive and metastatic drug-resistant tumor cells subpopulations, cancer recurrence and eventually, patient mortality. Thus, there is an urgent need to identify specific markers, by which the targeted imaging and/or therapeutic "guided missile"-like agents can specifically detect and/or eradicate all cancer cells within a heterogeneous tumor, while leaving the normal cells intact. As a member of heat shock protein 70 (HSP70) superfamily, glucose regulated protein 78 (GRP78) has been documented as a molecular chaperone in the endoplasmic reticulum (ER) which mainly responds to ER stresses in normal cells. There is over-expression of GRP78 on the surface of cancer cells and angiogenic endothelial cells, which makes it a promising target for different types of peptides and antibodies that can be employed for targeted cancer therapy or imaging. In this review, we discuss the biological processes, functional importance and translocation mechanisms of cell surface GRP78 (csGRP78) in tumor cells. As a cancer biomarker, we also review the potential applications of csGRP78 targeted therapy and imaging and finally we suggest a brief roadmap ahead of csGRP78 targeting for targeted theranostic implications.

Concurrent chemoradiation (CRT) as the standard treatment for unresectable stage III non-small cell lung cancer (NSCLC) has reached a therapeutic plateau. Variations in the chemotherapy backbone and addition of consolidation chemotherapy have failed to improve survival (1). Attempts to escalate radiation dose have been met with failure as well (2). As a result, the 5-year overall survival of locally advanced NSCLC has stagnated at 30–40% over the past 10–15 years. While there has been an explosion of new therapeutic strategies for metastatic NSCLC in recent years, locally advanced NSCLC is very much in need of innovation.