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PD-L1 status assessed by immunohistochemistry (IHC) has failed to reliably predict outcomes for patients with metastatic urothelial carcinoma (mUC) on immune checkpoint blockade (ICB). PD-L1 promoter methylation is an epigenetic mechanism that has been shown to regulate PD-L1 mRNA expression in various malignancies. The aim of our present study was to evaluate the predictive potential of PD-L1 promoter methylation status (mPD-L1) in ICB-treated mUC compared to conventional IHC-based PD-L1 assessment. We quantified mPD-L1 in formalin-fixed and paraffin-embedded tissue sections using an established quantitative methylation-specific PCR assay (qMSP) in a well-characterized multicenter ICB-treated cohort comprising N = 107 patients with mUC. Additionally, PD-L1 protein expression in tumor tissues was assessed using regulatory approved IHC protocols. The effect of pharmacological hypomethylation by the DNA methyltransferase inhibitor decitabine in combination with interferon-γ stimulation in urothelial carcinoma cell lines was investigated by IHC and FACS. mPD-L1 hypomethylation predicted objective response rate at the first staging on ICB. Patients with tumors categorized as PD-L1 hypomethylated (lower quartile) showed significantly prolonged progression-free (PFS) and overall survival (OS) after ICB initiation. In contrast, PD-L1 protein expression status neither correlated with response nor survival. In multivariable Cox regression analyses, PD-L1 promoter hypermethylation remained an independent predictor of unfavorable PFS and OS. In urothelial carcinoma cell lines, pharmacological demethylation led to an upregulation of membranous PD-L1 expression and an enhanced inducibility of PD-L1 expression by interferon γ. Hypomethylation of the PD-L1 promoter is a promising predictive biomarker for response to ICB in patients with mUC. [ABSTRACT FROM AUTHOR]

The analysis of peripheral blood mononuclear cells (PBMCs) by flow cytometry holds promise as a platform for immune checkpoint inhibition (ICI) biomarker identification. Our aim was to characterize the systemic immune compartment in resectable esophageal adenocarcinoma patients treated with neoadjuvant ICI therapy. In total, 24 patients treated with neoadjuvant chemoradiotherapy (nCRT) and anti-PD-L1 (atezolizumab) from the PERFECT study (NCT03087864) were included and 26 patients from a previously published nCRT cohort. Blood samples were collected at baseline, on-treatment, before and after surgery. Response groups for comparison were defined as pathological complete responders (pCR) or patients with pathological residual disease (non-pCR). Based on multicolor flow cytometry of PBMCs, an immunosuppressive phenotype was observed in the non-pCR group of the PERFECT cohort, characterized by a higher percentage of regulatory T cells (Tregs), intermediate monocytes, and a lower percentage of type-2 conventional dendritic cells. A further increase in activated Tregs was observed in non-pCR patients on-treatment. These findings were not associated with a poor response in the nCRT cohort. At baseline, immunosuppressive cytokines were elevated in the non-pCR group of the PERFECT study. The suppressive subsets correlated at baseline with a Wnt/β-Catenin gene expression signature and on-treatment with epithelial–mesenchymal transition and angiogenesis signatures from tumor biopsies. After surgery monocyte activation (CD40), low CD8+Ki67+ T cell rates, and the enrichment of CD206+ monocytes were related to early recurrence. These findings highlight systemic barriers to effective ICI and the need for optimized treatment regimens. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the top three malignant tumors in terms of morbidity, and the limited efficacy of existing therapies urges the discovery of potential treatment strategies. Immunotherapy gradually becomes a promising cancer treatment method in recent decades; however, less than 10% of CRC patients could really benefit from immunotherapy. It is pressing to explore the potential combination therapy to improve the immunotherapy efficacy in CRC patients. It is reported that Farnesoid X receptor (FXR) is deficiency in CRC and associated with immunity. Herein, we found that GW4064, a FXR agonist, could induce apoptosis, block cell cycle, and mediate immunogenic cell death (ICD) of CRC cells in vitro. Disappointingly, GW4064 could not suppress the growth of CRC tumors in vivo. Further studies revealed that GW4064 upregulated PD-L1 expression in CRC cells via activating FXR and MAPK signaling pathways. Gratifyingly, the combination of PD-L1 antibody with GW4064 exhibited excellent anti-tumor effects in CT26 xenograft models and increased CD8+ T cells infiltration, with 33% tumor bearing mice cured. This paper illustrates the potential mechanisms of GW4064 to upregulate PD-L1 expression in CRC cells and provides important data to support the combination therapy of PD-L1 immune checkpoint blockade with FXR agonist for CRC patients. [ABSTRACT FROM AUTHOR]

Due to the high variance in response rates concerning anti-PD1 immunotherapy (IT), there is an unmet need to discover innovative biomarkers to predict immune checkpoint inhibitor (ICI)-efficacy. Our study included 62 Caucasian advanced-stage non-small cell lung cancer (NSCLC) patients treated with anti-PD1 ICI. Gut bacterial signatures were evaluated by metagenomic sequencing and correlated with progression-free survival (PFS), PD-L1 expression and other clinicopathological parameters. We confirmed the predictive role of PFS-related key bacteria with multivariate statistical models (Lasso- and Cox-regression) and validated on an additional patient cohort (n = 60). We find that alpha-diversity showed no significant difference in any comparison. However, there was a significant difference in beta-diversity between patients with long- (>6 months) vs. short (≤6 months) PFS and between chemotherapy (CHT)-treated vs. CHT-naive cases. Short PFS was associated with increased abundance of Firmicutes (F) and Actinobacteria phyla, whereas elevated abundance of Euryarchaeota was specific for low PD-L1 expression. F/Bacteroides (F/B) ratio was significantly increased in patients with short PFS. Multivariate analysis revealed an association between Alistipes shahii, Alistipes finegoldii, Barnesiella visceriola, and long PFS. In contrast, Streptococcus salivarius, Streptococcus vestibularis, and Bifidobacterium breve were associated with short PFS. Using Random Forest machine learning approach, we find that taxonomic profiles performed superiorly in predicting PFS (AUC = 0.74), while metabolic pathways including Amino Acid Synthesis and Fermentation were better predictors of PD-L1 expression (AUC = 0.87). We conclude that specific metagenomic features of the gut microbiome, including bacterial taxonomy and metabolic pathways might be suggestive of ICI efficacy and PD-L1 expression in NSCLC patients. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are promising tools for drug delivery across different biological barriers. Here, we evaluated the potential of EVs-mediated delivery of CD38 siRNA on the immunosuppression of hepatocellular carcinoma (HCC). EVs were isolated from bone marrow mesenchymal stem cell culture medium and loaded with CD38 siRNA to prepare EVs/siCD38. Loss-of-function assays were conducted to investigate the biological functions of EVs/siCD38 in HCC cells. Xenograft mouse models were performed for further validation. High CD38 expression was found in HCC. EVs/siCD38 inhibited CD38 enzyme activity, decreased adenosine production, and promoted macrophage repolarization to M1 type, thus inhibiting HCC cell growth and metastasis in vitro as well as tumor growth in mice. Mechanistically, CD38 was upregulated in mice resistant to PD-1/PD-L1 inhibitor and EVs/siCD38 reversed the resistance of tumor to PD-1/PD-L1 inhibitor in vivo. Our results provide functional evidence for the use of EV-mediated delivery of CD38 siRNA to prevent immunosuppression feature of HCC. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade (ICB) is a powerful oncologic treatment modality for a wide variety of human malignancies, but the patient response rate to this treatment remains low, especially in patients with cutaneous squamous cell carcinoma (cSCC). 5-Aminoleuvulinic acid-photodynamic therapy (ALAPDT) is widely used to treat cancerous and precancerous skin diseases, but the value of ALA-PDT in the treatment of invasive cSCC is debatable. Our previous studies have shown that ALA-PDT can induce antitumor immune responses by promoting the immunogenic death of tumor cells. However, it is unclear whether ALA-PDT exerts synergistic effects with ICB in cSCC. Here, we report that PD-L1 blockade potentiates the antitumor effects of ALA-PDT both on primary and distant tumors, and optimizes the tumor microenvironment in cSCC. In this study, we first detected PD-L1 expression in patients with different grades of cSCC. Then we found the combination of anti-PD-L1 monoclonal antibody (mAb) and ALA-PDT killed tumor cells by apoptosis- and/or ferroptosis-mediated immunogenic cell death (ICD) and stimulated systemic immune response, as well as building the immunological memory response to prevent tumor recurrence. Furthermore, we found that combination therapy can be used to recruit tertiary lymphoid structure (TLS)-like intratumoral lymphoid aggregates, which may promote tumorinfiltrating lymphocyte (TIL)-mediated antitumor immunity. In summary, our work demonstrates that ICB treatment with an anti-PD-L1 antibody is a promising strategy that may potentiate the antitumor effects of ALA-PDT in cSCC. [ABSTRACT FROM AUTHOR]

The poor progress of immunotherapy on osteosarcoma patients requires deeper delineation of immune tolerance mechanisms in the osteosarcoma microenvironment and a new therapeutic strategy. Clearance of apoptotic cells by phagocytes, a process termed "efferocytosis," is ubiquitous in tumors and mediates the suppression of innate immune inflammatory response. Considering the massive infiltrated macrophages in osteosarcoma, efferocytosis probably serves as a potential target, but is rarely studied in osteosarcoma. Here, we verified M2 polarization and PD-L1 expression of macrophages following efferocytosis. Pharmacological inhibition and genetic knockdown were used to explore the underlying pathway. Moreover, tumor progression and immune landscape were evaluated following inhibition of efferocytosis in osteosarcoma model. Our study indicated that efferocytosis promoted PD-L1 expression and M2 polarization of macrophages. Ëfferocytosis was mediated by MerTK receptor in osteosarcoma and regulated the phenotypes of macrophages through the p38/STAT3 pathway. By establishing the murine osteosarcoma model, we emphasized that inhibition of MerTK suppressed tumor growth and enhanced the T cell cytotoxic function by increasing the infiltration of CD8+ T cells and decreasing their exhaustion. Our findings demonstrate that MerTK-mediated efferocytosis promotes osteosarcoma progression by enhancing M2 polarization of macrophages and PD-L1-induced immune tolerance, which were regulated through the p38/STAT3 pathway. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is characterized by an accumulation of monoclonal plasma cells within the bone marrow (BM). Macrophages are an abundant component of myeloma BM microenvironment and support survival of the malignant cells and foster myeloma development and progression by suppression of the immune response. In our previous study, we found that MM patients overexpress pro-inflammatory cytokine interleukin-32 (IL-32). The present study aimed to investigate the role of IL-32 in myeloma progression and mechanisms of IL-32 on macrophages functions. We discovered that the expression of IL-32 was associated with the disease stage in myeloma patients. MM-derived exosomes containing IL-32γ promoted the expression of programmed death-ligand 1(PD-L1) by macrophages, thus promoting immune evasion. Mechanistically, myeloma-secreted IL-32γ acted via proteinase 3 (PR3) to enhance 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) dependent glycolysis and subsequent PD-L1 expression. Moreover, the PFKFB3-Janus kinase 1 (JAK1) axis might contribute to the expression of PD-L1 by macrophages. To sum up, we concluded that IL-32 was a critical mediator in myeloma progression, and targeting IL-32 signaling might be used as a potential strategy for treating myeloma. [ABSTRACT FROM AUTHOR]

Recent studies have revealed that chemokine-like factor-like MARVEL transmembrane domain-containing family member 6 (CMTM6) promotes tumor progression and modulates tumor immunity by regulating programmed death-ligand 1 stability; however, its intrinsic functions and regulatory mechanisms in clear cell renal cell carcinoma (ccRCC) remain poorly understood. Here, we show that CMTM6 is upregulated in ccRCC tissues and is strongly associated with advanced tumor grades, early metastases, and a worse prognosis. CMTM6 depletion significantly impaired the proliferation, migration, and invasion of ccRCC cells in vitro and in xenograft mouse models in vivo. In addition, targeting CMTM6 promotes anti-tumor immunity, represented by increased infiltration of CD4+ and CD8+ T cells in syngeneic graft mouse models. Further research revealed that loss of CMTM6 triggered aberrant activation of DNA damage response, resulting in micronucleus formation and G2/M checkpoint arrest, finally leading to cellular senescence with robust upregulation of numerous chemokines and cytokines. Our findings show for the first time the novel role of CMTM6 in maintaining cancer genome stability and facilitating tumor-mediated immunosuppression, linking DNA damage signaling to the secretion of inflammatory factors. Targeting CMTM6 may improve the treatment of patients with advanced ccRCC. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade (ICB) therapy, while groundbreaking, must be improved to promote enhanced durable responses and to prevent the development of treatment-refractory disease. Cancer therapies that engage, enable, and expand the antitumor immune response will likely require rationally designed combination strategies. Targeting multiple immunosuppressive pathways simultaneously may provide additional therapeutic benefit over singular targeting. We therefore hypothesized that the use of two molecules which inhibit three independent, but overlapping, pathways (TIGIT:CD155, PD-1/PD-L1, and TGFβ) would provide significant antitumor efficacy in the syngeneic ICB resistant colorectal tumor model MC38 expressing human carcinoembryonic antigen (CEA) in CEA transgenic mice. This novel combination treatment strategy has significant antitumor activity and survival benefit in two models of murine carcinomas, MC38-CEA (CRC) and TC1 (HPV+ lung carcinoma). MC38-CEA mice that responded to αTIGIT and bintrafusp alfa combination therapy generated memory responses and were protected from rechallenge. These effects were dependent on CD4+ and CD8+ T cells, as well as increased immune infiltration into the TME. This combination induced production of tumor-specific CD8+ T cells, and an increase in activation and cytotoxicity resulting in an overall activated immune landscape in the tumor. Data presented herein demonstrate the αTIGIT and bintrafusp alfa combination has efficacy across multiple tumor models, including the checkpoint-resistant model of murine colon carcinoma, MC38- CEA and the HPV+ model TC-1. [ABSTRACT FROM AUTHOR]

Metabolic inhibition via PFKFB3 inhibition has demonstrated considerable tumor inhibitory effects in various studies; however, PFKFB3 inhibition did not show satisfactory tumor inhibition when used in clinical trials. PFKFB3 is a crucial metabolic enzyme that is highly upregulated in cancer cells and directly affects tumor glycolysis. Here, we showed that PFKFB3 inhibition suppresses tumors in vitro and in vivo in immune-deficient xenografts. However, this inhibition induces the upregulation of PD-L1 levels, which inactivated cocultured T-cells in vitro, compromises anti-tumor immunity in vivo, and reduced anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of PFKFB3 inhibition in immunocompetent and hu-PBMC NOG mouse models. Mechanistically, PFKFB3 inhibition increases phosphorylation of PFKFB3 at residue Ser461, which increases interaction with HIF-1a, and their colocalization into the nucleus, where HIF-1a transcriptionally upregulate PD-L1 expression and causes subsequent tumor immune evasion. Higher phos-PFKFB3 correlated with higher PD-L1 expression, lower CD8 and GRZMB levels, and shorter survival time in ESCC patients. [ABSTRACT FROM AUTHOR]

Immunostimulatory regimens are a game changer in the fight against cancer, but still only a minority of patients achieve clinical benefit. Combination with immunomodulatory drugs and agents converting otherwise non-immunogenic forms of cell death into bona fide "immunogenic cell death" (ICD) could improve the efficacy of these novel therapies. The aim of our study was to investigate conventional Amphotericin B (AmB) as an enhancer of antitumor immune responses. In tumor cell line models, AmB induced ICD with its typical hallmarks of calreticulin (CALR) expression and release of high mobility group box 1 (HMGB1) as well as Adenosine 5'-triphosphate (ATP). Interestingly, in contrast to non-ICD inducing treatments, ICD induction led to up-regulation of PD-L1-expression by ICD experiencing cells, resulting in decreased maturation of dendritic cells (DCs). Blocking this PD-L1 expression on tumor cells could unleash full ICD effects on antigen presenting cells. Even at sub-toxic concentrations, AmB was able to enhance CALR on leukemic blasts, particularly on phagocytic monoblastic THP-1 cells, which also showed features of "M1-like" differentiation after AmB exposure. The ability of AmB to increase the immunogenicity of tumor cells was confirmed in vivo in a mouse vaccination experiment. In conclusion, we demonstrate that AmB can promote antitumor immune responses in a dose-dependent manner by ICD induction, surface translocation of CALR on leukemic blasts even at sub-toxic concentrations, and "M1-like" polarization of phagocytic cells, making it noteworthy as potential booster for cancer immunotherapy. We additionally report for the first time that PD-L1 expression may be a feature of ICD, possibly as a negative feedback mechanism regulating the maturation status of DCs and thus indirectly affecting T-cell priming. [ABSTRACT FROM AUTHOR]

Blockade of checkpoint receptors with monoclonal antibodies against CTLA-4, PD-1 and PD-L1 has shown great clinical success in several cancer subtypes, yielding unprecedented responses albeit a significant number of patients develop resistance and remain refractory. Both PD-1/PD-L1 and HER-2 signaling pathway inhibitors have limited efficacy and exhibits significant toxicities that limit their use. Ongoing clinical studies support the need for rationale combination of immuno-oncology agents to make a significant impact in the lives of cancer patients. We introduce the development of a novel chimeric PDL1 B-cell peptide epitope vaccine (amino acid 130-147) linked to a "promiscuous" T cell measles virus fusion (MVF) peptide (MVF-PD-L1(130); PDL1-Vaxx) or linked to tetanus toxoid (TT3) TT3-PD-L1 (130) via a linker (GPSL). These vaccine constructs are highly immunogenic and antigenic in several syngeneic animal models. The PD-L1 vaccines elicited high titers of polyclonal antibodies that inhibit tumor growth in multiple syngeneic cancer models, eliciting antibodies of different subtypes IgG1, IgG2a, IgG2b and IgG3, induced PD-1/PD-L1 blockade, decreased proliferation, induced apoptosis and caused ADCC of tumor cells. The PDL1-Vaxx induces similar inhibition of tumor growth versus the standard anti-mouse PDL1 antibody in both syngeneic BALB/c and C57BL/6J mouse models. The combination of PDL1-Vaxx with HER-2 vaccine B-Vaxx demonstrated synergistic tumor inhibition in D2F2/E2 carcinoma cell line. The anti-PDL1-Vaxx block PD-1/PD-L1 interaction and significantly prolonged anti-tumor responses in multiple syngeneic tumor models. The combination of HER-2 vaccine (B-Vaxx) with either PDL1-Vaxx or PD1-Vaxx demonstrated synergistic tumor inhibition. PDL1-Vaxx is a promising novel safe checkpoint inhibitor vaccine. [ABSTRACT FROM AUTHOR]

Various predictive biomarkers are needed to select candidates for optimal and individualized treatments. Tumor-infiltrating immune cells have gained increasing interest in cancer research for the prediction of therapeutic response and survival. However, the role of dendritic cells (DCs) in PD-1 blockade immunotherapy remains unclear. In this study, we identified a population of PD-1+ DCs in the tumor microenvironment (TME) of cervical cancer (CC). The accumulation of PD-1+ DCs in cervical tumors was correlated with advanced stages, elevated preoperative squamous cell carcinoma antigen levels and lymph-vascular space invasion. PD-1 expression was induced on activated tumor-associated DCs (TADCs) in vitro compared with their resting counterparts. This PD-1+ DC population was characterized by reduced secretion of cytokines (IL-12, TNF-α, and IL-1β) and dysfunctional induction of T cell proliferation and cytotoxic reaction. PD-1 blockade significantly reinvigorated PD-1+ DCs to release IL-12, TNF-α, and IL-1β compared with PD-1-DCs. TILs from samples with higher PD-1+ DC infiltration could be induced to achieve a greater killing effect of PD-1 blockade treatment. Our findings suggested a role for PD-1+ DCs in immune surveillance dysfunction and CC progression. PD-1+ DC density in the TME may serve as a diagnostic factor for predicting the optimal beneficiaries of PD-1/PD-L1 blockade immunotherapy in CC. [ABSTRACT FROM AUTHOR]

The effect of anti-programmed cell death 1 (PD-1) antibody in Epstein-Barr virus-associated gastric cancer (EBVaGC) was debatable, and no predictive biomarkers for efficacy have been reported. Public reports on anti-PD-1 antibody monotherapy-treated EBVaGC with available programmed death ligand-1 (PD-L1) expression status were summarized and analyzed. Relevance with clinicopathologic characteristics of PD-L1 expression by immunohistochemistry was analyzed in 159 patients diagnosed with EBVaGC. Relevance with genomic transcriptome and mutation profile of PD-L1 status in EBVaGC was assessed with three datasets, the cancer genome atlas (TCGA), Gene Expression Omnibus (GEO) GSE51575, and GSE62254. Based on the data from 8 reports, patients with positive PD-L1 expression (n = 30) had significantly superior objective response rate (ORR) than patients with negative PD-L1 expression (n = 9) (63.3% vs. 0%, P = .001) in EBVaGC receiving anti-PD-1 antibody monotherapy. PD-L1 positivity was associated with less aggressive clinicopathological characteristics and was an independent predictor for a longer disease-free survival (hazard ratio [HR] and 95% CI: 0.45 [0.22-0.92], P = .03) and overall survival (HR and 95% CI: 0.17 [0.06-0.43], P < .001). Analysis of public EBVaGC transcriptome and mutation datasets revealed enhanced immune-related signal pathways in PD-L1high EBVaGC and distinct mutation patterns in PD-L1low EBVaGC. PD-L1 positivity indicates a subtype of EBVaGC with 'hot' immune microenvironment, lower aggressiveness, better prognosis, and higher sensitivity to anti-PD-1 immunotherapy. [ABSTRACT FROM AUTHOR]

Although PD-1/PD-L1 blockade therapy confers salutary effects across cancer types, their efficacy in Extranodal Natural killer/T-cell lymphoma (ENKTCL) patients is limited and unpredictable. Here, we comprehensively evaluated the expression profile of a panel of immune-regulatory makers to identify novel prognostic biomarkers and/or therapeutic targets for this malignancy. Using immunohistochemistry and multiplex immunofluorescence, we found that the expression of VISTA (88.1%) was predominantly in CD68+ macrophages and much higher than PD-L1 expression (68.7%) in ENKTCL. B7-H4 and HHLA2 proteins were not detected in ENKTCL. B7-H3 was expressed in minority of ENKTCL patients (13.7%) and mainly colocalized with CD31. A close correlation was detected between VISTA and PD-L1, but they were not co-expressed in the same cells. High expressions of VISTA or PD-L1 were significantly associated with detrimental clinicopathological characteristics, dismal prognosis, and high density of CD8+ TILs, and high VISTA expression was also significantly associated with high density of Foxp3+ TILs. VISTA combined with PD-L1 was an independent prognostic factor for PFS and OS. Moreover, the patients with high VISTA showed a poor response to PD-1 blockades in ENKTCL. In conclusion, these findings provide a rationale for VISTA as an ideal immunotherapeutic target next to PD-L1 for ENKTCL. [ABSTRACT FROM AUTHOR]

Perioperative chemotherapy enhances the survival rates for patients with esophageal and gastric (EG) adenocarcinoma, but not all patients benefit from this additional treatment. Chemotherapeutic agents have been demonstrated to alter the immune cell (IC) composition in the tumor microenvironment. Hence, there is a rationale to investigate the influence of neoadjuvant chemotherapy (NAC) on different IC subsets, to better understand and compare their utility as complementary prognostic or predictive biomarkers in a clinically relevant context. The density of T cells (CD8+ and FoxP3+), B cells (CD20+) and the expression of PD-L1 on ICs and tumor cells (TC) was assessed by immunohistochemistry on paired biopsies from primary tumors (PT) pre-NAC, and resected PT and lymph node metastases post-NAC. The cohort encompasses 148 patients with resectable EG adenocarcinoma, all of whom received NAC. The density of CD8+ cells was decreased and the density of FoxP3+ cells and CD20+ cells was increased in PT post-NAC. PD-L1 expression was not altered following NAC. In pre-NAC specimens, high FoxP3+ density and high PD-L1 expression on ICs were favorable prognostic factors, whereas high CD8+ density was an unfavorable prognostic factor. In post-NAC specimens, however, high FoxP3+ density was an unfavorable prognostic factor, and high PD-L1 expression on TC was associated with a shorter survival. There were no significant associations between IC density or PD-L1 expression in PT pre-NAC and histopathological regression. These findings propose that NAC might alter the density and prognostic impact of some IC subsets in EG adenocarcinoma. [ABSTRACT FROM AUTHOR]

Anti-PD1/PD-L1-directed immune checkpoint inhibitors are game changers in advanced non-small-cell lung cancer, but biomarkers are lacking. The aim of our study was to find clinically relevant biomarkers of the efficacy of ICI in non-squamous NSCLC. We conducted a retrospective study of patients receiving ICI for advanced non squamous NSCLC in two cohorts. For a subset of patients, RNAseq data were generated on tumor biopsy taken before ICI. The primary end point was progression-free survival under ICI. Secondary end point was overall survival from ICI initiation. In the cohort, we studied 231 patients. Clinico-pathological characteristics included KRAS mutant status (n = 88), TTF1-positive expression (n = 136), LIPI (Lung Immune Prognostic Index) score of 0 (n = 116). In our cohort, lack of TTF1 expression, LIPI score >0, line of treatment >1, and liver metastases were associated with poorer PFS. TTF1 and PD-L1 status could be used to stratify survival and improve the AUC for prediction of prognosis in comparison with the PD-L1 gold standard. Using an external cohort of 154 patients, we confirmed the independent prognostic role of TTF1. TTF1 expression and PD-L1 can be used to stratify risk and predict PFS and OS in patients treated with ICI for NS-NSCLC. [ABSTRACT FROM AUTHOR]

Only a small subset of colorectal cancer (CRC) patients benefits from immunotherapies, comprising blocking antibodies (Abs) against checkpoint receptor "programmed-cell-death-1" (PD1) and its ligand (PD-L1), because most cases lack the required mutational burden and neo-antigen load caused by microsatellite instability (MSI) and/or an inflamed, immune cell-infiltrated PD-L1+ tumor microenvironment. Peroxisome proliferator-activated-receptor-gamma (PPARγ), a metabolic transcription factor stimulated by anti-diabetic drugs, has been previously implicated in pre/clinical responses to immunotherapy. We therefore raised the hypothesis that PPARγ induces PD-L1 on microsatellite stable (MSS) tumor cells to enhance Ab-target engagement and responsiveness to PD-L1 blockage. We found that PPARγ-agonists upregulate PD-L1 mRNA/protein expression in human gastrointestinal cancer cell lines and MSS+ patient-derived tumor organoids (PDOs). Mechanistically, PPARγ bound to and activated DNA-motifs similar to cognate PPARγ-responsive-elements (PPREs) in the proximal -2 kb promoter of the human PD-L1 gene. PPARγ-agonist reduced proliferation and viability of tumor cells in co-cultures with PD-L1 blocking Ab and lymphokine-activated killer cells (LAK) derived from the peripheral blood of CRC patients or healthy donors. Thus, metabolic modifiers improved the antitumoral response of immune checkpoint Ab, proposing novel therapeutic strategies for CRC. [ABSTRACT FROM AUTHOR]

Background: Pegylated arginine deiminase (ADI-PEG 20) is a metabolism-based strategy that depletes arginine, resulting in tumoral stress and cytotoxicity. Preclinically, ADI-PEG 20 modulates T-cell activity and enhances the therapeutic efficacy of programmed death-1 (PD-1) inhibition. Methods: A phase 1b study, including a dose-escalation cohort and an expansion cohort, was undertaken to explore the effects of ADI-PEG 20 in combination with pembrolizumab, an anti-PD-1 antibody, for safety, pharmacodynamics, and response. CD3 levels and programmed death-ligand 1 (PD-L1) expression were assessed in paired biopsies collected prior to and after ADI-PEG 20 treatment but before pembrolizumab. Results: Twenty-five patients, nine in the dose-escalation cohort and sixteen in the expansion cohort, were recruited. Treatment was feasible with adverse events consistent with those known for each agent, except for Grade 3/4 neutropenia which was higher than expected, occurring in 10/25 (40%) patients. Mean arginine levels were suppressed for 1-3 weeks, but increased gradually. CD3+ T cells increased in 10/12 (83.3%) subjects following ADI-PEG 20 treatment, including in three partial responders (p = .02). PD-L1 expression was low and increased in 3/10 (30%) of subjects. Partial responses occurred in 6/25 (24%) heavily pretreated patients, in both argininosuccinate synthetase 1 proficient and deficient subjects. Conclusions: The immunometabolic combination was safe with the caveat that the incidence of neutropenia might be increased compared with either agent alone. ADI-PEG 20 treatment increased T cell infiltration in the low PD-L1 tumor microenvironment. The recommended phase 2 doses are 36 mg/m² weekly for ADI-PEG 20 and 200 mg every 3 weeks for pembrolizumab. [ABSTRACT FROM AUTHOR]

Anti-programmed cell death-1 (PD-1)/PD-ligand-1 (PD-L1) treatments are effective in a fraction of patients with advanced malignancies. However, the majority of patients do not respond to it. Resistance to cancer immunotherapy can be mediated by additional immune checkpoints. We hypothesized that co-targeting of PD-L1 and lymphocyte-activation gene 3 (LAG-3) could provide an alternative therapeutic approach. Here, we developed IBI323, a dual blockade bispecific antibody targeting PD-L1 and LAG-3. We assessed the binding affinity, blocking activity, cell bridging effect, and immunomodulation function of IBI323 using in vitro assays. We also evaluated, in two humanized mouse models, anti-tumor effects and antitumor T cell immunity induced by IBI323. IBI323 bound to PD-L1 and LAG-3 with similar potency as its parental antibodies and blocked the interaction of PD-1/PD-L1, CD80/PD-L1, and LAG-3/MHC-II. Moreover, IBI323 mediated the bridging of PD-L1+ cells and LAG-3+ cells and demonstrated superior immune stimulatory activity compared to each parent antibody in mixed leukocyte reaction. In PD-L1/LAG-3 double knock-in mice bearing human PD-L1 knock-in MC38 tumors, IBI323 showed stronger anti-tumor activity compared to each parental antibody. The better antitumor response correlated with increased tumor-specific CD8+ and CD4+ T cells. IBI323 also induced stronger anti-tumor effect against established A375 tumors compared with combination in mice reconstituted with human immune cells. Collectively, these data demonstrated that IBI323 preserved the blockade activities of parental anti-bodies while processing a novel cell bridging function. Based on the encouraging preclinical results, IBI323 has significant value in further clinical development. [ABSTRACT FROM AUTHOR]

Both pembrolizumab (P) and combination of pembrolizumab with platinum-based chemotherapy (PCT) represent standard 1st-line options for advanced non-small cell lung cancer (aNSCLC) with PD-L1 tumor proportion score (TPS) ≥50%. The two strategies have never been compared in a randomized trial. 256 consecutive patients with EGFR/ALK/ROS1 -wild-type PD-L1 TPS ≥50% aNSCLC receiving P (group P, n = 203) or PCT (group PCT, n = 53) as a 1st-line treatment were identified in the electronic databases of 4 Israeli cancer centers. Time-to-treatment discontinuation (TTD) and overall survival (OS) were assessed. Baseline characteristics were well balanced, except for age and ECOG PS differences in favor of group PCT. Median (m)TTD was 4.9 months (mo) (95% CI, 3.1-7.6) vs 8.0mo (95% CI, 4.7-15.6) (p-0.09), mOS was 12.5mo (95% CI, 9.8-16.4) vs 20.4mo (95% CI, 10.8-NR) (p-0.08), with P and PCT, respectively. In the propensity score matching analysis (n = 106; 53 patients in each group matched for age, sex and ECOG PS), mTTD was 7.9mo (95% CI, 2.8-12.7) vs 8.0mo (95% CI, 4.7-15.6) (p-0.41), and mOS was 13.3mo (95% CI, 6.8-20.3) vs 20.4mo (95% CI, 10.8-NR) (p-0.18), with P and PCT, respectively. Among various subgroups of patients examined, only in females (n = 86) mOS differed significantly between treatments (10.2mo (95% CI, 6.8-17.2) with P vs NR (95% CI, 11.4-NR) with PCT; p-0.02). In the real-world setting, no statistically significant differences in long-term outcomes with P vs PCT were observed; a prospective randomized trial addressing the comparative efficacy of P and PCT in different patient subgroups is highly anticipated. [ABSTRACT FROM AUTHOR]

The efficacy of PD-1/PD-L1 blockades is heterogeneous in different molecular subtypes of gastric cancer (GC). In this study, we analyzed relevant clinical trials to identify the molecular subtypes associated with the efficacy of PD-1/PD-L1 blockades, and public datasets, patient samples, and GC cell lines were used for investigating potential mechanisms. We found that GC with EBV-positive, MSI-H/dMMR, TMB-H or PIK3CA mutant subtype had enhanced efficacy of PD-L1/PD-1 blockades. Also, differentially expressed genes of these molecular subtypes shared the same gene signature and functional annotations related to immunity. Meanwhile, CIBERSORT identified that the overlapping landscapes of tumor-infiltrating immune cells in the four molecular subtypes were mainly M1-like macrophages (M1). The relationships between M1 and clinical characteristics, M1, and gene signatures associated with PD-1/PD-L1 blockades also revealed that M1 was associated with improved prognosis and required for the efficacy of PD-L1/PD-1 blockades in GC. We identified that tumor-infiltrating CD68+CD163- macrophages could represent M1 calculated by CIBERSORT in clinical application, and CXCL9, 10, 11/CXCR3 axis was involved in the mechanism of CD68+CD163- macrophages in the enhanced efficacy of PD-L1/PD-1 blockades. In conclusion, CD68+CD163- macrophages are required for the efficacy of PD-L1/PD-1 blockades and expand the applicable candidates in GC patients without the molecular subtypes. [ABSTRACT FROM AUTHOR]

Although previous studies suggest that cancer cachexia is a poor prognostic factor for immune checkpoint inhibitor monotherapy, the impact of cancer cachexia on chemoimmunotherapy is unclear. We investigated the impact of cancer cachexia on the therapeutic outcomes of chemoimmunotherapy for non-small cell lung cancer (NSCLC). We retrospectively analyzed patients' medical records with NSCLC who received chemoimmunotherapy in 12 institutions in Japan between January and November 2019. We defined cancer cachexia as weight loss exceeding 5% of the total body weight or a body mass index of < 20 kg/m² and weight loss of more than 2% of the total body weight within 6 months before chemoimmunotherapy initiation, with laboratory results exceeding reference values. This study enrolled 235 patients with NSCLC, among whom 196 were eligible for analysis, and 50 (25.5%) met the criteria for cachexia diagnosis. Patients with cancer cachexia had a significantly higher frequency of a programmed death-ligand 1 (PD-L1) expression of > 50% (48%, p = .01) and shorter progression-free survival (PFS; log-rank test: p = .04) than patients without cachexia. There was no significant difference in overall survival (OS) between the cachexia and no-cachexia groups (log-rank test: p = .14). In the PD-L1 > 50% population, there was no significant difference in PFS and OS (log-rank test: p = .19 and p = .79, respectively) between patients with NSCLC in the cachexia or no-cachexia groups. Cancer cachexia might be a poor prognostic factor in patients with NSCLC receiving chemoimmunotherapy. [ABSTRACT FROM AUTHOR]

Most patients with advanced or metastatic urothelial carcinoma do not benefit significantly from Immune checkpoint inhibitors (ICIs) use. A systematic review and meta-analysis of randomized controlled trials to assess the efficacy and activity of ICIs, in terms of Overall Survival (OS), Progression-free survival (PFS), and Objective Response Rate (ORR). We systematically searched for articles from PubMed, Cochrane Library, Embase, and Web of science from their inception to December 1, 2020 with no language restrictions. The search was performed to identify all clinical trials (phase I, phase II, phase III) of ICIs for treating urothelial carcinoma. The endpoints of the meta-analysis were OS, PFS, and ORR, compared unselected patients and in the subgroup of patients characterized by high expression of PD-L1 (PD-L1 selected patients). Sixteen studies comprising 5559 patients were identified, of which data for OS comparison were available from 4 RCTs (2342 patients), two studies for PFS (649 patients), and four RCTs were eligible for ORR analysis (2921 patients). Both pembrolizumab and atezolizumab have showed to improve OS compared to chemotherapy in unselected patients (HR 0.86, 95% CI 0.80-0.93, P = .0001, I² = 60%), while the difference was not significant in PD-L1 selected patients (HR 0.91, 95% CI 0.77-1.07, P = .23, I² = 0%). PFS difference was not observed in neither unselected population nor PD-L1 selected patients, the pooled HR of PFS for immunotherapy compared to control treatment was 1.05 (95% CI 0.74-1.49, P = .79, I² = 85%) and 0.84 (95% CI 0.68-1.03, P = .09, I² = 0%, respectively. Similar result was observed in ORR, the pooled HR of ORR for immunotherapy compared to control treatment was 1.45 (95% CI 0.53-3.98, P = .47, I² = 95%) and 2.19 (95% CI 0.79-6.08, P = .13, I² = 83%), respectively. Immunotherapy could significantly improve survival advantage in unselected patients but not in PD-L1 selected population, indicating that PD-L1 expression may not be a reliable marker in previously platinum-treated patients. [ABSTRACT FROM AUTHOR]

Co-stimulatory 4-1BB receptors on tumor-infiltrating T cells are a compelling target for overcoming resistance to immune checkpoint inhibitors, but initial clinical studies of 4-1BB agonist mAbs were accompanied by liver toxicity. We sought to engineer a tri-specific antibody-based molecule that stimulates intratumoral 4-1BB and blocks PD-L1/PD-1 signaling without systemic toxicity and with clinically favorable pharmacokinetics. Recombinant fusion proteins were constructed using scMATCH3 technology and humanized antibody single-chain variable fragments against PD-L1, 4-1BB, and human serum albumin. Paratope affinities were optimized using single amino acid substitutions, leading to design of the drug candidate NM21-1480. Multiple in vitro experiments evaluated pharmacodynamic properties of NM21-1480, and syngeneic mouse tumor models assessed antitumor efficacy and safety of murine analogues. A GLP multiple-dose toxicology study evaluated its safety in non-human primates. NM21-1480 inhibited PD-L1/PD-1 signaling with a potency similar to avelumab, and it potently stimulated 4-1BB signaling only in the presence of PD-L1, while exhibiting an EC50 that was largely independent of PD-L1 density. NM21-1480 exhibited high efficacy for co-activation of pre-stimulated T cells and dendritic cells. In xenograft models in syngeneic mice, NM21-1480 induced tumor regression and tumor infiltration of T cells without causing systemic T-cell activation. A GLP toxicology study revealed no evidence of liver toxicity at doses up to 140 mg/kg, and pharmacokinetic studies in non-human primates suggested a plasma half-life in humans of up to 2 weeks. NM21-1480 has the potential to overcome checkpoint resistance by co-activating tumor-infiltrating lymphocytes without liver toxicity. [ABSTRACT FROM AUTHOR]

Radiotherapy can boost the therapeutic response to immune checkpoint inhibitors (ICIs) by recruiting T lymphocytes and upregulating PD-L1 expression within the tumor microenvironment (TME). However, in some cases, tumor PD-L1 expression cannot be induced, even in the presence of abundant T lymphocytes, in locally advanced colorectal cancer patients who receive preoperative neoadjuvant concurrent chemoradiotherapy (CCRT). In this study, we found that PD-L1 promoter methylation is negatively correlated with tumor PD-L1 expression and is an independent biomarker for locally advanced colorectal cancer patients. PD-L1 methylation (mCD274) was significantly associated with shorter disease-free survival (cg15837913 loci, p = .0124). By multivariate Cox proportional hazards analyses including influent factors, mCD274 was classified as an independent prognostic factor for poor 5-year DFS [cg15837913, hazard ratio: HR = 4.06, 95% CI = 1.407-11.716, p = .01]. We found that the immunomodulatory agent DNA methyltransferase inhibitor (DNMTi) led to demethylation of the PD-L1 promoter and increased radiotherapy-induced PD-L1 upregulation via interferon β (IFNβ). DNMTi not only induced tumor PD-L1 expression but increased the expression of immune-related genes as well as intratumoral T cell infiltration in vivo. Furthermore, DNMTi strongly enhanced the response to combined treatment with radiotherapy and anti-PD-L1 inhibitors, and prolonged survival in microsatellite stability (MSS) colorectal model. Therefore, DNMTi remodeled the tumor microenvironment to improve the effect of radiotherapy and anti-PD-L1 immunotherapy by directly triggering tumor PD-L1 expression and eliciting stronger immune responses, which may provide potential clinical benefits to colorectal cancer patients in the future. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are considered to be related to the prognosis of cancer patients. CTC is a powerful indicator for recurrence or metastasis. The relationship, however, between the expression of programmed cell death receptor ligand 1 (PD-L1) on CTCs in peripheral blood and the prognosis, is still controversial. Here, we conducted a meta-analysis to evaluate its prognostic value. A total of 20 articles were screened from PubMed, Embase, Cochrane, China National Knowledge Internet (CNKI) and WanFang Database, and the Hazard Ratio (HR) along with 95% confidence intervals (CIs) of each article were combined to study the relationship between PD-L1 expression on CTCs and prognosis. The expression of PD-L1 on CTCs in the peripheral blood of cancer patients is associated with poor prognosis. The pooled HRs for overall survival (OS) in cancer patients were 1.85 (95% CI, 1.29-2.66, P = .001). The pooled HRs for progression-free survival (PFS) in cancer patients were 1.50 (95% CI, 1.12-2.01; P = .007). This is the first meta-analysis to clarify the expression of PD-L1 on CTCs at baseline affects the prognosis of cancer patients. Patients with CTCs expressing PD-L1 had a shorter survival time than patients with CTCs not expressing PD-L1. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a hematological malignancy characterized by the presence of clonal plasma cells in the bone marrow niche. Despite significant therapeutic advances, MM remains incurable for the majority of patients. Targeted radionuclide therapy (TRNT) has emerged as a promising treatment option to eradicate residual cancer cells. In this study, we developed and characterized single-domain antibodies (sdAbs) against the MM-antigen CS1 and evaluated its therapeutic potential in MM using TRNT. We first validated CS1 as potential target for TRNT. CS1 is expressed in normal and malignant plasma cells in different disease stages including progression and relapse. It is expressed in dormant as well as proliferating MM cells, while low expression could be observed in environmental cells. Biodistribution studies demonstrated the specific uptake of anti-CS1 sdAbs in tissues of 5TMM cell infiltration including bone, spleen and liver. TRNT using anti-CS1 sdAbs labeled with actinium-225 significantly prolonged survival of syngeneic, immunocompetent 5T33MM mice. In addition, we observed an increase in CD8+ T-cells and more overall PD-L1 expression on immune and non-immune cells, implying an interferon gamma signature using actinium-225 labeled CS1-directed sdAbs. In this proof-of-principle study, we highlight, for the first time, the therapeutic potential and immunomodulating effects of anti-CS1 radionuclide therapy to target residual MM cells. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most common and aggressive brain malignancy in adults and is currently incurable with conventional therapies. The use of chimeric antigen receptor (CAR) modified T cells has been successful in clinical treatment of blood cancers, except solid tumors such as GBM. This study generated two third-generation CARs targeting different epitopes of ephrin type-A receptor 2 (EphA2) and examined their anti-GBM efficacy in vitro and in tumor-bearing mice. We observed that these two types of T cells expressing CAR (CAR-T) targeting EphA2 could be activated and expanded by EphA2 positive tumor cells in vitro. The survival of tumor-bearing mice after EphA2 CAR-T cell treatment was significantly improved. T cells transduced with one of the two EphA2 CARs exhibited better anti-tumor activity, which is related to the upregulation of CXCR-1/2 and appropriate interferon-γ (IFN-γ) production. CAR-T cells expressed excessively high level of IFN-γ exhibited poor anti-tumor activity resulting from inducing the upregulation of PD-L1 in GBM cells. The combination of CAR-T cells with poor anti-tumor activity and PD1 blockade improved the efficacy in tumor-bearing mice. In conclusion, both types of EphA2 CAR-T cells eliminated 20%-50% of GBM in xenograft mouse models. The appropriate combination of IFN-γ and CXCR-1/2 levels is a key factor for evaluating the antitumor efficiency of CAR-T cells. [ABSTRACT FROM AUTHOR]

The optimization of adoptive transfer approaches of anti-tumor T cells requires both the functional improvement of the injected T cells and the modulation of the tumor microenvironment, favoring the recruitment of these T cells and their activation. We have recently shown the therapeutic benefit of two approaches tested individually in a melanoma model wich were on one hand the adoptive transfer of specific T cells deficient for the expression of the inhibitory receptor PD-1, and on the other hand PD-L1 targeted alpha therapy (TAT). In this study, we sought to investigate the efficacy of these two therapies combined, compared to each monotherapy, in order to evaluate the synergy between these two approaches, in the same melanoma model. Here we used melanoma-specific T-cell clones, previously validated for the edition of PDCD1 gene and with previously demonstrated superior anti-tumor activity than their wild-type counterparts, after adoptive transfer in NSG mice engrafted with PD-L1 expressing human melanoma tumors. We also used a previously validated TAT approach, using a 213Bi-anti-human-PD-L1 mAb, alone or in combination with adoptive cell transfer, in the same mouse model. We confirmed previous results obtained with each monotherapy and documented the safety and the superior ability of a combination between the adoptive transfer of PD-1 deficient T cells and TAT targeting PD-L1 to control the growth of melanoma tumors in NSG mice. This study provides the first proof-of-concept of the efficacy of a combination therapy using TAT, adoptive cell transfer and genomic editing of IC-coding genes. [ABSTRACT FROM AUTHOR]

Substantial evidence indicates that brain-derived neurotrophic factor (BDNF) plays an important role in tumorigenesis, in addition to its primary role in neuronal activity. Gastrointestinal stromal tumors (GISTs), which are the most common mesenchymal neoplasms of the gastrointestinal tract, contain multiple types of tumor-infiltrating lymphocytes (TILs) that express relevant immune checkpoint proteins. However, no data have been reported on the role of BDNF in GISTs. This study aimed to investigate the expression pattern and prognostic value of BDNF in GIST patients with different degrees of risk, as well as the relationship between BDNF expression and immune checkpoints. Immunohistochemistry (IHC) demonstrated that higher BDNF expression was more likely to be present in high-risk patients and suggested a poor prognosis. A similar phenomenon was demonstrated in plasma. Even more interesting was that a positive correlation was present between BDNF and PD-L1+ expression on TILs. Moreover, high BDNF expression levels in combination with a high PD-L1+ TIL count predict extremely poor survival. The combination of BDNF expression and TIL PD-L1+ expression as a single biomarker was a powerful significant independent predictor of prognosis. Taken together, BDNF expression may serve as a significant prognostic factor, as the combination of BDNF expression and the PD-L1+ TIL subset led to superior prediction of GIST prognosis. Furthermore, our research coupled a neurotrophin with immunity, which provides novel evidence of neural and immune regulation in a clinical study of GIST. [ABSTRACT FROM AUTHOR]

Avelumab is an IgG1 anti-programmed death ligand 1 (anti-PD-L1) monoclonal antibody that has been approved as a monotherapy for metastatic Merkel cell carcinoma and advanced urothelial carcinoma, and in combination with axitinib for advanced renal cell carcinoma. Avelumab is cleared faster and has a shorter half-life than other anti-PD-L1 antibodies, such as atezolizumab and durvalumab, but the mechanisms underlying these differences are unknown. IgG antibodies can be cleared through receptor- mediated endocytosis after binding of the antibody Fab region to target proteins, or via Fcγ receptor (FcγR)-mediated endocytosis. Unlike other approved anti-PD-L1 antibodies, avelumab has a native Fc region that retains FcγR binding capability. We hypothesized that the rapid clearance of avelumab might be due to the synergistic effect of both FcγR-mediated and PD-L1 target-mediated internalization. To investigate this, we performed in vitro and in vivo studies that compared engineered variants of avelumab and atezolizumab to determine mechanisms of cellular internalization. We found that both FcγR and PD-L1 binding contribute to avelumab internalization. While FcγR binding was the dominant mechanism of avelumab internalization in vitro, with CD64 acting as the most important FcγR, studies in mice and cynomolgus monkeys showed that both FcγR and PD-L1 contribute to avelumab elimination, with PD-L1 binding playing a greater role. These studies suggest that the rapid internalization of avelumab might be due to simultaneous binding of both PD-L1 and FcγR in trans. Our findings also provide a basis to alter the clearance and half-life of monoclonal antibodies in therapeutic development. [ABSTRACT FROM AUTHOR]

Immunotherapy targeting the CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint axis has emerged as a promising treatment strategy for various cancers. Experimental evidence suggests that phosphatidylinositol-4,5-bisphosphonate 3-kinase (PI3K) signaling may upregulate CD274 expression. Thus, we hypothesized that PIK3CA mutation, PTEN loss, or their combined status might be associated with CD274 overexpression in colorectal carcinoma. We assessed tumor CD274 and PTEN expression by immunohistochemistry and assessed PIK3CA mutation by pyrosequencing in 753 patients among 4,465 incident rectal and colon cancer cases that had occurred in two U.S.-wide prospective cohort studies. To adjust for potential confounders and selection bias due to tissue availability, inverse probability weighted multivariable ordinal logistic regression analyses used the 4,465 cases and tumoral data including microsatellite instability, CpG island methylator phenotype, KRAS and BRAF mutations. PIK3CA mutation and loss of PTEN expression were detected in 111 of 753 cases (15%) and 342 of 585 cases (58%), respectively. Tumor CD274 expression was negative in 306 (41%), low in 195 (26%), and high in 252 (33%) of 753 cases. PTEN loss was associated with CD274 overexpression [multivariable odds ratio (OR) 1.83; 95% confidence interval (CI), 1.22-2.75; P = .004]. PIK3CA mutation was statistically-insignificantly (P = .036 with the stringent alpha level of 0.005) associated with CD274 overexpression (multivariable OR, 1.54; 95% CI, 1.03-2.31). PIK3CA-mutated PTEN-lost tumors (n = 33) showed higher prevalence of CD274-positivity (82%) than PIK3CA-wild-type PTEN-lost tumors (n = 204; 70% CD274-positivity) and PTEN-expressed tumors (n = 147; 50% CD274-positivity) (P = .003). Our findings support the role of PI3K signaling in the CD274/PDCD1 pathway. [ABSTRACT FROM AUTHOR]

Cells in the tumor microenvironment of Follicular lymphoma (FL) express checkpoint molecules such as programmed death ligands 1 and 2 (PD-L1 and PD-L2) and are suppressing anti-tumor immune activity. Stimulation of peripheral blood mononuclear cells (PBMC) with PD-L1 (IO103) or PD-L2 (IO120) peptides can activate specific T cells inducing anti-regulatory functions including cytotoxicity against PD-L1/PD-L2-expressing cells. In this study, we vaccinated eight FL patients with PD-L1 and PD-L2 peptides following treatment with standard chemotherapy. Patients experienced grade 1-2 injection site reaction (5/8) and mild flu-like symptoms (6/8). One patient experienced neutropenia and thrombocytopenia during pseudo-progression. Enzyme-linked immunospot detected vaccine-specific immune responses in PBMC from all patients, predominately toward PD-L1. The circulating immune composition was stable during treatment; however, we observed a reduction regulatory T cells, however, not significant. One patient achieved a complete remission during vaccination and two patients had pseudo-progression followed by long-term disease regression. Further examination of these early signs of clinical efficacy of the dual-epitope vaccine in a larger study is warranted. [ABSTRACT FROM AUTHOR]

Metastatic clear cell renal cell carcinoma (mccRCC) benefits from several treatment options in the first-line setting with VEGFR inhibitors and/or immunotherapy including anti-PD-L1 or anti-PD1 agents. Identification of predictive biomarkers is highly needed to optimize patient care. Circulating markers could reflect the biology of metastatic disease. Therefore, we evaluated soluble forms of PD-L1 (sPD-L1) and PD-1 (sPD-1) in mccRCC patients. The levels of sPD-L1 and sPD-1 were evaluated from plasma samples of mccRCC patients before they received a first-line treatment (T0) by the VEGFR inhibitor sunitinib (50 patients) or by the anti-VEGF bevacizumab (37 patients). The levels of sPD-L1 and sPD-1 were correlated to clinical parameters and progression-free survival (PFS). High levels of sPD-1 or sPDL1 were not correlated to PFS under bevacizumab while they were independent prognostic factors of PFS in the sunitinib group. Patients with high T0 plasmatic levels of sPD-L1 had a shorter PFS (11.3 vs 22.5 months, p =.011) in the sunitinib group. Equivalent shorter PFS was found with high levels of sPD-1 (8.6 vs 14.1 months, p =.009). mccRCC patients with high plasmatic levels of sPD-L1 or sPD-1 are poor responders to sunitinib. sPD-L1 or sPD-1 could be a valuable tool to guide the optimal treatment strategy including VEGFR inhibitor. [ABSTRACT FROM AUTHOR]

In head and neck squamous cell carcinoma (HNSCC), data from studies using checkpoint-inhibiting antibodies that target programmed death 1 (PD-1) or its ligand the programmed death ligand 1 (PD-L1) demonstrated outstanding clinical activity. Translational investigations also suggested some correlations between therapeutic response and PD-L1 expression in tumor tissue. We comprehensively summarize results that have evaluated PD-L1 expression in HNSCC. We discuss flaws and strength of current PD-1/PD-L1 detection, quantification methods and the evaluation of PD-L1 as a prognostic and theragnostic biomarker. Understanding tumor microenvironment may help understanding resistance to checkpoint inhibitors, designing clinical trials that can exploit drug combinations. [ABSTRACT FROM AUTHOR]

Despite a proportion of renal cancer patients can experiment marked and durable responses to immune-checkpoint inhibitors, the treatment efficacy is widely variable and identifying the patient who will benefit from immunotherapy remains an issue. We performed a prospective study to investigate if soluble forms of the immune-checkpoints PD-1 (sPD-1), PD-L1 (sPD-L1), pan-BTN3As, BTN3A1, and BTN2A1, could be candidate to predict the response to immune-checkpoint blockade therapy. We evaluated the plasma levels in a learning cohort of metastatic clear cell renal carcinoma (mccRCC) patients treated with the anti-PD-1 agent nivolumab by ad hoc developed ELISA's. Using specific cut-offs determined through ROC curves, we showed that high baseline levels of sPD-1 (>2.11 ng/ml), sPD-L1 (>0.66 ng/ml), and sBTN3A1 (>6.84 ng/ml) were associated with a longer progression-free survival (PFS) to nivolumab treatment [median PFS, levels above thresholds: sPD-1, 20.7 months (p <.0001); sPD-L1, 19 months (p <.0001); sBTN3A1, 17.5 months (p =.002)]. High sPD-1 and sBTN3A1 levels were also associated with best overall response by RECIST and objective response of >20%. The results were confirmed in a validation cohort of 20 mccRCC patients. The analysis of plasma dynamic changes after nivolumab showed a statistically significant decrease of sPD-1 after 2 cycles (Day 28) in the long-responder patients. Our study revealed that the plasma levels of sPD-1, sPD-L1, and sBTN3A1 can predict response to nivolumab, discriminating responders from non-responders already at therapy baseline, with the advantages of non-invasive sample collection and real-time monitoring that allow to evaluate the dynamic changes during cancer evolution and treatment. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies against the PD-L1/PD-1 (programmed death ligand-1/programmed cell death protein-1) axis have achieved great successes in cancer treatments, but the development of small-molecule immunomodulators of the pathway has lagged far behind. We established a cellular coculture assay with two stable transfectant cell lines, a PD-L1-expressing tumor cell line PC9/PD-L1 and a PD-1-expressing T cell line Jurkat/PD-1. Western blotting analyses were used to monitor the PD-L1/PD-1 interaction and signaling. We analyzed PD-L1 glycosylation by lectin binding assay and glycosidase digestion, and examined subcellular localization of PD-L1 by immunocytochemical staining. Luciferase assay and real-time PCR were used to evaluate T cell activation in the coculture experiments. We found that coculturing of the PC9/PD-L1 cells with the Jurkat/PD-1 cells induced a lysosomal degradation of PD-1. A small-molecule PD-L1 inhibitor BMS1166 developed by Bristol-Myers Squibb inhibited the coculture-induced PD-1 degradation through a unique mechanism. BMS1166 specifically affected PD-L1 glycosylation and prevented transporting of the under-glycosylated form of PD-L1 from endoplasmic reticulum (ER) to Golgi, leading to accumulation of PD-L1 in ER. In doing so, BMS1166 blocked PD-L1/PD-1 signaling. Coculturing PD-L1-expressing cells with PD-1-expressing cells induced degradation of PD-1, which could be used as a readout to identify inhibitors of PD-L1/PD-1 signaling. The small-molecule PD-L1 inhibitor BMS1166 abolished the glycosylation and maturation of PD-L1 by blocking its exporting from ER to Golgi. Our study discovered a new strategy to identify inhibitors of the PD-L1/PD-1 signaling pathway and to develop new drugs for the treatment of cancer. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) patients have a high risk of developing lung cancer, with few treatment options available. Pirfenidone, an antifibrotic agent approved for the treatment of IPF, has been demonstrated to suppress the TGFβ signaling and modulate the expression of immune-related genes. However, for lung cancer patients with comorbid IPF, whether pirfenidone has any synergetic effect with immune checkpoint inhibitors has not been investigated. In this study, we showed that pirfenidone monotherapy attenuated tumor growth with an increased T cell inflammatory signature in tumors. Co-administration of pirfenidone with PD-L1 blockades significantly delayed the tumor growth and increased survival, compared with the effect of either treatment alone. Combination therapy promoted gene expression with a unique signature associated with innate and adaptive immune response resulted in the infiltration of immune cells and optimal T cell positioning. Furthermore, we showed a great benefit of combination therapy in alleviating the pulmonary fibrosis and reducing the tumor growth in a tumor-fibrosis model. Our results collectively demonstrated that pirfenidone facilitated antitumor immunity and enhanced the efficacy of PD-L1 blockades. It may act as an adjuvant to immunotherapy in cancer treatment, particularly, in lung cancer patients with preexisting IPF. [ABSTRACT FROM AUTHOR]

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is considered a novel anti-tumor target comparable to programmed cell death 1 ligand 1(PD-L1). However, little is known about Siglec-15. Our study aims to understand its expression signature, prognosis value, immune infiltration pattern, and biological function using multi-omic bioinformatics from public databases and verify them in lung cancer patients. Integrated analysis of The Cancer Genome Atlas and Genotype-Tissue Expression portals showed Siglec-15 was overexpressed across cancers. Genetic and epigenetic alteration analysis was performed using cBioportal and UALCAN, showed Siglec-15 was regulated at the genetic and epigenetic levels. Survival estimated using Kaplan–Meier plotter indicated high Siglec-15 expression correlated with favorable or unfavorable outcomes depending on the different type and subtype of cancer. Components of immune microenvironment were analyzed using CIBERSORT, and the correlation between immune cells and Siglec-15 was found to be distinct across cancer types. Based on Gene Set Enrichment Analysis, Siglec-15 was implicated in pathways involved in immunity, metabolism, cancer, and infectious diseases. Lung cancer patients with positive Siglec-15 expression showed significantly short survival rates in progression-free survival concomitant with reduced infiltration of CD20 + B, and dendritic cells by immunohistochemistry. Quantitative real-time PCR results indicated the overexpression of Siglec-15 was correlated with activation of the chemokine signaling pathway. In conclusion, Siglec-15 could serve as a vital prognostic biomarker and play an immune-regulatory role in tumors. These results provide us with clues to better understand Siglec-15 from the perspective of bioinformatics and highlight the importance of Siglec-15 in many types of cancer. [ABSTRACT FROM AUTHOR]

Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival (OS) and a dramatic improvement in patient quality of life. Despite the success of this approach, the number of benefitting patients is limited and there is a need for predictive biomarkers as well as a deeper mechanistic analysis of the cellular populations involved in clinical responses. With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients receiving pembrolizumab or nivolumab treatment at Karolinska University Hospital. Blood samples were collected before treatment and before administration of the second and fourth doses. Peripheral blood mononuclear cells were isolated and stained for flow cytometric analysis within 2 h of sample collection. Overall survival and progression-free survival (PFS) were inversely correlated with CD69 expression NK cells. In the myeloid compartment, high frequencies of non-classical monocytes and low frequencies of monocytic myeloid derived suppressor cells (MoMDSCs) correlated with response rates and OS. A deeper characterization of monocytic subsets showed that PD-L1 expression in MDSCs, non-classical and intermediate monocytes was significantly increased in patients with shorter PFS in addition to correlating inversely with OS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of activated NK cells and monocytic subsets are inversely correlated with survival and clinical benefit, suggesting that their role as predictive biomarkers should be further evaluated. [ABSTRACT FROM AUTHOR]

Hodgkin lymphoma (HL) is a unique type of hematopoietic cancer that has few tumor cells but a massive infiltration of immune cells. Findings on how the cancerous Hodgkin and Reed-Sternberg (HRS) cells survive and evade immune surveillance have facilitated the development of novel immunotherapies for HL. Trogocytosis is a fast process of intercellular transfer of membrane patches, which can significantly affect immune responses. In this review, we summarize the current knowledge of how trogocytosis contributes to the suppression of immune responses in HL. We focus on the ectopic expression of CD137 on HRS cells, the cause of its expression, and its implication on developing novel therapies for HL. Further, we review data demonstrating that similar mechanisms apply to CD30, PD-L1 and CTLA-4. [ABSTRACT FROM AUTHOR]

Subgroup analysis of clinical trials of PD-1/PD-L1 inhibitors have reported ethnic differences in outcomes. We systematically collected published data and performed a meta-analysis to compare therapeutic efficacy in Asian and non-Asian patients receiving PD-1/PD-L1 inhibitors. Eligible studies included phase II and III prospective clinical trials with available subgroup data on Asian versus non-Asian populations. Overall survival (OS) and progression-free survival (PFS) were used to evaluate differences in outcome between Asian versus non-Asian cancer patients. A total of 11,020 cancer patients from 19 prospective randomized controlled clinical trials were included. The overall estimated HR for OS was 0.69 with 95% CI of 0.61–0.77 in Asian versus 0.82 with 95% CI of 0.77–0.88 in non-Asian patients. The estimated hazard ratio (HR) for PFS measured 0.54 (95% CI, 0.32–0.76) and 0.69 (95% CI, 0.54–0.85) in Asian and non-Asian patients, respectively. Pooled ratios of OS HRs and PFS HRs reported in Asian versus non-Asian cancer patients were 0.84 (95% CI, 0.75–0.94) and 0.78 (95% CI, 0.59–0.97), respectively. This meta-analysis shows for the first time that Asian cancer patients have a significantly improved survival benefit than non-Asian patients receiving PD-1/PD-L1 inhibitor-based therapy. [ABSTRACT FROM AUTHOR]

Biomarkers predicting immunotherapy response in metastatic renal cell cancer (mRCC) are lacking. PD-L1 immunohistochemistry is a complementary diagnostic for immune checkpoint inhibitors (ICIs) in mRCC, but has shown minimal clinical utility and is not used in routine clinical practice. Tumor specimens from 56 patients with mRCC who received nivolumab were evaluated for PD-L1, cell proliferation (targeted RNA-seq), and outcome. For 56 patients treated with nivolumab as a standard of care, there were 2 complete responses and 8 partial responses for a response rate of 17.9%. Dividing cell proliferation into tertiles, derived from the mean expression of 10 proliferation-associated genes in a reference set of tumors, poorly proliferative tumors (62.5%) were more common than moderately (30.4%) or highly proliferative (8.9%) counterparts. Moderately proliferative tumors were enriched for PD-L1 positive (41.2%), compared to poorly proliferative counterparts (11.4%). Objective response for moderately proliferative (29.4%) tumors was higher than that of poorly (11.4%) proliferative counterparts, but not statistically significant (p =.11). When cell proliferation and negative PD-L1 tumor proportion scores were combined statistically significant results were achieved (p =.048), showing that patients with poorly proliferative and PD-L1 negative tumors have a very low response rate (6.5%) compared to moderately proliferative PD-L1 negative tumors (30%). Cell proliferation has value in predicting response to nivolumab in clear cell mRCC patients, especially when combined with PD-L1 expression. Further studies which include the addition of progression-free survival (PFS) along with sufficiently powered subgroups are required to further support these findings. [ABSTRACT FROM AUTHOR]

Checkpoint inhibitors have revolutionized cancer therapy and validated immunotherapy as an approach. Unfortunately, responses are seen in a minority of patients. Our objective is to use engineered adenoviruses designed to increase lymphocyte trafficking and cytokine production at the tumor, to assess if they increase the response rate to checkpoint inhibition, as these features have been regarded as predictive for the responses. When Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (an oncolytic adenovirus coding for TNFa and IL-2, also known as TILT-123) and checkpoint inhibitors were used together in fresh urological tumor histocultures, a significant shift toward immune activity (not only tumor necrosis alpha and interleukin-2 but also interferon gamma and granzyme B) and increased T-cell trafficking signals (CXCL10) was observed. In vivo, our viruses enabled an anti-PD-L1 (a checkpoint inhibitor) delivering complete responses in all the treated animals (hazard ratios versus anti-PD-L1 alone 0.057 [0.007; 0.451] or virotherapy alone 0.067 [0.011; 0.415]). To conclude, when an engineered oncolytic adenovirus was utilized to modify the tumor microenvironment towards what meta-analyses have pointed as predictive markers for checkpoint inhibitory therapy, the response to them increased synergistically. Of note, key findings were confirmed in fresh patient-derived tumor explants. [ABSTRACT FROM AUTHOR]

Despite some of the oncogenic driver mutations that have been associated with increased expression of programmed death-ligand 1 (PD-L1), the correlation between PD-L1 expression and ROS1 fusion in NSCLC cells, especially for those with Crizotinib resistance has not been fully addressed. The expression of PD-L1 in 30 primary NSCLC tumors with/without ROS1-fusion protein was evaluated by immunohistochemical (IHC) analysis. To assess the correlation between ROS1 fusion and PD-L1 expression, we down-regulated ROS1 with RNA interference or specific inhibitor (Crizotinib) in ROS1-fusion positive NSCLC cell line HCC78; or up-regulate ROS1-fusion gene in an immortalized human bronchial epithelial cell line (HBE). Mouse xenograft models were also used to determine the effect of ROS1 expression on PD-L1 expression in vivo. Crizotinib-resistant cell line was generated for measuring the association between Crizotinib resistance and PD-L1 expression. ROS1-rearrangement in primary NSCLC tumor was significantly associated with up-regulated PD-L1 expression. PD-L1 expression was significantly up-regulated in bronchial epithelial cells after forced expression of ROS1 fusion and was eliminated when HCC78 xenograft mouse models were treated with Crizotinib. We found PD-L1 expression was modulated by MEK-ERK pathway signaling in both parental and Crizotinib-resistant NSCLC cells with ROS1 fusion. The correlation between ROS1-fusion and PD-L1 overexpression suggested that PD-L1/PD-1 blockade could be the second-line treatment option for the Crizotinib-resistant NSCLC with ROS1 rearrangement. [ABSTRACT FROM AUTHOR]

Objective: To explore the relations between liver metastases (LM) and the efficacy of the treatments with programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitors. Method: Pubmed, Embase, American Society of Clinical Oncology and the European Society for Medical Oncology were searched to select eligible studies about PD-1 or PD-L1 inhibitors (Nivolumab, Pembrolizumab, Cemiplimab, Avelumab, Durvalumab, and Atezolizumab). We included only the original randomized controlled trials (RCTs), including the hazard ratios (HR) of death in both patients with LM and patients without LM. Then the data were extracted for the meta-analysis. Subgroup analyses of cancer types and drug types were also performed. Results: 5293 patients [1246 (24%) patients with LM, and 4047 (76%) patients without LM] from the eight RCTs were included for the final analysis. The pooled hazard ratio (HR) of death in the patients with LM was 0.82 (95% CI, 0.71 to 0.93, P =.003) while the pooled HR in the patients without LM was 0.72 (95% CI, 0.66 to 0.79, P <.001). Additionally, no significant difference was found between the two groups (P =.137). Conclusion: No statistically significant association of liver metastases with the efficacy of treatments with PD-1 or PD-L1 inhibitors in the treatment of advanced or metastatic cancer was found in the stratified analyses. Moreover, future studies about the safety of the PD-1 or PD-L1 inhibitors in patients with or without liver metastases are warranted. [ABSTRACT FROM AUTHOR]

This network meta-analysis (NMA), based on one phase II and nine phase III studies, involving 6,124 patients with metastatic NSCLC, indirectly compares Atezolizumab + Bevacizumab + chemotherapy (ABC), Atezolizumab + chemotherapy (AC), Pembrolizumab + chemotherapy (PC), Pembrolizumab alone, Bevacizumab + chemotherapy (BC) and chemotherapy alone. Each of these is recommended as front-line interventions, according to the US FDA and the European Medicines Agency (EMA) for advanced NSCLC without EGFR mutation or ALK rearrangement. Studies were identified through PubMed, EMBASE, the Cochrane Library, Medline, and abstracts found in oncology articles. Primary endpoints, i.e., progression-free survival (PFS) and overall survival (OS) with corresponding hazard ratios (HR), objective response rates (ORR) and adverse event (AEs) with odds risk (OR) were pooled according to frequentist network meta-analytical techniques. PD-L1 expression thresholds, as well as non-squamous/squamous were used to determine subgroups. Immunotherapy plus chemotherapy appeared superior to Pembrolizumab alone for PD-L1-high (i.e., TPS≥50%) NSCLC patients. BC might also be specifically recommended as an initial first-line treatment for PD-L1-high, non-squamous NSCLC patients, since BC was not inferior to Pembrolizumab alone. PC and ABC might be preferred for NSCLC patients with intermediate PD-L1 (1% ≤PD-L1, TPS<50%) expression. BC can also be tentatively recommended specifically for PD-L1-intermediate, non-squamous NSCLC patients. Combined immunotherapies can all be recommended for PD-L1-negative (i.e., TPS<1%) NSCLC patients, although especially the ABC combination for non-squamous NSCLC patients, which was superior to PC in regards of PFS. However, PC performed comparable to ABC in the whole population and in all subgroup save this one. More predictive biomarkers could be factored into further analyses to help identifying the most effective treatment regimens for specific patient groups. [ABSTRACT FROM AUTHOR]

ADAM10 and ADAM17 expression and soluble PD-L1 (sPD-L1) predict poor prognosis in many malignancies, including in patients treated with PD-(L)1 inhibitors. The mechanism of soluble PD-L1 production and its effects are unknown. Here we uncover a novel mechanism of ADAM10- and ADAM17-mediated resistance to PD-(L)1 inhibitors. ADAM10 and ADAM17 cleave PD-L1 from the surface of malignant cells and extracellular vesicles. This cleavage produces an active sPD-L1 fragment that induces apoptosis in CD8 + T cells and compromises the killing of tumor cells by CD8 + T cells. Reduced tumor site PD-L1 protein-to-mRNA ratios predict poor outcomes and are correlated with elevated ADAM10 and ADAM17 expression in multiple cancers. These results may explain the discordance between PD-L1 immunohistochemistry and PD-(L)1 inhibitor response. Thus, including ADAM10 and ADAM17 tissue staining may improve therapy selection. Furthermore, treatment with an ADAM10/ADAM17 inhibitor may abrogate PD-(L)1 inhibitor resistance and improve clinical responses to PD-(L)1 immunotherapy. [ABSTRACT FROM AUTHOR]