External validation of the lung-molGPA to predict survival in patients treated with stereotactic radiotherapy for brain metastases of non-small cell lung cancer.

BACKGROUND: In the era of personalized medicine, individualized prognostic models with tumor characteristics are needed to inform patients about survival. Before clinical use, external validation of such models by an independent group is needed. An updated version of the graded prognostic assessment (GPA) estimates survival in patients with brain metastases (BMs) of non-small cell lung cancer (NSCLC). This is the first external validation of the updated Lung-molGPA in patients treated with stereotactic radiotherapy (SRT) for one or more BMs. MATERIALS AND METHODS: Patients treated with SRT for BMs from NSCLC adenocarcinoma were retrospectively included. GPA score was calculated for each patient based on six prognostic factors including age, Karnofsky Performance Status, number of BMs, extracranial metastases, EGFR/ALK status, and PD-L1 expression. Kaplan-Meier analysis evaluated survival probability. Impact of individual prognostic factors on survival was assessed by univariate and multivariate analyses using the Cox proportional hazard model. Predictive performance was evaluated using discrimination (C-statistic) and calibration (Brier test). RESULTS: The cohort (n = 241) was divided into four prognostic groups. Overall median survival was 15 months. Predicted and observed median survival were similar between the original and validation cohorts, apart from the most favorable prognostic group. With adequate C-statistics and Brier scores, the Lung-molGPA provided accurate survival predictions. CONCLUSION: The Lung-molGPA accurately predicted survival in our European population, except for an overestimation of survival in the small most favorable prognostic group. This prognostic model was externally validated and is therefore useful for counseling of patients with BMs of NSCLC adenocarcinoma.

Predicting programmed death-ligand 1 (PD-L1) expression with fluorine-18 fluorodeoxyglucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT) metabolic parameters in resectable non-small cell lung cancer.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression is a predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). PD-L1 and glucose transporter 1 expression are closely associated, and studies demonstrate correlation of PD-L1 with glucose metabolism. AIM: The aim of this study was to investigate the association of fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG-PET/CT) metabolic parameters with PD-L1 expression in primary lung tumour and lymph node metastases in resected NSCLC. METHODS: We conducted a retrospective analysis of 210 patients with node-positive resectable stage IIB-IIIB NSCLC. PD-L1 tumour proportion score (TPS) was determined using the DAKO 22C3 immunohistochemical assay. Semi-automated techniques were used to analyse pre-operative [18F]FDG-PET/CT images to determine primary and nodal metabolic parameter scores (including max, mean, peak and peak adjusted for lean body mass standardised uptake values (SUV), metabolic tumour volume (MTV), total lesional glycolysis (TLG) and SUV heterogeneity index (HISUV)). RESULTS: Patients were predominantly male (57%), median age 70 years with non-squamous NSCLC (68%). A majority had negative primary tumour PD-L1 (TPS < 1%; 53%). Mean SUVmax, SUVmean, SUVpeak and SULpeak values were significantly higher (p < 0.05) in those with TPS ≥ 1% in primary tumour (n = 210) or lymph nodes (n = 91). However, ROC analysis demonstrated only moderate separability at the 1% PD-L1 TPS threshold (AUCs 0.58-0.73). There was no association of MTV, TLG and HISUV with PD-L1 TPS. CONCLUSION: This study demonstrated the association of SUV-based [18F]FDG-PET/CT metabolic parameters with PD-L1 expression in primary tumour or lymph node metastasis in resectable NSCLC, but with poor sensitivity and specificity for predicting PD-L1 positivity ≥ 1%. CLINICAL RELEVANCE STATEMENT: Whilst SUV-based fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography metabolic parameters may not predict programmed death-ligand 1 positivity ≥ 1% in the primary tumour and lymph nodes of resectable non-small cell lung cancer independently, there is a clear association which warrants further investigation in prospective studies. TRIAL REGISTRATION: Non-applicable KEY POINTS: • Programmed death-ligand 1 immunohistochemistry has a predictive role in non-small cell lung cancer immunotherapy; however, it is both heterogenous and dynamic. • SUV-based fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG-PET/CT) metabolic parameters were significantly higher in primary tumour or lymph node metastases with positive programmed death-ligand 1 expression. • These SUV-based parameters could potentially play an additive role along with other multi-modal biomarkers in selecting patients within a predictive nomogram.

Chimerization of the Anti-Viral CD8+ T Cell Response with A Broad Anti-Tumor T Cell Response Reverses Inhibition of Checkpoint Blockade Therapy by Oncolytic Virotherapy.

Although immune checkpoint inhibition (ICI) has produced profound survival benefits in a broad variety of tumors, a proportion of patients do not respond. Treatment failure is in part due to immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, we developed a vesicular stomatitis virus expressing interferon-ß (VSV-IFNß) as a viro-immunotherapy against HCC. Since HCC standard of care atezolizumab/bevacizumab incorporates ICI, we tested the hypothesis that pro-inflammatory VSV-IFNß would recruit, prime, and activate anti-tumor T cells, whose activity anti-PD-L1 ICI would potentiate. However, in a partially anti-PD-L1-responsive model of HCC, addition of VSV-IFNß abolished anti-PD-L1 therapy. Cytometry by Time of Flight showed that VSV-IFNß expanded dominant anti-viral effector CD8 T cells with concomitant, relative disappearance of anti-tumor T cell populations which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, the potent anti-viral response became amalgamated with an anti-tumor T cell response generating highly significant cures compared to anti-PD-L1 ICI alone. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, by chimerizing anti-viral and anti-tumor T cell responses through encoding tumor antigens within the virus, oncolytic virotherapy can be purposed for very effective immune driven tumor clearance and can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

Expression of Programmed Cell Death Ligand-1 and Mismatch Repair Status in Endometrial Carcinomas.

Background and Aims: Programmed death ligand-1 (PD-L1) is a co-regulatory molecule that suppresses local immunity, and mismatch repair (MMR) deficiency (dMMR) is reported to influence the response to anti-PD-L1-targeted therapy. This study was conducted to find the PD-L1 status, the occurrence of dMMR in endometrial carcinomas, and the association between them. Materials and Methods: The study included 35 resected specimens of endometrial carcinomas represented on formalin-fixed paraffin-embedded sections from January 2016 to July 2020. The clinicopathologic information including patient age, tumor histologic type, grade, stage, lymphovascular invasion, the extent of myometrial invasion, and the percentage of tumor-infiltrating lymphocytes (TILs) were obtained in all cases. The expression of PD-L1 and MMR antibodies including mutS homolog 2 (MSH-2), MSH-6, mutL homolog 1 (MLH-1) and MLH-3, and postmeiotic segregation 2 were assessed using immunohistochemistry. The statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) version 26. Results: PD-L1 expression was noted in 48.6% of the cases in tumor cells and 65.7% of the cases in TILs and MMR was deficient in 28.6% of endometrial carcinomas. A statistically significant relation was noted between dMMR and TILs, PD-L1 expression in tumor cells and TILs, PD-L1 expression in tumor cells, and extent of myometrial invasion. Although there was no statistically significant association between MMR status and PD-L1 expression in tumor cells or TILs, 60% of patients with dMMR were PD-L1 positive. Conclusion: Sixty percent of dMMR cases showed PD-L1 expression in tumor cells. We conclude, ECs that are MMR deficient might get better response to anti-PD-L1 therapy. This study also revealed the prognostic use of TILs in PD-L1-expressed tumors.

Tumor immune microenvironment and clinical outcomes in stage IV urothelial cancer: YODO study.

BACKGROUND: Bladder cancer is the 10th most common cancer globally, with a growing incidence in Japan. Evaluation of molecular, genetic, and cellular biomarkers that predict treatment response and prognosis in patients with metastatic urothelial carcinoma (mUC) may help optimize sequential treatment strategies with chemotherapy and immune checkpoint inhibitors (ICIs). METHODS: This multicenter, retrospective cohort study, evaluated programmed death-ligand 1 (PD-L1) expression, tumor mutational burden (TMB), and cancer-immune phenotype as predictive prognostic biomarkers following first-/second-line treatment in Japanese adult patients with mUC. The primary endpoint was prevalence of PD-L1 expression. Secondary endpoints were TMB, overall survival (OS), and progression-free survival (PFS) from initiation of first-line treatment, and exploratory endpoints were cancer-immune phenotype, OS, PFS, and treatment response according to potential biomarker status. RESULTS: Of the 143 patients included (mean age 71.7 years), PD-L1 expression was high in 29.4% of patients. Non-synonymous TMB was high in 33.6% and low in 66.4%. Cancer-immune phenotype was immune-desert in 62.9%, immune-excluded in 30.8%, and inflamed in 6.3%. Median OS and PFS following first-line treatment were 18.2 and 7.4 months, respectively. Overall response to second-line treatment was slightly better with high versus low/negative PD-L1 expression. PD-L1 expression and TMB were non-significant predictors of OS or PFS, whereas immune-excluded phenotype was associated with better OS in comparison with immune-desert phenotype. CONCLUSION: PD-L1 expression and TMB were non-significant predictors of prognosis after first-line treatment in Japanese patients with mUC, but cancer-immune phenotype may be an important prognostic factor in chemotherapy-ICI sequential treatment strategies. Clinical trial registration number UMIN000037727.

AR-A014418 regulates intronic polyadenylation and transcription of PD-L1 through inhibiting CDK12 and CDK13 in tumor cells.

BACKGROUND: Immune checkpoint molecules, especially programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), protect tumor cells from T cell-mediated killing. Immune checkpoint inhibitors, designed to restore the antitumor immunosurveillance, have exhibited significant clinical benefits for patients with certain cancer types. Nevertheless, the relatively low response rate and acquisition of resistance greatly limit their clinical applications. A deeper understanding of the regulatory mechanisms of PD-L1 protein expression and activity will help to develop more effective therapeutic strategies. METHODS: The effects of AR-A014418 and THZ531 on PD-L1 expression were detected by western blot, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. In vitro kinase assays with recombinant proteins were performed to confirm that AR-A014418 functioned as a CDK12 and CDK13 dual inhibitor. The roles of CDK12 and CDK13 in intronic polyadenylation (IPA) and transcription of PD-L1 were determined via RNA interference or protein overexpression. T-cell cytotoxicity assays were used to validate the activation of antitumor immunity by AR-A014418 and THZ531. RESULTS: AR-A014418 inhibits CDK12 to enhance the IPA, and inhibits CDK13 to repress the transcription of PD-L1. IPA generates a secreted PD-L1 isoform (PD-L1-v4). The extent of IPA was not enough to reduce full-length PD-L1 expression obviously. Only the superposition of enhancing IPA and repressing transcription (dual inhibition of CDK12 and CDK13) dramatically suppresses full-length PD-L1 induction by interferon-γ. AR-A014418 and THZ531 could potentiate T-cell cytotoxicity against tumor cells. CONCLUSIONS: Our work identifies a new regulatory pathway for PD-L1 expression and discovers CDK12 and CDK13 as promising drug targets for immune modulation and combined therapeutic strategies.

ANXA1-derived peptide for targeting PD-L1 degradation inhibits tumor immune evasion in multiple cancers.

BACKGROUND: Immune checkpoint inhibitors (ICIs) therapy targeting programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) shows promising clinical benefits. However, the relatively low response rate highlights the need to develop an alternative strategy to target PD-1/PD-L1 immune checkpoint. Our study focuses on the role and mechanism of annexin A1 (ANXA1)-derived peptide A11 degrading PD-L1 and the effect of A11 on tumor immune evasion in multiple cancers. METHODS: Binding of A11 to PD-L1 was identified by biotin pull-down coupled with mass spectrometry analysis. USP7 as PD-L1's deubiquitinase was found by screening a human deubiquitinase cDNA library. The role and mechanism of A11 competing with USP7 to degrade PD-L1 were analyzed. The capability to enhance the T cell-mediated tumor cell killing activity and antitumor effect of A11 via suppressing tumor immune evasion were investigated. The synergistic antitumor effect of A11 and PD-L1 mAb (monoclonal antibody) via suppressing tumor immune evasion were also studied in mice. The expression and clinical significance of USP7 and PD-L1 in cancer tissues were evaluated by immunohistochemistry. RESULTS: A11 decreases PD-L1 protein stability and levels by ubiquitin proteasome pathway in breast cancer, lung cancer and melanoma cells. Mechanistically, A11 competes with PD-L1's deubiquitinase USP7 for binding PD-L1, and then degrades PD-L1 by inhibiting USP7-mediated PD-L1 deubiquitination. Functionally, A11 promotes T cell ability of killing cancer cells in vitro, inhibits tumor immune evasion in mice via increasing the population and activation of CD8+ T cells in tumor microenvironment, and A11 and PD-1 mAb possess synergistic antitumor effect in mice. Moreover, expression levels of both USP7 and PD-L1 are significantly higher in breast cancer, non-small cell lung cancer and skin melanoma tissues than those in their corresponding normal tissues and are positively correlated in cancer tissues, and both proteins for predicting efficacy of PD-1 mAb immunotherapy and patient prognosis are superior to individual protein. CONCLUSION: Our results reveal that A11 competes with USP7 to bind and degrade PD-L1 in cancer cells, A11 exhibits obvious antitumor effects and synergistic antitumor activity with PD-1 mAb via inhibiting tumor immune evasion and A11 can serve as an alternative strategy for ICIs therapy in multiple cancers.

PD-L1 Upregulation by the mTOR Pathway in VEGFR-TKI-Resistant Metastatic Clear Cell Renal Cell Carcinoma.

PURPOSE: Tyrosine kinase inhibitors (TKI) targeting vascular endothelial growth factor receptor (VEGFR) signaling pathways have been used for metastatic clear cell renal cell carcinoma (mCCRCC), but resistance to the drug develops in most patients. We aimed to explore the underlying mechanism of the TKI resistance with regard to programmed death-ligand 1 (PD-L1) and to investigate signaling pathway associated with the resistant mechanism. MATERIALS AND METHODS: To determine the mechanism of resistance, 10 mCCRCC patients from whom tumor tissues were harvested at both the pretreatment and the TKI-resistant post-treatment period were included as the discovery cohort, and their global gene expression profiles were compared. A TKI-resistant renal cancer cell line was established by long-term treatment with sunitinib. RESULTS: Among differentially expressed genes in the discovery cohort, increased PD-L1 expression in post-treatment tissues was noted in four patients. Pathway analysis showed that PD-L1 expression was positively correlated with the mammalian target of rapamycin (mTOR) signaling pathway. The TKI-resistant renal cancer cells showed increased expression of PD-L1 and mTOR signaling proteins and demonstrated aggressive tumoral behaviour. Treatment with mTOR inhibitors down-regulated PD-L1 expression and suppressed aggressive tumoral behaviour, which was reversed with stimulation of the mTOR pathway. CONCLUSION: These results showed that PD-L1 expression may be increased in a subset of VEGFR-TKI-resistant mCCRCC patients via the mTOR pathway.

Ipilimumab plus nivolumab in avelumab-refractory Merkel cell carcinoma: a multicenter study of the prospective skin cancer registry ADOREG.

Merkel cell carcinoma is a rare, highly aggressive skin cancer with neuroendocrine differentiation. Immune checkpoint inhibition has significantly improved treatment outcomes in metastatic disease with response rates to programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) inhibition of up to 62%. However, primary and secondary resistance to PD-1/PD-L1 inhibition remains a so far unsolved clinical challenge since effective and safe treatment options for these patients are lacking.Fourteen patients with advanced (non-resectable stage III or stage IV, Union international contre le cancer 2017) Merkel cell carcinoma with primary resistance to the PD-L1 inhibitor avelumab receiving subsequent therapy (second or later line) with ipilimumab plus nivolumab (IPI/NIVO) were identified in the prospective multicenter skin cancer registry ADOREG. Five of these 14 patients were reported previously and were included in this analysis with additional follow-up. Overall response rate, progression-free survival (PFS), overall survival (OS) and adverse events were analyzed.All 14 patients received avelumab as first-line treatment. Thereof, 12 patients had shown primary resistance with progressive disease in the first tumor assessment, while two patients had initially experienced a short-lived stabilization (stable disease). Six patients had at least one systemic treatment in between avelumab and IPI/NIVO. In total, 7 patients responded to IPI/NIVO (overall response rate 50%), and response was ongoing in 4 responders at last follow-up. After a median follow-up of 18.85 months, median PFS was 5.07 months (95% CI 2.43-not available (NA)), and median OS was not reached. PFS rates at 12 months and 24 months were 42.9% and 26.8 %, respectively. The OS rate at 36 months was 64.3%. Only 3 (21%) patients did not receive all 4 cycles of IPI/NIVO due to immune-related adverse events.In this multicenter evaluation, we observed high response rates, a durable benefit and promising OS rates after treatment with later-line combined IPI/NIVO. In conclusion, our patient cohort supports our prior findings with an encouraging activity of second-line or later-line IPI/NIVO in patients with anti-PD-L1-refractory Merkel cell carcinoma.

Combination cancer immunotherapy targeting TNFR2 and PD-1/PD-L1 signaling reduces immunosuppressive effects in the microenvironment of pancreatic tumors.

BACKGROUNDS: In advanced pancreatic ductal adenocarcinoma (PDAC), immune therapy, including immune checkpoint inhibitors, has limited efficacy, encouraging the study of combination therapy. METHODS: Tumor necrosis factor receptor 2 (TNFR2) was analyzed via immunohistochemistry, immunofluorescence, western blotting, and ELISAs. The in vitro mechanism that TNFR2 regulates programmed cell death 1 ligand 1 (PD-L1) was investigated using immunofluorescence, immunohistochemistry, flow cytometry, western blotting, and chromatin immunoprecipitation (ChIP). In vivo efficacy and mechanistic studies, using C57BL/6 mice and nude mice with KPC cell-derived subcutaneous and orthotopic tumors, employed antibodies against TNFR2 and PD-L1. Survival curves were constructed for the orthotopic model and a genetically engineered PDAC model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre). Mass cytometry, immunohistochemistry, and flow cytometry analyzed local and systemic alterations in the immunophenotype. RESULTS: TNFR2 showed high expression and is a prognostic factor in CD8+ T cell-enriched pancreatic cancer. TNFR2 promotes tumorigenesis and progression of pancreatic cancer via dual effect: suppressing cancer immunogenicity and partially accelerating tumor growth. TNFR2 positivity correlated with PD-L1, and in vitro and in vivo, it could regulate the expression of PDL1 at the transcription level via the p65 NF-κB pathway. Combining anti-TNFR2 and PD-L1 antibodies eradicated tumors, prolonged overall survival in pancreatic cancer, and induced strong antitumor immune memory and secondary prevention by reducing the infiltration of Tregs and tumor-associated macrophages and inducing CD8+ T cell activation in the PDAC microenvironment. Finally, the antitumor immune response derived from combination therapy is mainly dependent on CD8+ T cells, partially dependent on CD4+ T cells, and independent of natural killer cells. CONCLUSIONS: Anti-TNFR2 and anti-PD-L1 combination therapy eradicated tumors by inhibiting their growth, relieving tumor immunosuppression, and generating robust memory recall.

IFN-γ-induced PD-L1 expression on human melanocytes is impaired in vitiligo.

Mounting evidence shows that the PD-1/PD-L1 axis is involved in tumor immune evasion. This is demonstrated by anti-PD-1 antibodies that can reverse tumor-associated PD-L1 to functionally suppress anti-tumor T-cell responses. Since type I and II interferons are key regulators of PD-L1 expression in melanoma cells and IFN-γ-producing CD8+ T cells and IFN-α-producing dendritic cells are abundant in vitiligo skin, we aimed to study the role of PD-1/PD-L1 signalling in melanocyte destruction in vitiligo. Moreover, impaired PD-1/PD-L1 function is observed in a variety of autoimmune diseases. It is, therefore, hypothesized that manipulating PD-1/PD-L1 signalling might have therapeutic potential in vitiligo. The PD-1+ T cells were abundantly present in situ in perilesional vitiligo skin, but expression of PD-L1 was limited and confined exclusively to dermal T cells. More specifically, neither melanocytes nor other epidermal skin cells expressed PD-L1. Exposure to IFN-γ, but also type I interferons, increased PD-L1 expression in primary melanocytes and fibroblasts, derived from healthy donors. Primary human keratinocytes only showed increased PD-L1 expression upon stimulation with IFN-γ. More interestingly, melanocytes derived from non-lesional vitiligo skin showed no PD-L1 upregulation upon IFN-γ exposure, while other skin cells displayed significant PD-L1 expression after exposure. In a vitiligo skin explant model, incubation of non-lesional vitiligo skin with activated (IFN-γ-producing) T cells from vitiligo lesions was previously described to induce melanocyte apoptosis. Although PD-L1 expression was induced in epidermal cells in these explants, this induction was completely absent in melanocytes. The lack of PD-L1 upregulation by melanocytes in the presence of IFN-γ-producing T cells shows that melanocytes lack protection against T-cell attack during vitiligo pathogenesis. Manipulating PD-1/PD-L1 signalling may, therefore, be a therapeutic option for vitiligo patients.

Immunoexpression of PD-L1, CD4+ and CD8+ cell infiltrates and tumor-infiltrating lymphocytes (TILs) in the microenvironment of actinic cheilitis and lower lip squamous cell carcinoma

Abstract Lower lip squamous cell carcinomas (LLSCC) could be associated with a previous history of potentially malignant oral diseases (PMOD), especially actinic cheilitis (AC), with high sun exposure being a well-described risk factor. Immune evasion mechanisms, such as the PD-1/PD-L1 (programmed cell death protein 1/programmed death-ligand 1) pathway has been gaining prominence since immunotherapy with immune checkpoint inhibitors showed a positive effect on the survival of patients with different types of neoplasms. Concomitant with the characterization of the tumor microenvironment, the expression of either or both PD-1 and PD-L1 molecules may estimate mutual relations of progression or regression of the carcinoma and prognostic values of the patient. Objective: Considering the importance of tumor microenvironment characterization, this study aims to determine the immunoexpression of PD-L1 and correlate with the frequency of CD4+ and CD8+ cells in AC and LLSCC lesions and with tumor-infiltrating lymphocytes (TILs) in LLSCC and its relationship with histopathological characteristics. Methodology: This sample includes 33 cases of AC and 17 cases of LLSCC. The cases were submitted to histopathological analysis and to CD4+, CD8+, and PD-L1+ cell determination by immunohistochemistry. Results: There was a significant difference among the frequencies of CD4+, CD8+, and PD-L1+ cells between AC and LSCC cases, higher in the last group. Moreover, histopathological and atypical changes in AC and LLSCC were correlated with the frequencies of PD-L1+, CD4+, and CD8+ cells. In AC, PD-L1+ cases had a low frequency of CD4+ cells, but on the other hand, PD-L1+ cases of LLSCC had a higher frequency of CD4+ and CD8+ cells. Conclusion: Therefore, the PD-L1 molecule may be a potential escape route for the immune response in oral lesions, but the mechanisms differ between AC and LLSCC. Future studies related to immune evasion and immunotherapy in oral lesions should consider the analysis of inflammatory infiltrate and TILs.

SAMHD1 inhibits PD-L1 expression in lung adenocarcinoma cells / 国际肿瘤学杂志

Objective:To explore the correlation between SAM domain and HD domain-containing protein 1 (SAMHD1) and programmed death-ligand 1 (PD-L1) expression in lung adenocarcinoma.Methods:The expression of SAMHD1 in lung adenocarcinoma and its effect on prognosis were analyzed by online database GEPIA and Kaplan-Meier Plotter. The expression of SAMHD1 in lung adenocarcinoma cell lines was detected by quantitative real-time PCR (qPCR) and Western blotting. SAMHD1 gene was silenced in H1975, H1299 and LLC cells by small interfering RNA transfection and lentivirus infection, respectively. The mRNA and protein expression levels of PD-L1 in lung adenocarcinoma cells of control group, siSAMHD1-1 group and siSAMHD1-2 group were detected by qPCR and Western blotting. The membrane PD-L1 level was detected by flow cytometry. A mouse lung adenocarcinoma xenograft model was constructed. The PD-L1 levels in the tumor tissues of control group and shSAMHD1 group were detected by immunohistochemistry. Cell proliferation activities of the control, siSAMHD1-1 and siSAMHD1-2 groups were detected by CCK-8 assays.Results:The GEPIA database results showed that the mRNA expression of SAMHD1 in lung adenocarcinoma was lower than that in normal lung tissue (4.81±0.90 vs. 5.99±0.76, t=20.67, P<0.001) . The median overall survival time of patients with high SAMHD1 expression was significantly longer than that of patients with low SAMHD1 expression (109.0 months vs. 87.7 months, χ2=26.83, P=0.002) . The relative mRNA expression levels of SAMHD1 in A549, PC9, H1299 and H1975 cells were 1.00±0.02, 0.75±0.05, 3.49±0.19 and 7.25±0.38 ( F=589.00, P<0.001) , and the relative protein expression levels were 1.00±0.06, 0.34±0.07, 1.67±0.22 and 2.11±0.63 ( F=15.79, P=0.001) . In H1975 cells, the relative mRNA levels of PD-L1 in the control, siSAMHD1-1 and siSAMHD1-2 groups were 1.00±0.00, 1.54±0.26 and 2.89±0.13 ( F=102.30, P<0.001) , and the relative protein expression levels were 1.00±0.01, 1.50±0.10 and 1.52±0.33 ( F=6.65, P=0.030) . In H1299 cells, the relative mRNA levels of PD-L1 in the three groups were 1.00±0.08, 1.63±0.03 and 2.14±0.03 ( F=368.80, P<0.001) , and the relative protein levels of PD-L1 were 1.00±0.07, 1.88±0.35 and 2.05±0.38 ( F=10.66, P=0.011) . The expression level of PD-L1 in the siSAMHD1-1 and siSAMHD1-2 groups was higher than that in the control group (all P<0.05) . Flow cytometry results showed that in H1975 cells, the fluorescence intensity of membrane PD-L1 in the control, siSAMHD1-1 and siSAMHD1-2 groups were 246.83±27.59, 325.60±8.00 and 308.93±7.60 ( F=17.56, P=0.003) , and in H1299 cells, the fluorescence intensity of membrane PD-L1 in the three groups were 959.00±6.25, 1 084.33±7.64 and 1 085.33±21.22 ( F=86.74, P<0.001) . The fluorescence intensity of PD-L1 in the siSAMHD1-1 group and siSAMHD1-2 group was higher than that in the control group (all P<0.05) . In xenograft mouse model, the H-SCORE of PD-L1 in the shSAMHD1 group was higher than that in the control group (7.99±1.10 vs. 4.49±0.43, t=5.13, P=0.007) . The proliferative activities of H1975 cells in the control group, siSAMHD1-1 group and siSAMHD1-2 group at 72 h were 0.50±0.02, 0.75±0.05 and 0.73±0.06 ( F=25.01, P=0.001) . The proliferative activities of H1299 cells in the three groups at 72 h were 0.80±0.01, 1.00±0.04 and 0.93±0.07 ( F=13.90, P=0.006) . The cell proliferation activity in the siSAMHD1-1 group and siSAMHD1-2 group was higher than that in the control group (all P<0.05) . Conclusion:SAMHD1 silencing induces PD-L1 expression in lung adenocarcinoma.

Spatiotemporal depletion of tumor-associated immune checkpoint PD-L1 with near-infrared photoimmunotherapy promotes antitumor immunity.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) is a new modality for treating cancer, which uses antibody-photoabsorber (IRDye700DX) conjugates that specifically bind to target tumor cells. This conjugate is then photoactivated by NIR light, inducing rapid necrotic cell death. NIR-PIT needs a highly expressed targeting antigen on the cells because of its reliance on antibodies. However, using antibodies limits this useful technology to only those patients whose tumors express high levels of a specific antigen. Thus, to propose an alternative strategy, we modified this phototechnology to augment the anticancer immune system by targeting the almost low-expressed immune checkpoint molecules on tumor cells. METHODS: We used programmed death-ligand 1 (PD-L1), an immune checkpoint molecule, as the target for NIR-PIT. Although the expression of PD-L1 on tumor cells is usually low, PD-L1 is almost expressed on tumor cells. Intratumoral depletion with PD-L1-targeted NIR-PIT was tested in mouse syngeneic tumor models. RESULTS: Although PD-L1-targeted NIR-PIT showed limited effect on tumor cells in vitro, the therapy induced sufficient antitumor effects in vivo, which were thought to be mediated by the 'photoimmuno' effect and antitumor immunity augmentation. Moreover, PD-L1-targeted NIR-PIT induced antitumor effect on non-NIR light-irradiated tumors. CONCLUSIONS: Local PD-L1-targeted NIR-PIT enhanced the antitumor immune reaction through a direct photonecrotic effect, thereby providing an alternative approach to targeted cancer immunotherapy and expanding the scope of cancer therapeutics.

PD-L1 quantification across tumor types using the reverse phase protein microarray: implications for precision medicine.

BACKGROUND: Anti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely used to assess PD-L1 expression, their clinical utility remains controversial due to the partial predictive value and lack of standardized cut-offs across antibody clones. Using a high throughput immunoassay, the reverse phase protein microarray (RPPA), coupled with a fluorescence-based detection system, this study compared the performance of six anti-PD-L1 antibody clones on 666 tumor samples. METHODS: PD-L1 expression was measured using five antibody clones (22C3, 28-8, CAL10, E1L3N and SP142) and the therapeutic antibody atezolizumab on 222 lung, 71 ovarian, 52 prostate and 267 breast cancers, and 54 metastatic lesions. To capture clinically relevant variables, our cohort included frozen and formalin-fixed paraffin-embedded samples, surgical specimens and core needle biopsies. Pure tumor epithelia were isolated using laser capture microdissection from 602 samples. Correlation coefficients were calculated to assess concordance between antibody clones. For two independent cohorts of patients with lung cancer treated with nivolumab, RPPA-based PD-L1 measurements were examined along with response to treatment. RESULTS: Median-center PD-L1 dynamic ranged from 0.01 to 39.37 across antibody clones. Correlation coefficients between the six antibody clones were heterogeneous (range: -0.48 to 0.95) and below 0.50 in 61% of the comparisons. In nivolumab-treated patients, RPPA-based measurement identified a subgroup of tumors, where low PD-L1 expression equated to lack of response. CONCLUSIONS: Continuous RPPA-based measurements capture a broad dynamic range of PD-L1 expression in human specimens and heterogeneous concordance levels between antibody clones. This high throughput immunoassay can potentially identify subgroups of tumors in which low expression of PD-L1 equates to lack of response to treatment.

THADA drives Golgi residency and upregulation of PD-L1 in cancer cells and provides promising target for immunotherapy.

BACKGROUND: The abnormal upregulation of programmed death-ligand 1 (PD-L1) in cancer cells inhibits T cell-mediated cytotoxicity, but the molecular mechanisms that drive and maintain PD-L1 expression are still incompletely understood. METHODS: Combined analyses of genomes and proteomics were applied to find potential regulators of PD-L1. In vitro experiments were performed to investigate the regulatory mechanism of PD-L1 by thyroid adenoma associated gene (THADA) using human colorectal cancer (CRC) cells. The prevalence of THADA was analyzed using CRC tissue microarrays by immunohistochemistry. T cell killing assay, programmed cell death 1 binding assay and MC38 transplanted tumor models in C57BL/6 mice were developed to investigate the antitumor effect of THADA. RESULTS: THADA is critically required for the Golgi residency of PD-L1, and this non-redundant, coat protein complex II (COPII)-associated mechanism maintains PD-L1 expression in tumor cells. THADA mediated the interaction between PD-L1 as a cargo protein with SEC24A, a module on the COPII trafficking vesicle. Silencing THADA caused absence and endoplasmic reticulum (ER) retention of PD-L1 but not major histocompatibility complex-I, inducing PD-L1 clearance through ER-associated degradation. Targeting THADA substantially enhanced T cell-mediated cytotoxicity, and increased CD8+ T cells infiltration in mouse tumor tissues. Analysis on clinical tissue samples supported a potential role of THADA in upregulating PD-L1 expression in cancer. CONCLUSIONS: Our data reveal a crucial cellular process for PD-L1 maturation and maintenance in tumor cells, and highlight THADA as a promising target for overcoming PD-L1-dependent immune evasion.

[Current landscape of biomarker development for immune checkpoint inhibitors targeting PD-1/PD-L1 pathway in oncology]. / Un tour d'horizon du développement de biomarqueurs pour les anticorps PD-1 et PD-L1 en cancérologie.

The immune checkpoints inhibitors targeting PD-1 or PD-L1 represent a new paradigm in the cancer treatment strategy. However, some populations of patients do not benefit from these agents. The identification of predictive biomarkers appears as an essential step for the treatment pathway, to guarantee the access to an evidence-based medicine accounting for the potential toxicity profile, the cost for the healthcare system and the clinical benefit eventually provided by these new drugs. In this review, we propose, based on scientific literature and industrial communications, an overview of the current landscape of predictive biomarkers related to PD-1 or PD-L1 inhibitors efficacy, validated or under development, their evidence level, and limits accounting for identified or potential confounding factors. Our paper shows that, despite the important amount of work performed in this field, there is not yet a validated and efficient solution for the prediction of the activity and/or the toxicity of anti-PD-1 and anti-PD-L1 antibodies.

CX-072 (pacmilimab), a Probody PD-L1 inhibitor, in combination with ipilimumab in patients with advanced solid tumors (PROCLAIM-CX-072): a first-in-human, dose-finding study.

BACKGROUND: Probody® therapeutics are antibody prodrugs designed to be activated by tumor-associated proteases. This conditional activation restricts antibody binding to the tumor microenvironment, thereby minimizing 'off-tumor' toxicity. Here, we report the phase 1 data from the first-in-human study of CX-072 (pacmilimab), a Probody immune checkpoint inhibitor directed against programmed death-ligand 1 (PD-L1), in combination with the anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) antibody ipilimumab. METHODS: Adults (n=27) with advanced solid tumors (naive to PD-L1/programmed cell death protein 1 or CTLA-4 inhibitors) were enrolled in the phase 1 combination therapy dose-escalation portion of this multicenter, open-label, phase 1/2 study (NCT03013491). Dose-escalation pacmilimab/ipilimumab followed a standard 3+3 design and continued until the maximum tolerated dose (MTD) was determined. Pacmilimab+ipilimumab was administered intravenously every 3 weeks for four cycles, followed by pacmilimab administered every 2 weeks as monotherapy. The primary objective was identification of dose-limiting toxicities and determination of the MTD. Other endpoints included the rate of objective response (Response Evaluation Criteria In Solid Tumors v.1.1). RESULTS: Twenty-seven patients were enrolled in pacmilimab (mg/kg)+ipilimumab (mg/kg) dose-escalation cohorts: 0.3+3 (n=6); 1+3 (n=3); 3+3 (n=3); 10+3 (n=8); 10+6 (n=6); and 10+10 (n=1). Dose-limiting toxicities occurred in three patients, one at the 0.3+3 dose level (grade 3 dyspnea/pneumonitis) and two at the 10+6 dose level (grade 3 colitis, grade 3 increased aspartate aminotransferase). The MTD and recommended phase 2 dose was pacmilimab 10 mg/kg+ipilimumab 3 mg/kg administered every 3 weeks. Pacmilimab-related grade 3-4 adverse events (AEs) and grade 3-4 immune-related AEs were reported in nine (33%) and six (22%) patients, respectively. Three patients (11%) discontinued treatment because of AEs. The overall response rate was 19% (95% CI 6.3 to 38.1), with one complete (anal squamous cell carcinoma) and four partial responses (cancer of unknown primary, leiomyosarcoma, mesothelioma, testicular cancer). Responses lasted for >12 months in four patients. CONCLUSIONS: The MTD and recommended phase 2 dose of pacmilimab (10 mg/kg)+ipilimumab (3 mg/kg) every 3 weeks is active and has a favorable tolerability profile.

CX-072 (pacmilimab), a Probody ® PD-L1 inhibitor, in advanced or recurrent solid tumors (PROCLAIM-CX-072): an open-label dose-finding and first-in-human study.

BACKGROUND: Probody® therapeutics are antibody prodrugs that are activated in the tumor microenvironment by tumor-associated proteases, thereby restricting the activity to the tumor microenvironment and minimizing 'off-tumor' toxicity. We report dose-escalation and single-agent expansion phase data from the first-in-human study of CX-072 (pacmilimab), a Probody checkpoint inhibitor directed against programmed death-ligand 1 (PD-L1). METHODS: In the dose-escalation phase of this multicenter, open-label study (NCT03013491), adults with advanced solid tumors (naive to programmed-death-1/PD-L1 or cytotoxic T-lymphocyte-associated antigen 4 inhibitors) were enrolled into one of seven dose-escalation cohorts, with pacmilimab administered intravenously every 14 days. The primary endpoints were safety and determination of the maximum tolerated dose (MTD). In the expansion phase, patients with one of six prespecified malignancies (triple-negative breast cancer [TNBC]; anal squamous cell carcinoma [aSCC]; cutaneous SCC [cSCC]; undifferentiated pleomorphic sarcoma [UPS]; small bowel adenocarcinoma [SBA]; and thymic epithelial tumor [TET]); or high tumor mutational burden (hTMB) tumors were enrolled. The primary endpoint was objective response (Response Evaluation Criteria In Solid Tumors v.1.1). RESULTS: An MTD was not reached with doses up to 30 mg/kg. A recommended phase 2 dose (RP2D) of 10 mg/kg was chosen based on pharmacokinetic and pharmacodynamic findings in the expansion phase. Ninety-eight patients enrolled in the expansion phase: TNBC (n=14), aSCC (n=14), cSCC (n=14), UPS (n=20), SBA (n=14), TET (n=8), and hTMB tumors (n=14). Of 114 patients receiving pacmilimab at the RP2D, grade ≥3 treatment-related adverse events (TRAEs) were reported in 10 patients (9%), serious TRAEs in six patients (5%), and treatment discontinuation due to TRAEs in two patients (2%). Grade ≥3 immune-related AEs occurred in two patients (rash, myocarditis). High PD-L1 expression (ie, >50% Tumor Proportion Score) was observed in 22/144 (19%) patients. Confirmed objective responses were observed in patients with cSCC (n=5, including one complete response), hTMB (n=4, including one complete response), aSCC (n=2), TNBC (n=1), UPS (n=1), and anaplastic thyroid cancer (n=1). CONCLUSIONS: Pacmilimab can be administered safely at the RP2D of 10 mg/kg every 14 days. At this dose, pacmilimab had a low rate of immune-mediated toxicity and showed signs of antitumor activity in patients not selected for high PD-L1 expression. TRIAL REGISTRATION NUMBER: NCT03013491.

PD-1, PD-L1, and PD-L2 Gene Expression and Tumor Infiltrating Lymphocytes in Canine Melanoma.

Melanoma in humans and dogs is considered highly immunogenic; however, the function of tumor-infiltrating lymphocytes (TILs) is often suppressed in the tumor microenvironment. In humans, current immunotherapies target checkpoint molecules (such as PD-L1, expressed by tumor cells), inhibiting their suppressive effect over TILs. The role of PD-L2, an alternative PD-1 ligand also overexpressed in malignant tumors and in patients with anti-PD-L1 resistance, remains poorly understood. In the current study, we evaluated the expression of checkpoint molecule mRNAs in canine melanoma and TILs. Analysis of checkpoint molecule gene expression was performed by RT-qPCR (real-time quantitative polymerase chain reaction) using total RNA isolated from formalin-fixed and paraffin-embedded melanomas (n = 22) and melanocytomas (n = 9) from the Virginia Tech Animal Laboratory Services archives. Analysis of checkpoint molecule expression revealed significantly higher levels of PDCD1 (PD-1) and CD274 (PD-L1) mRNAs and an upward trend in PDCD1LG2 (PD-L2) mRNA in melanomas relative to melanocytomas. Immunohistochemistry revealed markedly increased numbers of CD3+ T cells in the highest PD-1-expressing subgroup of melanomas compared to the lowest PD-1 expressors, whereas densities of IBA1+ cells (macrophages) were similar in both groups. CD79a+ cell numbers were low for both groups. As in human melanoma, overexpression of the PD-1/PD-L1/PD-L2 axis is a common feature of canine melanoma. High expression of PD-1 and PD-L1 correlates with increased numbers of CD3+ cells. Additionally, the high level of IBA1+ cells in melanomas with low PD-1 expression and low CD3+ cells levels suggest that the expression of checkpoint molecules is modulated by interactions between T cells and cancer cells rather than histiocytes.