Immune Checkpoint Inhibitor Immunotherapy Research Articles

Single-Cell Analyses of a Novel Mouse Urothelial Carcinoma Model Reveal a Role of Tumor-Associated Macrophages in Response to Anti-PD-1 Therapy.

Simple SummaryImmune checkpoint inhibitors (ICI) have been standard care for advanced bladder cancer patients who fail or are not eligible for chemotherapy. There is an urgent need for preclinical models to study immunotherapy responses because the majority of patients with advanced bladder cancer do not respond to ICI therapy. To develop an authentic bladder cancer preclinical model in immunocompetent mice, we generated a stable organoid and xenograft model by the ex vivo transduction of adenovirus-expressing Cre recombinase into normal mouse urothelial organoid cells isolated from mice engineered with LoxP sites flanking the Trp53, Pten, and Rb1 genes. The triple knockout (TKO) urothelial organoids developed into high-grade urothelial carcinomas of the basal subtype, both in vitro and in vivo. To study ICI anti-PD-1 treatment responses, the TKO tumors were treated with an anti-PD-1 antibody or a control IgG2a. A mixed pattern of treatment responses was observed. Single-cell analyses of immune cells revealed significantly different infiltration of immune cells between non-responders and responders. A higher percentage of immune cell infiltration, including macrophage and T cell tumor infiltration, was detected in responders compared to non-responders. Overall, these findings suggest that this preclinical TKO model will be a useful tool to study the factors influencing bladder cancer immunotherapy responses and suggest that modulating tumor-associated macrophages may help overcome ICI immunotherapy resistance.Approximately 80% of patients with advanced bladder cancer do not respond to immune checkpoint inhibitor (ICI) immunotherapy. Therefore, there is an urgent unmet need to develop clinically relevant preclinical models so that factors governing immunotherapy responses can be studied in immunocompetent mice. We developed a line of mouse triple knockout (TKO: Trp53, Pten, Rb1) urothelial carcinoma organoids transplanted into immunocompetent mice. These bladder tumors recapitulate the molecular phenotypes and heterogeneous immunotherapy responses observed in human bladder cancers. The TKO organoids were characterized in vivo and in vitro and compared to the widely used MB49 murine bladder cancer model. RNAseq analysis of the TKO tumors demonstrated a basal subtype. The TKO xenografts demonstrated the expression of urothelial markers (CK5, CK7, GATA3, and p63), whereas MB49 subcutaneous xenografts did not express urothelial markers. Anti-PD-1 immunotherapy resulted in a mixed pattern of treatment responses for individual tumors. Eight immune cell types were identified (basophils, B cells, dendritic cells, macrophages, monocytes, neutrophils, NK cells, and T cells) in ICI-treated xenografts. Responder xenografts displayed significantly increased immune cell infiltration (15.3%, 742 immune cells/4861 total cells) compared to the non-responder tumors (10.1%, 452 immune cells/4459 total cells, Fisher Exact Test p < 0.0001). Specifically, there were more T cells (1.0% vs. 0.4%, p = 0.002) and macrophages (8.6% vs. 6.4%, p = 0.0002) in responder xenografts than in non-responder xenografts. In conclusion, we have developed a novel preclinical model that exhibits a mixed pattern of response to anti-PD-1 immunotherapy. The higher percentage of macrophage tumor infiltration in responders suggests a potential role for the innate immune microenvironment in regulating ICI treatment responses.

MP06-09 SINGLE CELL ANALYSES OF MURINE UROTHELIAL CARCINOMA CELLS REVEAL A ROLE OF TUMOR-ASSOCIATED MACROPHAGES IN RESPONSE TO ANTI-PD1 THERAPY

You have accessJournal of UrologyCME1 May 2022MP06-09 SINGLE CELL ANALYSES OF MURINE UROTHELIAL CARCINOMA CELLS REVEAL A ROLE OF TUMOR-ASSOCIATED MACROPHAGES IN RESPONSE TO ANTI-PD1 THERAPY Dongbo Xu, Li Wang, Yali Zhang, Yanqing Wang, Xiaojing Zhang, Yue Wu, Gary Smith, Bo Xu, Prashant Singh, Yuesheng Zhang, David Goodrich, Jianmin Wang, and Qiang Li Dongbo XuDongbo Xu More articles by this author , Li WangLi Wang More articles by this author , Yali ZhangYali Zhang More articles by this author , Yanqing WangYanqing Wang More articles by this author , Xiaojing ZhangXiaojing Zhang More articles by this author , Yue WuYue Wu More articles by this author , Gary SmithGary Smith More articles by this author , Bo XuBo Xu More articles by this author , Prashant SinghPrashant Singh More articles by this author , Yuesheng ZhangYuesheng Zhang More articles by this author , David GoodrichDavid Goodrich More articles by this author , Jianmin WangJianmin Wang More articles by this author , and Qiang LiQiang Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Approximately 80% of patients with advanced bladder cancer do not respond to immune checkpoint inhibitor (ICI) immunotherapy. The molecular mechanism of ICI treatment response remains unknown largely due to the lack of clinically relevant immunocompetent mouse models. METHODS: We developed a syngeneic triple knockout (TKO: Trp53/Pten/Rb1) bladder cancer model in immunocompetent C57 BL/6J mice. Mouse urothelial cells (Trp53f/f, Ptenf/f, Rb1f/f) were transfected ex vivo with Ad5CMVCre (Adenovirus Cre recombinase driven by a CMV promoter). The TKO urothelial cells were subcutaneously injected into immunocompetent C57 BL/6J for tumor formation. The tumors were treated with an anti-PD1 antibody (BioXcell, RMP1-14, 200 μg, intraperitoneally twice weekly) (n=9) or a control IgG (n=9). Single cell analyses were performed for tumor xenografts from control IgG (n=2), responders (30% less than the median of control tumors) (n=2) and non-responders (n=2) using PTPRC (also named CD45) expression as an immune cell marker. Seurat package was used for data analysis including dimension reduction, cell clustering, and identification of cell markers. SingleR package was utilized to identify the cell types. Immunohistochemistry (IHC) of CD8 and F4/80 was performed to identify T cells and macrophages respectively. RESULTS: Anti-PD-1 immunotherapy resulted in a mixed pattern of treatment responses in individual tumors. Eight clusters of immune cells were identified (basophils, B cells, dendritic cells, macrophages, monocytes, neutrophils, NK cell, and T cells) in ICI treated xenografts. Responder xenografts displayed significantly increased immune cell infiltration (15.2%, 742 immune cells/4861 total cells) compared to the non-responder tumors (10.1%, 452 immune cells/4459 total cells, Fisher Exact Test p<0.0001). Specifically, there were more T cells (46/4861 vs 18/4459, p=0.002), and macrophages (420/4861 vs 287/4459, p=0.002) in responder xenografts than in non-responder xenografts. Compared to control IgG tumors, a near complete response tumor exhibited an immune-inflamed phenotype with significant infiltration of T cells and macrophages, whereas non-responder tumors showed no significant change of T cells and macrophages infiltration. CONCLUSIONS: The higher percentage of T cells and macrophages tumor infiltration in responders suggests a potential role of innate immune microenvironment for the ICI treatment response. Modulating tumor associated macrophage may overcome the resistance to ICI immunotherapy. Source of Funding: This research work was supported in part by NIH Grants, K08CA252161(Li, Q), P30CA016056 (NCI Cancer Center Core Support Grant), the Roswell Park Alliance Foundation and the Friends of Urology Foundation © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e80 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Dongbo Xu More articles by this author Li Wang More articles by this author Yali Zhang More articles by this author Yanqing Wang More articles by this author Xiaojing Zhang More articles by this author Yue Wu More articles by this author Gary Smith More articles by this author Bo Xu More articles by this author Prashant Singh More articles by this author Yuesheng Zhang More articles by this author David Goodrich More articles by this author Jianmin Wang More articles by this author Qiang Li More articles by this author Expand All Advertisement PDF DownloadLoading ...

Development of a multiplex microbead-based immunoassay for quantitative analysis of mouse immune checkpoint proteins

Abstract Immune checkpoint protein research plays a significant role in improving antitumor immune response as well as achieving a deeper understanding of the immune system and the disease states of inflammation, sepsis, and autoimmunity. Various mouse models have contributed to the clinical successes of immune checkpoint inhibitor immunotherapies and the understanding of checkpoint molecule-related inflammatory processes. Here we report the assay verifications and applications of a newly developed Luminex bead-based mouse immune-oncology checkpoint protein multiplex immunoassay for the quantitative profiling of 28 mouse immune checkpoint molecules and immune regulators (PD-1, PD-L1, PD-L2, CTLA-4/CD152, TIM-3, LAG-3, B7-H2/ICOSL, B7-H3/CD276, BTLA, GITR, HVEM, CD27, CD40, CD80/B7-1, CD226/DNAM-1, 4-1BBL/CD137L/TNFSF9, CD137/4-1BB, 5’-NT/CD73, BCA-1/CXCL-13, CD25/IL-2Rα, Granzyme B, E-cadherin, Galectin-1, Galectin-3, IFNγ, IL-10, TLR-2, and TNFα). We present quantitative protein biomarker profiling of mouse serum/plasma samples and conditioned media and lysates from mouse tumor cell lines, including LLC, B16-F10, MCA205, ID8, MB49, AT3, CT-2A, and YUMM1.7, as well as DC2.4 (dendritic cell line), C2C12 (myoblast cell line), and bEnd.3 (endothelial cell line). Our results demonstrate that this mouse immune checkpoint protein multiplex immunoassay is a powerful research tool for the quantitation of key immune regulator proteins and has potential application in biomarker discovery and translational research.

Dietary low-fiber promotes resistance to immune checkpoint inhibitor immunotherapy in the LSL-KrasG12D Lung Cancer Model

Abstract Dietary fiber interaction with the gut microbiota impact gut microbiota composition and is likely to play a large role in affecting the efficacy of immune checkpoint inhibitors (ICIs) therapy. Here we show that a diet with low-fiber promoted lung tumor development and reduced tumor-bearing animal long-term survival in both prophylactic early disease and established late disease of murine LSL-KrasG12D lung cancer model, which displays low neoantigen burden and is refractory to anti-PD-1 antibody. In this model, the administration of anti-PD-1 in the presence of IL-17 neutralization resulted in tumor suppression, indicating that limited intrinsic neoantigen alone was not the primary determinant of resistance to ICIs therapy. Interestingly, dietary low-fiber led to reduced anti-tumor effect of anti-PD-1 and anti-IL-17 partially in gut microbiota-dependent manner. Fecal microbiota transplantation from mice fed low-fiber diet also enhanced lung tumor burden. An altered immune response with significantly reduced gut lamina propria RORgt+IL-17A+ Th17 cells, lung Foxp3+Treg cells, enhanced lung Th17 cells, but no alteration of lung gdTCR+ cells were found in mice fed low-fiber diet. Furthermore, a low-fiber diet led to significant changes in metabolites including a high level of serine in the plasma of animals. Mice fed normal chow with drinking water containing serine increased lung tumorigenesis. Our study demonstrates a functional interconnection between dietary fiber and spontaneous lung tumor development. Supported by grant from NIH/NIGMS(P20-GM135004)

Combination of interventional oncology local therapies and immunotherapy for the treatment of hepatocellular carcinoma.

Interventional oncology (IO) local therapies of hepatocellular carcinoma (HCC) can activate anti-cancer immunity and it is potentially leading to an anti-cancer immunity throughout the body. For the development of an effective HCC treatment regime, great emphasis has been dedicated to different IO local therapy mediated immune modulation and possible combinations with immune checkpoint inhibitor immunotherapy. In this review paper, we summarize the status of combination of IO local therapy and immunotherapy, as well as the prospective role of therapeutic carriers and locally administered immunotherapy in advanced HCC.

G6PD functions as a metabolic checkpoint to regulate granzyme B expression in tumor-specific cytotoxic T lymphocytes

BackgroundGranzyme B is a key effector of cytotoxic T lymphocytes (CTLs), and its expression level positively correlates with the response of patients with mesothelioma to immune checkpoint inhibitor immunotherapy. Whether...

Current status and future perspective of immune checkpoint inhibitors in colorectal cancer.

Immune checkpoint inhibitors (ICIs), as a subverter of immunotherapy in oncology, are changing all aspects of therapy for malignant tumors, especially their remarkable effects on melanoma and non-small cell lung cancer (NSCLC). For colorectal cancer (CRC), only a small number of patients with high immunogenicity (microsatellite instability-high/mismatch-repair deficient (MSI-H/dMMR)) benefit greatly from ICIs treatment, and most CRC patients with low immunogenicity (microsatellite instability-low/mismatch-repair proficient (MSI-L/pMMR)) do not. Currently, numerous clinical trials are ongoing to improve CRC patients' response to ICIs immunotherapy through better patient selection and novel combination strategies. Thus, this review discusses the current status and latest progress of ICIs treatment in CRC. We expect that these studies can change the pattern of CRC immunotherapy in the future.

Short-Course Palliative Radiotherapy Combined With Immunotherapy for Recurrent/Advanced/Metastatic Head and Neck Cancer

To evaluate tumor response rates in patients treated with a combination of palliative short-course radiotherapy (SCRT) and immune checkpoint inhibitor immunotherapy (IO) in patients with recurrent, advanced or metastatic (R/A/M) head and neck cancer (HNC).We reviewed the medical records of patients treated with short-course palliative intent radiation therapy (RT) (hypofractionated RT in 4-5 fractions) for R/A/M HNC between 2014-2020. Patient, disease, and treatment factors were abstracted from the medical record. Patients were categorized as having been treated with combined immunotherapy and radiotherapy (IO+RT) if the RT was given after at least 1 cycle of IO with at least 1 cycle of IO afterward. Treated gross tumor volume (GTV) was measured prior to and after RT and percent change in GTV volume from baseline to best overall response (BOR) was calculated. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method.In total, 19 patients were treated with SCRT, 7 of whom were treated with combined IO+RT. Primary site included oral cavity (n = 6), larynx/hypopharynx (5), oropharynx (3), sinonasal (2), cutaneous (2), and thyroid (1). Squamous cell histology was most frequent (16/19) with the other 3 comprised of sinonasal intestinal-type adenocarcinoma, mucosal melanoma, and Hürthle cell carcinoma. Disease status included 6 patients with a recurrence, 6 were metastatic, 3 locally advanced/incurable, and 3 recurrent/metastatic. Prior treatments included RT (n = 9) and chemotherapy (6); ECOG performance status was 1-2 in 13 patients and 3-4 in 6 patients. Seventeen patients were treated with "quad-shot" radiotherapy (14.4-14.8 Gy in 4 fractions delivered twice daily) and 2 were treated with 20 Gy in 5 fractions. Median (range) number of IO cycles given pre- and post-RT were 7 (1-11) and 2 (1-21), respectively. Of the 17 evaluable patients for treatment response, 13 patients responded or were stable while 4 had progression. Median pre-RT target volume in the RT cohort was 37.2 (6.14-486.21) cc and 145.28 (24.21-219.51) cc in the IO+RT cohort. Median post-RT target volume in the RT cohort was 28.00 (17.23, 57.630 cc and 58.44 (0.00, 246.56) cc in the IO+RT cohort. Median change in target volume in RT cohort was -42% (range: -100, 259) cc and -54% (-100, 12) cc in the IO+RT cohort. 1-year PFS and OS were 5% and 21%, respectively. 1-year OS was numerically higher for IO+RT patients (43% v. 8%, P = 0.13).In this select cohort of patients with R/A/M HNC, combined IO+SCRT was feasible and showed an improved tumor response as evidenced by greater decrement in post-treatment tumor volume. An ongoing clinical trial (NCT04454489) aims to prospectively evaluate the potential benefit of combined quad-shot radiotherapy with Pembrolizumab to improve response rates to IO alone.

Pan-cancer analysis reveals homologous recombination deficiency score as a predictive marker for immunotherapy responders.

The immune context of the tumor microenvironment (TME) is critical for effective immunotherapy. Nonetheless, DNA-based biomarkers for the immune-sensitive TME and the identification of immune checkpoint inhibitor (ICI) responders are under-explored. This study aims to comprehensively landscape the homologous recombination deficiency (HRD) score, an emerging hallmark for tumor genome instability that triggers immune responsiveness across major cancer types, and to unveil their link to the TME and immunotherapeutic response. The HRD-associated genomic scars were characterized in 9088 tumor samples across 32 cancer types from TCGA. We evaluated the HRD score's performance in classifying ICI responders using an independent breast cancer cohort (GSE87049) and 11 in vivo murine mammary tumor models treated with anti-PD1/CTLA4 regimen (GSE124821). This study revealed a broad association between HRD-high genotype and neoantigenesis in the major cancer types including bladder cancer, breast cancer, head and neck squamous carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, and sarcoma. Tumors with high HRD score bears increased leukocyte infiltration and lymphocyte fraction and demonstrated immune-sensitive microenvironment. The tumor immune dysfunction and exclusion (TIDE) model further confirmed HRD score-high genotype as a potential predictor for ICI immunotherapy responders in breast cancer. In conclusion, tumors with high HRD score exhibit an immune-sensitive TME. The HRD-high genotype is a promising marker for identifying ICI therapy responders among breast cancer patients.

Current Status of Immune Checkpoint Inhibitor Immunotherapy for Lung Cancer.

Immunotherapy is a major breakthrough in the treatment of cancer in recent years. Immune checkpoint inhibitors (ICIs) including programmed death-ligand 1 (PD-L1)/programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) have been used for different histologic types of cancer including primary lung cancer that represents the most common and fatal cancer globally. Among ICI immunotherapy agents, atezolizumab, durvalumab, ipilimumab, nivolumab, and pembrolizumab are currently used as standard-of-care (SOC) treatment for metastatic or earlier stages of lung cancer. Major issues of ICI immunotherapy in lung cancer comprise the use of immune biomarkers prior to ICI therapy, selection of ICI agents, combination of ICIs/chemotherapy, combination of ICIs/radiotherapy, sequence of tyrosine kinase inhibitor (TKI) targeted therapy and ICI immunotherapy, sequence of chemotherapy and ICI immunotherapy, treatment duration of ICI regimen and ICI therapy for different histopathology, stage, PD-L1, and performance status. Based on the contemporary major clinical trials and authoritative guidelines, the objective of this review is to present an overview of the current status of ICI immunotherapy in lung cancer.

Survival Outcomes of Salvage Metastasectomy After Failure of Modern-Era Systemic Therapy for Melanoma.

Metastasectomy for selected patients with melanoma was associated with improved survival in the era before effective systemic therapy. Emerging evidence shows that these benefits persist even in this era of BRAF-targeted therapy and immune checkpoint inhibitor immunotherapy. This study aimed to evaluate the outcomes of salvage metastasectomy after failure of systemic therapy. Stage 3 or 4 melanoma patients with extracranial disease progression after at least 4 weeks of systemic treatment between 2009 and 2020 were identified and categorized as resected to no evidence of disease (NED), non-progressive residual disease (NPRD), or progressive residual disease (PRD). Systemic therapy was stratified into BRAF-targeted therapy, immune checkpoint inhibitor immunotherapy, or both. The end points of overall survival (OS), progression-free survival (PFS), and locoregional disease control (LRC) were assessed using Kaplan-Meier curves. Uni- and multivariable Cox regression procedures were used to examine factors associated with OS, PFS and LRC. The study enrolled 190 patients. Among all the patients, the 5-year OS from metastatectomy was 52%, the 3-year PFS was 21%, and the 5-year LRC was 61%. After resection to NED, NPRD, and PRD, the 5-year OS values were 69%, 62% and 8%, respectively. Fewer lines of preoperative therapy, use of preoperative immunotherapy, and resection to NED were predictors of improved OS. After resection to NED, NPRD, and PRD, the 3-year PFS values were 23%, 24% and 10%, and the 5-year LRC values were 61%, 72% and 34%, respectively. Salvage metastasectomy was associated with durable survival and disease control, particularly after resection to NED, preoperative immunotherapy, and fewer lines of preoperative systemic therapy.

WDR5-H3K4me3 epigenetic axis regulates OPN expression to compensate PD-L1 function to promote pancreatic cancer immune escape

BackgroundDespite PD-L1 (Programmed death receptor ligand-1) expression on tumor cells and cytotoxic T lymphocytes tumor infiltration in the tumor microenvironment, human pancreatic cancer stands out as one of the human...

Precise medicine of programmed cell death-1/programmed cell death 1 ligand 1 inhibitor immunotherapy combined radiotherapy for inoperable advanced lung cancer: A protocol for systematic review and meta-analysis.

Background:Programmed cell death-1/programmed cell death 1 ligand 1 (PD-1/PD-L1) inhibitors are a group of immune checkpoint inhibitors immunotherapy for cancer treatment. These immune checkpoint inhibitors are becoming first-line treatments for several types of cancer. Radiotherapy for cancer is a traditional treatment and the therapeutic effect is not satisfactory due to the side effect of chemotherapeutic drugs. This study aims to evaluate the efficacy and safety of PD1/PD-L1 inhibitor immunotherapy combined chemotherapy for inoperable advanced lung cancer.Methods:We will utilize PubMed, PubMed Central, EMbase, Medline, CNKI, WAN FANG Database, and Web of Science to screen eligible studies published from January 1, 2015 to December 30, 2020. Two reviewers will extract data and evaluate the risk of bias independently. The quality of the included studies will be evaluated using the RevMan 5.3 software for data analysis.Results:This review will summarize high-quality evidence of trials to evaluate the precise medicine efficacy and safety of PD1/PD-L1 inhibitor combined radiotherapy for inoperable advanced lung cancer.Conclusions:The findings of the systematic review will provide scientific evidence of the efficacy and safety of PD1/PD-L1 inhibitor combined radiotherapy for inoperable advanced lung cancer to guide the clinician's drug use.Ethics and dissemination:Not applicable.INPLASY registration number:INPLASY202140123.

Hepatocellular carcinoma in viral and autoimmune liver diseases: Role of CD4+ CD25+ Foxp3+ regulatory T cells in the immune microenvironment.

More than 90% of cases of hepatocellular carcinoma (HCC) occurs in patients with cirrhosis, of which hepatitis B virus and hepatitis C virus are the leading causes, while the tumor less frequently arises in autoimmune liver diseases. Advances in understanding tumor immunity have led to a major shift in the treatment of HCC, with the emergence of immunotherapy where therapeutic agents are used to target immune cells rather than cancer cells. Regulatory T cells (Tregs) are the most abundant suppressive cells in the tumor microenvironment and their presence has been correlated with tumor progression, invasiveness, as well as metastasis. Tregs are characterized by the expression of the transcription factor Foxp3 and various mechanisms ranging from cell-to-cell contact to secretion of inhibitory molecules have been implicated in their function. Notably, Tregs amply express checkpoint molecules such as cytotoxic T lymphocyte-associated antigen 4 and programmed cell-death 1 receptor and therefore represent a direct target of immune checkpoint inhibitor (ICI) immunotherapy. Taking into consideration the critical role of Tregs in maintenance of immune homeostasis as well as avoidance of autoimmunity, it is plausible that targeting of Tregs by ICI immunotherapy results in the development of immune-related adverse events (irAEs). Since the use of ICI becomes common in oncology, with an increasing number of new ICI currently under clinical trials for cancer treatment, the occurrence of irAEs is expected to dramatically rise. Herein, we review the current literature focusing on the role of Tregs in HCC evolution taking into account their opposite etiological function in viral and autoimmune chronic liver disease, and we discuss their involvement in irAEs due to the new immunotherapies.

Association of progression free survival with the timing of genomic testing in non-small cell lung cancer: Evidence from the GuardantInform clinic-genomic database.

e21143 Background: The clinical utility of ctDNA analysis for biomarker identification in advanced non-small cell lung cancer (NSCLC) has been established, supporting clinical adoption of this testing modality. All FDA-approved, immune-checkpoint inhibitor (ICI) immunotherapies require the prerequisite of negativity for ALK rearrangement and EGFR mutation, or prior toxicity or progression on oral therapies targeting mutations in these genes. Despite these recommendations and support from the American Society of Clinical Oncology and National Comprehensive Cancer Network, broad-panel marker testing for targeted therapy options in NSCLC continues to be underperformed. Data are even limited on when those tests are typically conducted and the effects of timing on the treatment outcomes. Methods: The GuardantINFORM™ clinic-genomic database was interrogated for patients over the age of 18 having a Guardant360 test positive for EGFR mutations of L858R or exon 19 after 2016 and known to have a confirmed diagnosis of lung cancer one year prior to testing. Patients had to have at least three claims prior to, and 90 days of follow-up after, their test. Patients were stratified into three groups by pre- and post-test treatment: 1) patients treated with an EGFR tyrosine kinase inhibitor (TKI) as a first-line treatment after the test, with no other chemotherapy, ICI immunotherapy (IO), or targeted therapy before the test; 2) patients with other first-line treatments prior to the test and then EGFR TKI immediately following the test; and 3) patients treated with ICI or chemotherapy after the test and before EGFR TKI or patients not treated after the test. Real-world time to next treatment (rwTTN) was defined as the index date to the treatment different than the index therapy. Progression free survival (PFS) time was defined as rwTTN or time to death, whichever came earlier. Patients who experienced adverse events (AEs) associated with chemotherapy or IO were reported as a percentage (p-value&lt;0.001). Results: Among the 3 cohorts of patients (384 in each group who were matched on age with difference &lt; 3, gender, and follow-up time with difference &lt; 4 months) identified, a statistically significant difference in PFS was discovered between group 1 ( EGFR TKI as first-line treatment) and the other two groups based on the Cox proportional hazard model [hazard ratio=1.8, p-value&lt;0.001, median survival time for group 1= 26 months (95% CI 23-29) vs. 17 months (95% CI 14-19)]. No difference existed in PFS between groups 2 and 3. The proportion of patients experiencing treatment-associated AEs was lowest in group 1 (13.5% vs 15.9% in group 2 vs 30.2% in group 3 for ICI-related AEs, 16.9% vs 23.2% vs 38.5% for chemo-related AEs, p-value&lt;0.001). Conclusions: Performing genomic testing sooner, as early as first-line treatment, may improve the treatment response for patients with NSCLC.

Multi-Omics Perspective Reveals the Different Patterns of Tumor Immune Microenvironment Based on Programmed Death Ligand 1 (PD-L1) Expression and Predictor of Responses to Immune Checkpoint Blockade across Pan-Cancer.

Immune checkpoint inhibitor (ICI) therapies have shown great promise in cancer treatment. However, the intra-heterogeneity is a major barrier to reasonably classifying the potential benefited patients. Comprehensive heterogeneity analysis is needed to solve these clinical issues. In this study, the samples from pan-cancer and independent breast cancer datasets were divided into four tumor immune microenvironment (TIME) subtypes based on tumor programmed death ligand 1 (PD-L1) expression level and tumor-infiltrating lymphocyte (TIL) state. As the combination of the TIL Z score and PD-L1 expression showed superior prediction of response to ICI in multiple data sets compared to other methods, we used the TIL Z score and PD-L1 to classify samples. Therefore, samples were divided by combined TIL Z score and PD-L1 to identify four TIME subtypes, including type I (3.24%), type II (43.24%), type III (6.76%), and type IV (46.76%). Type I was associated with favorable prognosis with more T and DC cells, while type III had the poorest condition and composed a higher level of activated mast cells. Furthermore, TIME subtypes exhibited a distinct genetic and transcriptional feature: type III was observed to have the highest mutation rate (77.92%), while co-mutations patterns were characteristic in type I, and the PD-L1 positive subgroup showed higher carbohydrates, lipids, and xenobiotics metabolism compared to others. Overall, we developed a robust method to classify TIME and analyze the divergence of prognosis, immune cell composition, genomics, and transcriptomics patterns among TIME subtypes, which potentially provides insight for classification of TIME and a referrable theoretical basis for the screening benefited groups in the ICI immunotherapy.

Characterization of the Prognostic m6A-Related lncRNA Signature in Gastric Cancer.

N6-methyladenosine (m6A) is a common form of mRNA modification regulated by m6A RNA methylation regulators and play an important role in the progression of gastric cancer (GC). However, the prognostic role of m6A-related lncRNA in gastric cancer has not been fully explored. This study aims at exploring the biological function and prognostic roles of the m6A-related lncRNA signature in gastric cancer. A total of 800 m6A-related lncRNAs were identified through Pearson correlation analysis between m6A regulators and all lncRNAs. Eleven m6A-related lncRNA signatures were identified through a survival analysis and the Kaplan-Meier (KM) curve analysis results suggest that patients in the low-risk group have a better overall survival (OS) and disease-free survival (DFS) outcome than the high-risk group. Also, the lncRNA signature can serve as an independent prognostic factor for OS and DFS. The gene set enrichment analysis (GSEA) result suggests that patients in the high-risk group were mainly enriched in the ECM receptor interaction, focal adhesion, and cytokine-cytokine receptor interaction pathway, while the low-risk group was characterized by the base excision repair pathway. We further constructed an individualized prognostic prediction model via the nomogram based on the independent prognostic factor for the OS and DFS, respectively. In addition, some candidate drugs aimed at GC risk group differentiation were identified using the Connective Map (CMAP) database. Lastly, four subgroups (C1, C2, C3, and C4) were identified based on the m6A-related lncRNA expression, through a consensus clustering algorithm. Among them, C1 and C2 have a greater likelihood to respond to immune checkpoint inhibitor immunotherapy, suggesting that the C1 and C2 subgroup might benefit from immunotherapy. In conclusion, the m6A-related lncRNA signature can independently predict the OS and DFS of GC and may aid in development of personalized immunotherapy strategies.

Regulatory T-cell Transcriptomic Reprogramming Characterizes Adverse Events by Checkpoint Inhibitors in Solid Tumors.

Immune checkpoint inhibitors (ICI), which target immune regulatory pathways to unleash antitumor responses, have revolutionized cancer immunotherapy. Despite the remarkable success of ICI immunotherapy, a significant proportion of patients whose tumors respond to these treatments develop immune-related adverse events (irAE) resembling autoimmune diseases. Although the clinical spectrum of irAEs is well characterized, their successful management remains empiric. This is in part because the pathogenic mechanisms involved in the breakdown of peripheral tolerance and induction of irAEs remain elusive. Herein, we focused on regulatory T cells (Treg) in individuals with irAEs because these cells are vital for maintenance of peripheral tolerance, appear expanded in the peripheral blood of individuals with cancer, and abundantly express checkpoint molecules, hence representing direct targets of ICI immunotherapy. Our data demonstrate an intense transcriptomic reprogramming of CD4+CD25+CD127- Tregs in the blood of individuals with advanced metastatic melanoma who develop irAEs following ICI immunotherapy, with a characteristic inflammatory, apoptotic, and metabolic signature. This inflammatory signature was shared by Tregs from individuals with different types of cancer developing irAEs and individuals with autoimmune diseases. Our findings suggest that inflammatory Treg reprogramming is a feature of immunotherapy-induced irAEs, and this may facilitate translational approaches aiming to induce robust antitumor immunity without disturbing peripheral tolerance.

Phenotypic and Proteomic Analysis Identifies Hallmarks of Blood Circulating Extracellular Vesicles in NSCLC Responders to Immune Checkpoint Inhibitors.

Simple SummaryPurpose of this study was to investigate the prognostic and predictive role of blood circulating extracellular vesicles (EVs) in patients with advanced non-small cell lung cancer treated with immunotherapy. A newly optimized flow cytometry protocol was applied for identification and subtyping of blood circulating EVs in a total cohort of 59 NSCLC patients, which included 31 patients treated with anti-PD-1/PD-L1 agents and 28 patients treated with traditional chemotherapy. Our results show that pre-treatment concentration of blood circulating endothelial-derived EVs was correlated with overall survival and clinical response in patients treated with immunotherapy. Additionally, proteomic analysis of purified blood circulating EVs indicated differences in EV protein cargo between responders and non-responders to immunotherapy. These findings may pave the way to the identification of novel immunotherapy biomarkers in patients with advanced NSCLC.Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the prognostic and predictive value of circulating endothelial and leukocyte-derived extracellular vesicles (EV) in patients with advanced NSCLC treated with anti-PD-1/PD-L1 agents. In addition, the relationship between total blood circulating EV proteome and response to ICIs was investigated. An optimized flow cytometry method was employed for the identification and subtyping of blood circulating EVs in 59 patients with advanced NSCLC. Blood samples were collected from patients receiving anti-PD-1/PD-L1 inhibitors (n = 31) or chemotherapy (n = 28). An exploratory proteomic analysis of sorted blood EVs was conducted in a subset of patients. Our results show that a low blood concentration of circulating endothelial-derived EVs before treatment was strongly associated to longer overall survival (p = 0.0004) and higher disease control rate (p = 0.045) in patients treated with ICIs. Interestingly, shotgun proteomics revealed that EVs of responders to anti-PD-1 therapy had a specific protein cargo before treatment. In addition, EV protein cargo was specifically modulated during immunotherapy. We identified a previously unknown association between circulating endothelial-derived extracellular vesicle concentration and immunotherapy-related clinical outcomes. We also observed differences in circulating extracellular vesicle proteome according to anti-PD-1-based treatment response in NSCLC patients. Overall, these results may contribute to the identification of novel circulating biomarkers for rational immunotherapy approaches in patients affected by NSCLC.

The Periphery of Salivary Gland Carcinoma Tumors Reveals a PD-L1/PD-1 Biomarker Niche for the Evaluation of Disease Severity and Tumor-Immune System Interplay.

The treatment options for patients with advanced salivary gland cancers (SGCs) are limited. Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. However, the response to ICI immunotherapy is largely driven by the immune cell signatures within the tumor tissue and the para-tumoral tissue compartments. To date, there are no data on the expression of programed cell death protein-1/programed cell death protein-ligand 1 (PD-1/PD-L1) in SGC, which may enable the implementation of ICI immunotherapy for this disease. Thus, we performed an immunohistochemical analysis of PD-1 and PD-L1 expression in tumor cells and tumor-infiltrating immune cells (TIICs) in the tumor center and periphery of 62 SGC patients. The tumor periphery showed significantly higher expression of PD-L1 in tumor cells than in TIICs. Moreover, peripheral TIICs had significantly higher PD-1 expression than peripheral tumor cells. PD-1-positive tumor cells were detected exclusively in the tumor center of high-grade tumors, and most importantly, the presence of lymph node (LN) metastases and primary tumor stage significantly correlated with the presence of PD-L1-positive tumor cells in the tumor periphery. The PD-1/PD-L1 molecular signatures in SGC are clustered predominantly in the tumor periphery, reflect disease severity, and may predict the response to ICI immunotherapy in SGC patients.