Digital gene expression analysis of NSCLC-patients reveals strong immune pressure, resulting in an immune escape under immunotherapy

Background Immune checkpoint inhibitors (ICIs) are currently one of the most promising therapy options in the field of oncology. Although the first pivotal ICI trial results were published in 2011, few biomarkers exist to predict their therapy outcome. PD-L1 expression and tumor mutational burden (TMB) were proven to be sometimes-unreliable biomarkers. We have previously suggested the analysis of processing escapes, a qualitative measurement of epitope structure alterations under immune system pressure, to provide predictive information on ICI response. Here, we sought to further validate this approach and characterize interactions with different forms of immune pressure. Methods We identified a cohort consisting of 48 patients with advanced non-small cell lung cancer (NSCLC) treated with nivolumab as ICI monotherapy. Tumor samples were subjected to targeted amplicon-based sequencing using a panel of 22 cancer-associated genes covering 98 mutational hotspots. Altered antigen processing was predicted by NetChop, and MHC binding verified by NetMHC. The NanoString nCounter® platform was utilized to provide gene expression data of 770 immune-related genes. Patient data from 408 patients with NSCLC were retrieved from The Cancer Genome Atlas (TCGA) as a validation cohort. Results The two immune escape mechanisms of PD-L1 expression (TPS score) (n = 18) and presence of altered antigen processing (n = 10) are mutually non-exclusive and can occur in the same patient (n = 6). Both mechanisms have exclusive influence on different genes and pathways, according to differential gene expression analysis and gene set enrichment analysis, respectively. Interestingly, gene expression patterns associated with altered processing were enriched in T cell and NK cell immune activity. Though both mechanisms influence different genes, they are similarly linked to increased immune activity. Conclusion Pressure from the immune system will lay the foundations for escape mechanisms, leading to acquisition of resistance under therapy. Both PD-L1 expression and altered antigen processing are induced similarly by pronounced immunoactivity but in different context. The present data help to deepen our understanding of the underlying mechanisms behind those immune escapes.

Conserved angio-immune subtypes of the cancer microenvironment predict response to immune checkpoint blockade therapy

Tumor microenvironment (TME) shapes the tumor progression and therapy outcome. Particularly, tumor angiogenesis and immunity impact the effect of immune checkpoint blockade (ICB) therapy. Here, we analyzed the transcriptome from 11,069 patients from The Cancer Genome Atlas (TCGA) to assess 91 functional gene sets corresponding to endothelial and T-cell activity. Intriguingly, TME across 30 non-hematological tumors can be classified into three distinct conserved angio-immune subtypes: high angiogenesis with low immune activity, low angiogenesis with high immune activity, and the one in-between. Remarkably, patients displaying TME with poor angiogenic activity with corresponding high immune activity show the most significant responses to ICB therapy in many cancer types. Notably, re-evaluation of the Javelin Renal 101, renal cell carcinoma clinical trial, provided compelling evidence that the baseline angiogenic state is critical in determining responses to checkpoint blockade. This study offers a clear rationale for incorporating baseline angiogenic state for ICB treatment decision-making.

Plant Virus Nanoparticles for Anti-cancer Therapy

Plant virus nanoparticles (VNPs) are inexpensive to produce, safe, biodegradable and efficacious as treatments. The applications of r plant virus nanoparticles range from epitope carriers for vaccines to agents in cancer immunotherapy. Both VNPs and virus-like particles (VLPs) are highly immunogenic and are readily phagocytosed by antigen presenting cells (APCs), which in turn elicit antigen processing and display of pathogenic epitopes on their surfaces. Since the VLPs are composed of multiple copies of their respective capsid proteins, they present repetitive multivalent scaffolds which aid in antigen presentation. Therefore, the VLPs prove to be highly suitable platforms for delivery and presentation of antigenic epitopes, resulting in induction of more robust immune response compared to those of their soluble counterparts. Since the tumor microenvironment poses the challenge of self-antigen tolerance, VLPs are preferrable platforms for delivery and display of self-antigens as well as otherwise weakly immunogenic antigens. These properties, in addition to their diminutive size, enable the VLPs to deliver vaccines to the draining lymph nodes in addition to promoting APC interactions. Furthermore, many plant viral VLPs possess inherent adjuvant properties dispensing with the requirement of additional adjuvants to stimulate immune activity. Some of the highly immunogenic VLPs elicit innate immune activity, which in turn instigate adaptive immunity in tumor micro-environments. Plant viral VLPs are nontoxic, inherently stable, and capable of being mass-produced as well as being modified with antigens and drugs, therefore providing an attractive option for eliciting anti-tumor immunity. The following review explores the use of plant viruses as epitope carrying nanoparticles and as a novel tools in cancer immunotherapy.

Pattern Recognition Receptors (PRRs) in Macrophages Possess Prognosis and Immunotherapy Potential for Melanoma

BackgroundPattern recognition receptors (PRRs) family plays a vital role in the initial stage of innate immune response and the subsequent activation of adaptive immunity. Increasing evidences have indicated that several PRRs play critical roles in the progress of inflammation and tumorigenesis. However, the comprehensive significance of PRRs family in clinical prognosis of different cancers is still elusive.MethodsWe analyzed expression of 20 canonical PRRs in tumor samples from 9502 patients of 33 tumor types. Next, we used expression profiles of PRRs in skin cutaneous melanoma (SKCM) to build a Cox prognosis model. Then, we analyzed immune infiltration features and immune activity of high risk score and low risk score patients. Finally, we analyzed the single-cell sequencing data of different cancers and detected the expression of PRRs in mouse melanoma model to identify PRRs-expressing cell types.ResultsWe found PRRs had a significantly positive correlation with prognosis in SKCM rather than other tumors, and PRR-based Cox model had a much better prognosis potential than any single PRR. Further analysis shows risk score could indicate immunocyte infiltration and immune activity in SKCM. We also found the expressions of some PRR genes were highly correlated with the expression of immune checkpoints molecules in SKCM, indicating they could be indicators for clinical immune therapy. Finally, we found only in SKCM samples, the expression of PRRs is especially high in a subpopulation of macrophages with a trait of CD206 low expression, probably explaining why PRRs have prognosis potential in melanoma.ConclusionsOur study reveals PRR family in macrophages has a positive prognosis potential in melanoma and could be valuable for clinical prognosis and immune therapy.

47 Tertiary lymphoid structures (TLS) in desmoplastic melanomas (DM) differ from non-DM-associated TLS by their intratumoral location and enhanced immune activity

BackgroundTertiary lymphoid structures (TLS) are ectopic lymphoid organs that are localized near tumors and other sites of inflammation, and are commonly believed to support antitumor immunity. We previously published studies that show that most desmoplastic melanomas contain TLS, and that TLS in cutaneous metastatic melanomas varied widely in maturation state, in proportions of proliferating T and B cells, and in markers of B cell function, including AID and CD21. Thus, we hypothesized that there may be diversity in TLS function, or immunologic activity, among melanomas. To address this hypothesis, we evaluated TLS in primary desmoplastic melanomas (DM), and non-desmoplastic melanomas (non-DM) for markers of cell proliferation which are indicative of early immune activity.MethodsDM and non-DM tumor specimens, which included primary melanomas (PM), and cutaneous metastatic melanomas (CMM), were evaluated for TLS by multiplex Immunofluorescence histology, by staining for CD20, CD8, PNAd, Ki67, FoxP3, and DAPI. Lymphoid aggregates were identified in 20x spectrally unmixed images by visual inspection and identified as TLS if possessing organized T-cell and B-cell regions in addition to high endothelial venule-like vasculature (PNAd+). TLS were identified in 30 out of 64 screened (48%) CMM, 4/4 non-DM PM, and 8 out of 11 screened (73%) DM. Immune cells localized in TLS were enumerated using Halo software (Indica Labs). Mann-Whitney tests were used for statistical assessments.ResultsDM commonly contain a dense network of fibroblasts and associated stroma, which are not typical for other non-DM (PM and CMM). TLS in DM are located throughout the tumors, intratumorally, in sharp distinction from the peritumoral location of TLS in non-DM. Furthermore, when compared to TLS of non-DM (PM and CMM), TLS of DM contain increased densities of CD20+ B cells (PM p=0.007; CMM p<0.0001) and CD8+ T cells (PM p=0.017; CMM p=0.0006), and a higher proportion of proliferating (Ki67+) CD20+ B cells (PM p=0.04; CMM p=0.009).ConclusionsRecently published studies have identified tumor-associated fibroblasts as the likely initiating cells for TLS formation in murine melanomas. The intratumoral location of TLS in DM puts them in close proximity to the dense fibroblasts and desmoplastic stroma in these tumors, which may be responsible for their intratumoral location. The increased density of B and T cells, and higher proportion of proliferating (Ki67+) B cells, in DM than in non-DM, suggests that there may be greater immune activation, increased germinal center maturation, or less regulation in TLS of DM.Ethics ApprovalApproval was obtained for these studies under IRB protocol #’s 10598 and 19694.

A Six-Immune-Related Genes Prognostic Signature for Glioblastoma

Background: Glioblastoma (GBM) multiforme is a common malignant brain tumor with high mortality. It is urgently necessary to develop a new treatment because traditional approaches have reached a bottleneck.Purpose: Here we created an immune-related gene (IRGs)-based prognostic signature to comprehensively define the prognosis of glioblastoma (GBM).Methods: Glioblastoma samples were abstracted from the Chinese Glioma Genome Atlas (CGGA) and the Gene Expression Omnibus (GEO). We retrieved IRGs from the ImmProt data resource. Univariate Cox analysis was adopted to determine the prognostically remarkable IRGs for individual with GBM. The prognostically optimal IRGs were determined via LASSO regression, and predictive model created. Besides, the association of specific factors with the overall survival (OS) of individuals with GBM was explored via multivariate Cox-regression. Lastly, we constructed a predictive nomogram integrating the independent predictive factors to determine the one-, two-, and three-year OS likelihoods of individuals with GBM. Additionally,gene set enrichment analysis(GSEA) and single sample GSEA(ssGSEA) were performed to understand the correlation between the risk score and immune activity.Results: Overall, 273 IRGs which exhibited differential expression were identified in GBM tumor in contrast with the non-malignant samples. Of these 273 IRGs, only six were remarkably linked to OS of individuals with GBM, which were employed in constructing the predictive signature. The GBM were categorized into either the high-risk GBM group or the low-risk GBM group. There were remarkable differences between the high-risk GBM and the low-risk GBM groups regarding OS. The AUC for predicting one-,two-, and three-year OS in training set was 0.610,0.698 and 0.694.In line with the AUC of validation set was 0.608,0.692 and 0.678.Besides,the results of ssGSEA showed the score of prognostic signature is closely related to immune activity.Conclusion: Herein, a robust predictive model based on IRGs was created to estimate the diversity of OS likelihoods in GBM patients, as well as aid future clinical research.