Prognostic Immunophenotyping Clusters of Clear Cell Renal Cell Carcinoma Defined by the Unique Tumor Immune Microenvironment

Background: The tumor microenvironment affects the occurrence and development of cancers, including clear cell renal cell carcinoma (ccRCC). However, how the immune contexture interacts with the cancer phenotype remains unclear.Methods: We identified and evaluated immunophenotyping clusters in ccRCC using machine-learning algorithms. Analyses for functional enrichment, DNA variation, immune cell distribution, association with independent clinicopathological features, and predictive responses for immune checkpoint therapies were performed and validated.Results: Three immunophenotyping clusters with gradual levels of immune infiltration were identified. The intermediate and high immune infiltration clusters (Clusters B and C) were associated with a worse prognosis for ccRCC patients. Tumors in the immune-hot Clusters B and C showed pro-tumorigenic immune infiltration, and these patients showed significantly worse survival compared with patients in the immune-cold Cluster A in the training and testing cohorts (n = 422). In addition to distinct immune cell infiltrations of immunophenotyping, we detected significant differences in DNA variation among clusters, suggesting a high degree of genetic heterogeneity. Furthermore, expressions of multiple immune checkpoint molecules were significantly increased. Clusters B and C predicted favorable outcomes in 64 ccRCC patients receiving immune checkpoint therapies from the FUSCC cohort. In 360 ccRCC patients from the FUSCC validation cohort, Clusters B and C significantly predicted worse prognosis compared with Cluster A. After immunophenotyping of ccRCC was confirmed, significantly increased tertiary lymphatic structures, aggressive phenotype, elevated glycolysis and PD-L1 expression, higher abundance of CD8+ T cells, and TCRn cell infiltration were found in the immune-hot Clusters B and C.Conclusion: This study described immunophenotyping clusters that improved the prognostic accuracy of the immune contexture in the ccRCC microenvironment. Our discovery of the novel independent prognostic indicators in ccRCC highlights the relationship between tumor phenotype and immune microenvironment.

Case Report: Response to Immunotherapy, Can Radiotherapy Be a Troublemaker?

Immunotherapy has dramatically changed the treatment landscape for several tumor types. However, the impact of previous radiotherapy (RT) on response to immunotherapy is still unknown. We report the case of a 58-year-old female diagnosed with a squamous anal cell carcinoma previously treated with RT and having a dissociated response to anti-PD1 agent. An extensive analysis of the immune contexture performed on the tissue collected from both previously RT-treated and RT-untreated lesions confirmed differences on immune microenvironment, highlighting the potential impact of radiotherapy on the immune response.

Carbon ion radiotherapy boosts anti-tumour immune responses by inhibiting myeloid-derived suppressor cells in melanoma-bearing mice

Numerous studies have shown that carbon ion radiotherapy (CIRT) induces anti-cancer immune responses in melanoma patients, yet the mechanism remains elusive. The abundance of myeloid-derived suppressor cells (MDSC) in the tumour microenvironment is associated with therapeutic efficacy and disease outcome. This study analysed the changes in the immune contexture in response to the carbon ion treatment. The murine melanoma B16, MelanA, and S91 tumour models were established in syngeneic immunocompetent mice. Then, the tumours were irradiated with carbon ion beams, and flow cytometry was utilised to observe the immune contexture changes in the bone marrow, peripheral blood, spleen, and tumours. The immune infiltrates in the tumour tissues were further assessed using haematoxylin/eosin staining and immunohistochemistry. The immunoblot detected the expression of proteins associated with the JAK/STAT signalling pathway. The secretion of immune-related cytokines was examined using ELISA. Compared to conventional radiotherapy, particle beams have distinct advantages in cancer therapy. Here, the use of carbon ion beams (5 GyE) for melanoma-bearing mice was found to reduce the population of MDSC in the bone marrow, peripheral blood, and spleen of the animals via a JAK2/STAT3-dependent mechanism. The percentage of CD3+, CD4+, CD8+ T cells, macrophages, and natural killer cells increased after radiation, resulting in reduced tumour growth and prolonged overall survival in the three different mouse models of melanoma. This study, therefore, substantiated that CIRT boosts anti-tumour immune responses via the inhibition of MDSC.

IMMU-43. IMMUNE CONTEXTURE OF ISOCITRATE DEHYDROGENASE STRATIFIED HUMAN GLIOMAS REVEALED BY SINGLE-CELL TRANSCRIPTOMICS AND ACCESSIBLE CHROMATIN

The immune cell composition of isocitrate dehydrogenase wild type (IDH-wt) glioma patients significantly differs compared to IDH-mutant (IDH-mut) yet a detailed and unbiased understanding of their transcriptomic and epigenetic landscapes remains elusive. To this end, we performed single-cell RNA-sequencing (scRNA-seq) and single-cell Assay for Transposase-Accessible Chromatin using sequencing (sc-ATAC-seq) on ~100,000 tumor-associated immune cells from seventeen IDH mutation classified primary and recurrent human gliomas and non-glioma brains (NGBs). Our analyses revealed sixty-two transcriptionally distinct myeloid and lymphoid cell states within and across glioma subtypes and we noted microglial attrition with increasing disease severity concomitant with invading monocyte-derived cells (MDCs) and lymphocytes. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells. As tissue macrophages exhibit multifaceted polarization in response to microenvironmental cues, we clarify the existence of microglia/macrophage functional states beyond M1/M2 paradigms exemplified by the presence of palmitic-, oleic- acid, and glucocorticoid responsive polarized states. We identified cytotoxic T cells with poly-functional cytolytic states mostly in recurrent IDH-wt gliomas. Furthermore, ligand-receptor interactome analyses showed a preponderance of antigen presentation/phagocytosis over the checkpoint axis in IDH-wt compared to IDH-mut gliomas. Additionally, our sc-ATAC-seq analyses revealed differences in regulatory networks in NGBs, IDH-mut, and IDH-wt glioma-associated immune cells. In particular, we noted reduced microglial usage of an iron recycling SPIC transcription factor and Interferon Regulatory Factors (IRFs); IRF1 and IRF2 in IDH-wt relative to IDH-mut and NGBs. Unique features such as amplification of 11-Zinc Finger Protein accessibility were restricted to MDCs. Finally, sc-ATAC-seq profiles of CD8+ exhausted T cells from IDH-wt showed strong enhancer accessibility on CTLA-4, Layilin, and TIM-3 but no enrichment on PD1 was seen. In summary, our study provides unprecedented granular detail of transcriptionally and epigenomically defined glioma-specific immune contexture that can be exploited for immunotherapy applications.

828 Quantifying perivascular immune cells in the stroma of human triple negative breast tumors using deep learning spatial analytics

BackgroundThe spatial organization and density of the immune infiltrate in the tumor microenvironment, referred to as immune contexture, can yield information relevant to prognosis and prediction of response to immunotherapy in cancer. Specifically, a distinct subset of tumor-associated macrophages (TAMs) accumulate around blood vessels where they stimulate tumor angiogenesis and limit tumor responses to frontline anti-cancer therapies like irradiation and chemotherapy.MethodsIn this study we leveraged the NeoGenomics MultiOmyx Multiplex Immunofluorescence platform alongside artificial intelligence (AI) based quantitative image analysis. This AI platform was ultimately used to investigate the distribution of perivascular (PV) TAMs, CD4+ and CD8+ T cells, and CD4+FOXP3+ regulatory T cells (Tregs) of 40 human triple negative breast carcinomas (TNBCs), and how this changed following neoadjuvant chemotherapy. During the multiplexing phase, eleven rounds of paired antibody staining were performed in sequence on tumor sections. After each round of staining, high resolution images were captured for regions of interests (ROIs) selected by a pathologist. We used AI models to segment and classify cells for each biomarker and classify regions as tumor cell islands (TCIs) or stroma. First, each nucleus was segmented out using a convolutional neural network combined with watershed thresholding on the DAPI (diamidino-2-phenylindole) immunofluorescent image. From the resulting nuclear segmentation mask, a pixel dilation on cells classified as non-tumor was employed to generate a cellular segmentation mask. A list of neighbours within a specified distance for each cell was generated by radially expanding from the cellular segmentation mask. Finally, cell neighbour information was combined with the marker expression information to quantify the cell clusters of interest.ResultsWe discovered that in the PV areas, up to 30% of PD1-LAG3-CD3+CD8+ T cells formed direct contact with both CD163+TIM3+ TAMs and CD4+FOXP3+ Tregs. Furthermore, these immune cell triads preferentially accumulated in the PV stroma regions. It is likely that close interaction with immunosuppressive TAMs and Tregs would supress the function of T cells as they enter the PV region to reach the TCIs.ConclusionsUsing an advanced analytics platform, we invented a new method to quantify clusters of cells within various regions of a tumor section. Using this platform, we detected specific immune cell triads, the frequency and location of which could correlate with the efficacy of T-cell based immunotherapies in TNBC. These analyses will enable further investigation of numerous complex cell interactions in TMEs.

672 The effect of chemoradiotherapy and tumor histology on the immune contexture of tumor-draining lymph nodes in NSCLC

BackgroundRecently, the concept of locally delivered immune modulatory agents (re-)invigorating sub-optimally primed tumor-specific T cells and lifting suppression in the tumor microenvironment (TME) and tumor-draining lymph nodes (TDLN) has gained attention. TDLN play an important role in the induction of tumor-specific effector T cells. It is here that specialized dendritic cell (DC) subsets present tumor-derived antigens to naïve T cells and start effective adaptive immune responses to cancer. Unfortunately, TDLN are also rapidly targeted by tumors for immune suppression, which may impair the efficacy of immunotherapy. Currently, there is limited knowledge on the immune contexture of TDLN in non-small cell lung cancer (NSCLC), differences between types of tumor histology, and the influence of standard treatment.MethodsIn an exploratory study, we collected and analyzed viable cells from TDLN from patients with NSCLC, scheduled for surgical resection. To date, we have analyzed 43 TDLN from a total of 10 patients with multiparameter flowcytometry panels, either untreated or after neoadjuvant chemoradiotherapy (nCRT).ResultsOur analyses reveal differences between squamous cell carcinoma (SCC) and adenocarcinoma (AC), discernable even within this small cohort. In AC, higher levels of PD-L1 on CD11c+CD1c- LN-resident macrophages and CD1a+ migratory DC were accompanied by a lower activation state of CD8+ T cells by PD-1, CTLA-4 and CD69 expression levels. Furthermore, we found decreased activation of LN-resident DCs (by PD-L1 and CD83 levels) and a striking decrease in PD-1 and CD69 on CD8+ T cells, a decrease in effector and central memory CD8+ T cells, and an increase in naïve CD8+ T cells and Treg subsets after nCRT treatment, the current standard treatment of stage III NSCLC patients.ConclusionsThese AC/SCC –related differences and nCRT-induced alterations in the immune status of hold clues for future patient stratification and combinatorial design of CRT with immunotherapy.Ethics ApprovalThis study was approved by the Medical Ethics Committee; 2017.545

Determinants of renal cell carcinoma invasion and metastatic competence

Metastasis is the principal cause of cancer related deaths. Tumor invasion is essential for metastatic spread. However, determinants of invasion are poorly understood. We addressed this knowledge gap by leveraging a unique attribute of kidney cancer. Renal tumors invade into large vessels forming tumor thrombi (TT) that migrate extending sometimes into the heart. Over a decade, we prospectively enrolled 83 ethnically-diverse patients undergoing surgical resection for grossly invasive tumors at UT Southwestern Kidney Cancer Program. In this study, we perform comprehensive histological analyses, integrate multi-region genomic studies, generate in vivo models, and execute functional studies to define tumor invasion and metastatic competence. We find that invasion is not always associated with the most aggressive clone. Driven by immediate early genes, invasion appears to be an opportunistic trait attained by subclones with diverse oncogenomic status in geospatial proximity to vasculature. We show that not all invasive tumors metastasize and identify determinants of metastatic competency. TT associated with metastases are characterized by higher grade, mTOR activation and a particular immune contexture. Moreover, TT grade is a better predictor of metastasis than overall tumor grade, which may have implications for clinical practice.

Interleukin-30 feeds breast cancer stem cells via CXCL10 and IL23 autocrine loops and shapes immune contexture and host outcome

BackgroundBreast cancer (BC) progression to metastatic disease is the leading cause of death in women worldwide. Metastasis is driven by cancer stem cells (CSCs) and signals from their microenvironment. Interleukin (IL) 30 promotes BC progression, and its expression correlates with disease recurrence and mortality. Whether it acts by regulating BCSCs is unknown and could have significant therapeutic implications.MethodsHuman (h) and murine (m) BCSCs were tested for their production of and response to IL30 by using flow cytometry, confocal microscopy, proliferation and sphere-formation assays, and PCR array. Immunocompetent mice were used to investigate the role of BCSC-derived IL30 on tumor development and host outcome. TCGA PanCancer and Oncomine databases provided gene expression data from 1084 and 75 hBC samples, respectively, and immunostaining unveiled the BCSC microenvironment.ResultshBCSCs constitutively expressed IL30 as a membrane-anchored glycoprotein. Blocking IL30 hindered their proliferation and self-renewal efficiency, which were boosted by IL30 overexpression. IL30 regulation of immunity gene expression in human and murine BCSCs shared a significant induction of IL23 and CXCL10. Both immunoregulatory mediators stimulated BCSC proliferation and self-renewal, while their selective blockade dramatically hindered IL30-dependent BCSC proliferation and mammosphere formation. Orthotopic implantation of IL30-overexpressing mBCSCs, in syngeneic mice, gave rise to poorly differentiated and highly proliferating MYC+KLF4+LAG3+ tumors, which expressed CXCL10 and IL23, and were infiltrated by myeloid-derived cells, Foxp3+ T regulatory cells and NKp46+RORγt+ type 3 innate lymphoid cells, resulting in increased metastasis and reduced survival. In tumor tissues from patients with BC, expression of IL30 overlapped with that of CXCL10 and IL23, and ranked beyond the 95th percentile in a Triple-Negative enriched BC collection from the Oncomine Platform. CIBERSORTx highlighted a defective dendritic cell, CD4+ T and γδ T lymphocyte content and a prominent LAG3 expression in IL30highversus IL30low human BC samples from the TCGA PanCancer collection.ConclusionsConstitutive expression of membrane-bound IL30 regulates BCSC viability by juxtacrine signals and via second-level mediators, mainly CXCL10 and IL23. Their autocrine loops mediate much of the CSC growth factor activity of IL30, while their paracrine effect contributes to IL30 shaping of immune contexture. IL30-related immune subversion, which also emerged from computational analyses, strongly suggests that targeting IL30 can restrain the BCSC compartment and counteract BC progression.