Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers.

The characterization of the microenvironment of human tumors led to the description of tertiary lymphoid structures (TLS) characterized by mature dendritic cells in a T-cell zone adjacent to B-cell follicle including a germinal center. TLS represent sites of lymphoid neogenesis that develop in most solid cancers. Analysis of the current literature shows that the TLS presence is associated with a favorable clinical outcome for cancer patients, regardless of the approach used to quantify TLS and the stage of the disease. Using several approaches that combine immunohistochemistry, gene expression assays, and flow cytometry on large series of lung tumors, our work demonstrated that TLS are important sites for the initiation and/or maintenance of the local and systemic T- and B-cell responses against tumors. Surrounded by high endothelial venules, they represent a privileged area for the recruitment of lymphocytes into tumors and generation of central-memory T and B cells that circulate and limit cancer progression. TLS can be considered as a novel biomarker to stratify the overall survival risk of untreated cancer patients and as a marker of efficient immunotherapies. The induction and manipulation of cancer-associated TLS using drug agonists and/or biotherapies should open new avenues to treat cancer patients.

The clinical role of the TME in solid cancer.

The highly complex and heterogenous ecosystem of a tumour not only contains malignant cells, but also interacting cells from the host such as endothelial cells, stromal fibroblasts, and a variety of immune cells that control tumour growth and invasion. It is well established that anti-tumour immunity is a critical hurdle that must be overcome for tumours to initiate, grow and spread and that anti-tumour immunity can be modulated using current immunotherapies to achieve meaningful anti-tumour clinical responses. Pioneering studies in melanoma, ovarian and colorectal cancer have demonstrated that certain features of the tumour immune microenvironment (TME)-in particular, the degree of tumour infiltration by cytotoxic T cells-can predict a patient's clinical outcome. More recently, studies in renal cell cancer have highlighted the importance of assessing the phenotype of the infiltrating T cells to predict early relapse. Furthermore, intricate interactions with non-immune cellular players such as endothelial cells and fibroblasts modulate the clinical impact of immune cells in the TME. Here, we review the critical components of the TME in solid tumours and how they shape the immune cell contexture, and we summarise numerous studies evaluating its clinical significance from a prognostic and theranostic perspective.

Association of IL-36γ with tertiary lymphoid structures and inflammatory immune infiltrates in human colorectal cancer.

IL-1 family cytokines play a dual role in the gut, with different family members contributing either protective or pathogenic effects. IL-36γ is an IL-1 family cytokine involved in polarizing type-1 immune responses. However, its function in the gut, including in colorectal cancer pathogenesis, is not well appreciated. In a murine model of colon carcinoma, IL-36γ controls tertiary lymphoid structure formation and promotes a type-1 immune response concurrently with a decrease in expression of immune checkpoint molecules in the tumor microenvironment. Here, we demonstrate that IL-36γ plays a similar role in driving a pro-inflammatory phenotype in human colorectal cancer. We analyzed a cohort of 33 primary colorectal carcinoma tumors using imaging, flow cytometry, and transcriptomics to determine the pattern and role of IL-36γ expression in this disease. In the colorectal tumor microenvironment, we observed IL-36γ to be predominantly expressed by M1 macrophages and cells of the vasculature, including smooth muscle cells and high endothelial venules. This pattern of IL-36γ expression is associated with a CD4+ central memory T cell infiltrate and an increased density of B cells in tertiary lymphoid structures, as well as with markers of fibrosis. Conversely, expression of the antagonist to IL-36 signaling, IL-1F5, was associated with intratumoral expression of checkpoint molecules, including PD-1, PD-L1, and CTLA4, which can suppress the immune response. These data support a role for IL-36γ in the physiologic immune response to colorectal cancer by sustaining inflammation within the tumor microenvironment.

Transcriptomic analysis of the tumor microenvironment to guide prognosis and immunotherapies.

Tumors are highly heterogeneous tissues where malignant cells are surrounded by and interact with a complex tumor microenvironment (TME), notably composed of a wide variety of immune cells, as well as vessels and fibroblasts. As the dialectical influence between tumor cells and their TME is known to be clinically crucial, we need tools that allow us to study the cellular composition of the microenvironment. In this focused research review, we report MCP-counter, a methodology based on transcriptomic markers that assesses the proportion of several immune and stromal cell populations in the TME from transcriptomic data, and we highlight how it can provide a way to decipher the complex mechanisms at play in tumors. In several malignancies, MCP-counter scores have been used to show various prognostic impacts of the TME, which we also show to be linked with the mutational burden of tumors. We also compared established molecular classifications of colorectal cancer and clear-cell renal cell carcinoma with the output of MCP-counter, and show that molecular subgroups have different TME profiles, and that these profiles are consistent within a given subgroup. Finally, we provide insights as to how knowing the TME composition may shape patient care in the near future.

Implications of the tumor immune microenvironment for staging and therapeutics.

Characterizing the tumor immune microenvironment enables the identification of new prognostic and predictive biomarkers, the development of novel therapeutic targets and strategies, and the possibility to guide first-line treatment algorithms. Although the driving elements within the tumor microenvironment of individual primary organ sites differ, many of the salient features remain the same. The presence of a robust antitumor milieu characterized by an abundance of CD8+ cytotoxic T-cells, Th1 helper cells, and associated cytokines often indicates a degree of tumor containment by the immune system and can even lead to tumor elimination. Some of these features have been combined into an 'Immunoscore', which has been shown to complement the prognostic ability of the current TNM staging for early stage colorectal carcinomas. Features of the immune microenvironment are also potential therapeutic targets, and immune checkpoint inhibitors targeting the PD-1/PD-L1 axis are especially promising. FDA-approved indications for anti-PD-1/PD-L1 are rapidly expanding across numerous tumor types and, in certain cases, are accompanied by companion or complimentary PD-L1 immunohistochemical diagnostics. Pathologists have direct visual access to tumor tissue and in-depth knowledge of the histological variations between and within tumor types and thus are poised to drive forward our understanding of the tumor microenvironment. This review summarizes the key components of the tumor microenvironment, presents an overview of and the challenges with PD-L1 antibodies and assays, and addresses newer candidate biomarkers, such as CD8+ cell density and mutational load. Characteristics of the local immune contexture and current pathology-related practices for specific tumor types are also addressed. In the future, characterization of the host antitumor immune response using multiplexed and multimodality biomarkers may help predict which patients will respond to immune-based therapies.

Tertiary lymphoid structures in cancer and beyond.

Tertiary lymphoid structures (TLS) are ectopic lymphoid formations found in inflamed, infected, or tumoral tissues. They exhibit all the characteristics of structures in the lymph nodes (LN) associated with the generation of an adaptive immune response, including a T cell zone with mature dendritic cells (DC), a germinal center with follicular dendritic cells (FDC) and proliferating B cells, and high endothelial venules (HEV). In this review, we discuss evidence for the roles of TLS in chronic infection, autoimmunity, and cancer, and address the question of whether TLS present beneficial or deleterious effects in these contexts. We examine the relationship between TLS in tumors and patient prognosis, and discuss the potential role of TLS in building and/or maintaining local immune responses and how this understanding may guide therapeutic interventions.

Shaping of an effective immune microenvironment to and by cancer cells.

A high density of intratumoral effector memory CD8+/Th1 T cells is associated with favorable prognosis in most cancers and may be induced or increased by immunotherapy. Efficient adaptive immune reactions are shaped in tumor adjacent tertiary lymphoid structures, which exhibit all characteristics of immunity generating lymphoid formations in reactive lymph nodes. Malignant tumor cells impact favorably or deleteriously their immune microenvironment if they bear genetic mutations that result in neo-antigens or by producing chemokines and cytokines that recruit lymphocytes and myeloid cells or increase inflammation and neo-angiogenesis. This intricate network of interactions results in control or escape of tumors, and its understanding will help define goals to monitor efficiency of immunotherapies.

Comprehensive Molecular Characterization of Gallbladder Carcinoma and Potential Targets for Intervention.

PURPOSE: Gallbladder carcinoma (GBC) is an uncommon and aggressive disease, which remains poorly defined at a molecular level. Here, we aimed to characterize the molecular landscape of GBC and identify markers with potential prognostic and therapeutic implications. EXPERIMENTAL DESIGN: GBC samples were analyzed using the MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) platform (targeted NGS assay that analyzes 505 cancer-associated genes). Variants with therapeutic implications were identified using OncoKB database. The associations between recurrent genetic alterations and clinicopathologic characteristics (Fisher exact tests) or overall survival (univariate Cox regression) were evaluated. P values were adjusted for multiple testing. RESULTS: Overall, 244 samples (57% primary tumors and 43% metastases) from 233 patients were studied (85% adenocarcinomas, 10% carcinomas with squamous differentiation, and 5% neuroendocrine carcinomas). The most common oncogenic molecular alterations appeared in the cell cycle (TP53 63% and CDKN2A 21%) and RTK_RAS pathways (ERBB2 15% and KRAS 11%). No recurrent structural variants were identified. There were no differences in the molecular landscape of primary and metastasis samples. Variants in SMAD4 and STK11 independently associated with reduced survival in patients with metastatic disease. Alterations considered clinically actionable in GBC or other solid tumor types (e.g., NTRK1 fusions or oncogenic variants in ERBB2, PIK3CA, or BRCA1/2) were identified in 35% of patients; 18% of patients with metastatic disease were treated off-label or enrolled in a clinical trial based on molecular findings. CONCLUSIONS: GBC is a genetically diverse malignancy. This large-scale genomic analysis revealed alterations with potential prognostic and therapeutic implications and provides guidance for the development of targeted therapies.

Evaluating the impact of age on immune checkpoint therapy biomarkers.

Both tumors and aging alter the immune landscape of tissues. These interactions may play an important role in tumor progression among elderly patients and may suggest considerations for patient care. We leverage large-scale genomic and clinical databases to perform comprehensive comparative analysis of molecular and cellular markers of immune checkpoint blockade (ICB) response with patient age. These analyses demonstrate that aging is associated with increased tumor mutational burden, increased expression and decreased promoter methylation of immune checkpoint genes, and increased interferon gamma signaling in older patients in many cancer types studied, all of which are expected to promote ICB efficacy. Concurrently, we observe age-related alterations that might be expected to reduce ICB efficacy, such as decreases in T cell receptor diversity. Altogether, these changes suggest the capacity for robust ICB response in many older patients, which may warrant large-scale prospective study on ICB therapies among patients of advanced age.

Spatial UMAP and Image Cytometry for Topographic Immuno-oncology Biomarker Discovery.

Multiplex immunofluorescence (mIF) can detail spatial relationships and complex cell phenotypes in the tumor microenvironment (TME). However, the analysis and visualization of mIF data can be complex and time-consuming. Here, we used tumor specimens from 93 patients with metastatic melanoma to develop and validate a mIF data analysis pipeline using established flow cytometry workflows (image cytometry). Unlike flow cytometry, spatial information from the TME was conserved at single-cell resolution. A spatial uniform manifold approximation and projection (UMAP) was constructed using the image cytometry output. Spatial UMAP subtraction analysis (survivors vs. nonsurvivors at 5 years) was used to identify topographic and coexpression signatures with positive or negative prognostic impact. Cell densities and proportions identified by image cytometry showed strong correlations when compared with those obtained using gold-standard, digital pathology software (R2 > 0.8). The associated spatial UMAP highlighted "immune neighborhoods" and associated topographic immunoactive protein expression patterns. We found that PD-L1 and PD-1 expression intensity was spatially encoded-the highest PD-L1 expression intensity was observed on CD163+ cells in neighborhoods with high CD8+ cell density, and the highest PD-1 expression intensity was observed on CD8+ cells in neighborhoods with dense arrangements of tumor cells. Spatial UMAP subtraction analysis revealed numerous spatial clusters associated with clinical outcome. The variables represented in the key clusters from the unsupervised UMAP analysis were validated using established, supervised approaches. In conclusion, image cytometry and the spatial UMAPs presented herein are powerful tools for the visualization and interpretation of single-cell, spatially resolved mIF data and associated topographic biomarker development.

Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD-1 blockade.

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this "AstroPath" whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti-programmed cell death-1 (PD-1)-based therapy, including CD163+PD-L1- myeloid cells and CD8+FoxP3+PD-1low/mid T cells. These features were combined to stratify long-term survival after anti-PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution.

Integrating histopathology, immune biomarkers, and molecular subgroups in solid cancer: the next step in precision oncology.

For many years, the gold standard cancer grading and staging had focused on the characteristics of the cancer cells and often disregarded the non-tumoral cell compartments. The expansion of research on the tumor immune microenvironment, the successes and dissemination of immunotherapies to treat cancer, and the open access to large -omic databases have allowed the development of novel powerful immune-based prognostic and theranostic biomarkers. Although they often correlate with histopathologic characteristics and TNM staging, in many instances, they are independently associated with, and potentially superior predictors of, the patient's prognosis and response to immunotherapies. As pathologists in the era of precision medicine, we are uniquely positioned to participate in the integration of these histologic and molecular features of the tumor microenvironment to provide the best prognostic information to clinicians and patients. In this review, we summarize some of the most important immune-related prognostic biomarkers in solid cancer, how they integrate with traditional histopathologic (i.e., staging and grading) and novel molecular stratification systems, and their potential role as predictors to response to agents blocking the PD-1/PD-L1 axis.

Tumor Cells Hijack Macrophage-Produced Complement C1q to Promote Tumor Growth.

Clear-cell renal cell carcinoma (ccRCC) possesses an unmet medical need, particularly at the metastatic stage, when surgery is ineffective. Complement is a key factor in tissue inflammation, favoring cancer progression through the production of complement component 5a (C5a). However, the activation pathways that generate C5a in tumors remain obscure. By data mining, we identified ccRCC as a cancer type expressing concomitantly high expression of the components that are part of the classical complement pathway. To understand how the complement cascade is activated in ccRCC and impacts patients' clinical outcome, primary tumors from three patient cohorts (n = 106, 154, and 43), ccRCC cell lines, and tumor models in complement-deficient mice were used. High densities of cells producing classical complement pathway components C1q and C4 and the presence of C4 activation fragment deposits in primary tumors correlated with poor prognosis. The in situ orchestrated production of C1q by tumor-associated macrophages (TAM) and C1r, C1s, C4, and C3 by tumor cells associated with IgG deposits, led to C1 complex assembly, and complement activation. Accordingly, mice deficient in C1q, C4, or C3 displayed decreased tumor growth. However, the ccRCC tumors infiltrated with high densities of C1q-producing TAMs exhibited an immunosuppressed microenvironment, characterized by high expression of immune checkpoints (i.e., PD-1, Lag-3, PD-L1, and PD-L2). Our data have identified the classical complement pathway as a key inflammatory mechanism activated by the cooperation between tumor cells and TAMs, favoring cancer progression, and highlight potential therapeutic targets to restore an efficient immune reaction to cancer.

Immune-based identification of cancer patients at high risk of progression.

Tumors are highly heterogeneous structures where malignant cells interact with a large variety of cell populations, including a clinically-relevant immune component. We review and compare the most recent methods designed to analyze and quantify the composition of immune and stromal microenvironment of tumors and discuss their use in identification of patients for high risk of progression. If the impact of the various immune components on patient's relapse share common rules in most malignancies, clear cell renal cell tumors behave differently with regards to immunity. We focus on this specific pathology to show how the tumor interacts with the host's immune system and how this intricate relationship shapes the clinical outcome.

T cells responding to Trypanosoma cruzi detected by membrane TNF-α and CD154 in chagasic patients.

INTRODUCTION: Chagas disease is a parasitic infection whose pathogenesis is related to parasite persistence and a dysfunctional cellular immune response. Variability in cytokine secretion among chronic Trypanosoma cruzi-infected patients might preclude the identification of the pool of antigen specific T cells. The goal of this study was to determine the fraction of T cells responding to T. cruzi antigen measured by the expression of membrane TNF-α and CD154. METHODS: A total of 21 chagasic patients, 11 healthy and 5 non-chagasic cardiomyopathy controls were analyzed. PBMCs were short-term cultured in the presence of anti-CD28, anti-CD49d, anti-TNF-α, and TACE (TNF-α converting enzyme) inhibitor either under T. cruzi-lysate or polyclonal stimuli. Cells were stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD154, and analyzed with flow cytometry. RESULTS: CD4+ and CD8+ T cells in chagasic patients displayed higher percentages of membrane-bound TNF-α+ and CD154+ compared with controls after T. cruzi-antigen stimulation. Both markers displayed a positive correlation in the T cell subpopulations analyzed. Symptomatic chagasic patients were differentiated from asymptomatic patients based on the expression of CD154 and membrane TNF-α in TCD4+ and TCD8+ compartments, respectively. CONCLUSIONS: These results show that both markers could be useful for assessing the pool of antigen-specific T cells in chronic chagasic patients.

Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab.

BACKGROUND: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified. METHODS: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1. RESULTS: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm2, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 µm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1. CONCLUSIONS: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, Tregs, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.

Tumor-Infiltrating and Peripheral Blood T-cell Immunophenotypes Predict Early Relapse in Localized Clear Cell Renal Cell Carcinoma.

Purpose: The efficacy of PD-1 checkpoint blockade as adjuvant therapy in localized clear cell renal cell carcinoma (ccRCC) is currently unknown. The identification of tumor microenvironment (TME) prognostic biomarkers in this setting may help define which patients could benefit from checkpoint blockade and uncover new therapeutic targets.Experimental Design: We performed multiparametric flow cytometric immunophenotypic analysis of T cells isolated from tumor tissue [tumor-infiltrating lymphocytes (TIL)], adjacent non-malignant renal tissue [renal-infiltrating lymphocytes (RIL)], and peripheral blood lymphocytes (PBL), in a cohort of patients (n = 40) with localized ccRCC. Immunophenotypic data were integrated with prognostic and histopathologic variables, T-cell receptor (TCR) repertoire analysis of sorted CD8+PD-1+ TILs, tumor mRNA expression, and digital quantitative immunohistochemistry.Results: On the basis of TIL phenotypic characterization, we identified three dominant immune profiles in localized ccRCC: (i) immune-regulated, characterized by polyclonal/poorly cytotoxic CD8+PD-1+Tim-3+Lag-3+ TILs and CD4+ICOS+ cells with a Treg phenotype (CD25+CD127-Foxp3+/Helios+GITR+), that developed in inflamed tumors with prominent infiltrations by dysfunctional dendritic cells and high PD-L1 expression; (ii) immune-activated, enriched in oligoclonal/cytotoxic CD8+PD-1+Tim-3+ TILs, that represented 22% of the tumors; and (iii) immune-silent, enriched in TILs exhibiting RIL-like phenotype, that represented 56% of patients in the cohort. Only immune-regulated tumors displayed aggressive histologic features, high risk of disease progression in the year following nephrectomy, and a CD8+PD-1+Tim-3+ and CD4+ICOS+ PBL phenotypic signature.Conclusions: In localized ccRCC, the infiltration with CD8+PD-1+Tim-3+Lag-3+ exhausted TILs and ICOS+ Treg identifies the patients with deleterious prognosis who could benefit from adjuvant therapy with TME-modulating agents and checkpoint blockade. This work also provides PBL phenotypic markers that could allow their identification. Clin Cancer Res; 23(15); 4416-28. ©2017 AACR.

Cancer immune contexture and immunotherapy.

The immune contexture that characterizes the density, the location, the organization and the functional orientation of tumor-infiltrating immune cells in cancers has a clinical impact on patient's outcome. It is, in great part, shaped by the malignant cells, as in a given cancer type, tumors presenting different oncogenic processes have different immune contextures. Moreover, the immune contexture in metastatic sites reflects that of the corresponding primary tumors. Finally, the components forming the immune contexture represent targets and markers of efficient anti-cancer immunotherapies.

Tertiary Lymphoid Structures in Cancers: Prognostic Value, Regulation, and Manipulation for Therapeutic Intervention.

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that reflect lymphoid neogenesis occurring in tissues at sites of inflammation. They are detected in tumors where they orchestrate local and systemic anti-tumor responses. A correlation has been found between high densities of TLS and prolonged patient's survival in more than 10 different types of cancer. TLS can be regulated by the same set of chemokines and cytokines that orchestrate lymphoid organogenesis and by regulatory T cells. Thus, TLS offer a series of putative new targets that could be used to develop therapies aiming to increase the anti-tumor immune response.

Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression.

We introduce the Microenvironment Cell Populations-counter (MCP-counter) method, which allows the robust quantification of the absolute abundance of eight immune and two stromal cell populations in heterogeneous tissues from transcriptomic data. We present in vitro mRNA mixture and ex vivo immunohistochemical data that quantitatively support the validity of our method's estimates. Additionally, we demonstrate that MCP-counter overcomes several limitations or weaknesses of previously proposed computational approaches. MCP-counter is applied to draw a global picture of immune infiltrates across human healthy tissues and non-hematopoietic human tumors and recapitulates microenvironment-based patient stratifications associated with overall survival in lung adenocarcinoma and colorectal and breast cancer.