Tumor-infiltrating lymphocytes in the immunotherapy era

The clinical success of cancer immune checkpoint blockade (ICB) has refocused attention on tumor-infiltrating lymphocytes (TILs) across cancer types. The outcome of immune checkpoint inhibitor therapy in cancer patients has been linked to the quality and magnitude of T cell, NK cell, and more recently, B cell responses within the tumor microenvironment. State-of-the-art single-cell analysis of TIL gene expression profiles and clonality has revealed a remarkable degree of cellular heterogeneity and distinct patterns of immune activation and exhaustion. Many of these states are conserved across tumor types, in line with the broad responses observed clinically. Despite this homology, not all cancer types with similar TIL landscapes respond similarly to immunotherapy, highlighting the complexity of the underlying tumor-immune interactions. This observation is further confounded by the strong prognostic benefit of TILs observed for tumor types that have so far respond poorly to immunotherapy. Thus, while a holistic view of lymphocyte infiltration and dysfunction on a single-cell level is emerging, the search for response and prognostic biomarkers is just beginning. Within this review, we discuss recent advances in the understanding of TIL biology, their prognostic benefit, and their predictive value for therapy

INVESTIGATING THE ROLES OF TAP63 AND TAP73 IN CUTANEOUS SQUAMOUS CELL CARCINOMA AND LUNG ADENOCARCINOMA

TP63 and TP73 (which encode p63 and p73, respectively) are highly conserved transcription factors with important roles in development and tissue homeostasis. Similar to their homolog, p53, both p63 and p73 have been shown to mediate tumor suppression in multiple tissue types. Interestingly, however, both genes are expressed as multiple isoforms, which appear to have different and, in many cases, antagonistic functions. Through the use of isoform-specific null alleles of p63 and p73 our lab and others have shown that the full-length N-terminal isoforms of p63 and p73 (referred to as TAp63 and TAp73, respectively) exhibit distinct functions in development, metabolism and tumor suppression. My thesis research focuses on understanding the conserved functions of TAp63 and TAp73 in tumorigenesis. To do so, I have utilized multiple model systems to investigate the contributions of TAp63 and TAp73 in the development and progression of cutaneous squamous cell carcinoma (cuSCC) and lung adenocarcinoma (LUAC). I have found that TAp63-null (TAp63-/-) mice exhibit increased susceptibility to UVR-induced cuSCC. To identify TAp63-regulated transcriptional programs relevant to the pathophysiology cuSCC, we performed RNA sequencing of cuSCC and normal tissues from wild-type (WT) and TAp63-/- mice. My data revealed significant perturbations in global miRNA and mRNA expression in TAp63-deficient tumors, which resembled similar transcriptional changes in human cuSCC, including the downregulation of miR-30c-2* and miR-497. Proteomic profiling of cuSCC cell lines and subsequent validation experiments led to the discovery of novel direct targets of miR-497 and miR-30c-2*. The most relevant of these proteins exhibited pro-oncogenic functions, including roles in cell cycle and mitotic progression. My data establishes TAp63 as an essential regulator of miRNA expression during skin carcinogenesis and reveals a previously undescribed network of miRNAs and mRNAs, which include viable, yet previously unexplored targets for therapeutic intervention. Along with TAp63-mediated tumor suppression in the skin, I have also developed a novel TAp73 conditional knockout mouse, which includes a conditional fluorescent reporter (TAp73fltd) that allows us to follow the deletion event at the cellular level. Using this model we have found that the loss of TAp73 accelerates the initiation and progression of oncogenic Krasdriven LUAC. Our data suggests that TAp73 may be necessary for anti-tumor immune responses. In addition, we have found that the loss of TAp73 promotes migration and invasion in human LUAC cell lines. These studies suggest that TAp73 can suppress tumor development and progression through both cell-autonomous and non-cell-autonomous mechanisms. Together, the studies described in this dissertation provide insights into the genetic and epigenetic mechanisms that contribute to cuSCC and LUAC development, which may, in turn, inform future biomarker and drug discovery studies

Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front.

Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains

Tumor immunology & the application of immunotherapy in ovarian carcinoma

It has become abundantly clear that a successful anti-tumor immune response in cancer requires the presence, activation, and co-stimulation of all lymphoid components of the immune system, including CD8+ T cells, CD4+ T cells and B cells. This thesis elucidates on the immune environment and its importance in the application of immunotherapy in ovarian cancer. Thus far, immunotherapy is moderately successful in the treatment of ovarian cancer compared to e.g. melanoma and lung cancer. To improve clinical outcome it is essential to combine the right therapies for the right patient and to administer the treatment at the right window-of-opportunity. From our data we conclude that CD8+CD103+ TRM have a strong predictive value and quantification can play an important role in determining treatment strategy for different patient groups (high vs low TIL). Furthermore, upregulation of MHC-I expression in NACT patients may restore antigen presentation and the prognostic effect of TILs, which could eventually lead to improved response to immunotherapy in this group of patients. Finally, combining vaccination strategy with chemotherapy and/or ICI could improve the overall response rates in HGSOC patients

Egr2/Egr3 are essential tumour suppressor genes for lymphomagenesis

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Non-Hodgkin’s lymphoma is the fifth most common cancer in the UK, accounting for 4% of all new cases. The control of lymphomagenesis still remains a challenge. Early growth response gene (Egr) 2 and 3 are zinc finger transcription factors. Egr2 plays an important role in the development of both central nervous system and lymphocytes. However the mechanism of action in lymphocytes is still unknown. In order to fully understand the function of Egr2, in lymphocytes, we developed Egr2 and 3 double knockout mice (Egr2-/-Egr3-/-) by crossbreeding lymphocyte specific Egr2 knockout mice (CD2-Egr2-/-) with Egr3 knockout mice (Egr3-/-), as previous reports suggested that Egr3 compensates for the role of Egr2. In the absence of Egr2 and 3, the homeostasis of T cells is dysregulated with hyper-homeostatic proliferation of effector like phenotype cells. More importantly the development of spontaneous B and T cell lymphoma was found in more than 70% of Egr2-/-Egr3-/- mice. The lymphoma cells from Egr2-/-Egr3-/- mice were highly proliferative and metastatically spread into other non-lymphoid organs, such as lung, liver and kidney. In additional to this lymphoma development the Egr2-/-Egr3-/- mice showed signs of chronic inflammatory disorder. This inflammatory disorder was characterised by glomerulonephritis and an increase in serum cytokines, which may provide the microenvironment for the lymphoma development. To explore the molecular mechanism of tumour development in Egr2-/-Egr3-/- mice, the transcriptional profile of Egr2 was studied by microarray and ChIP-on-chip. We found firstly that Egr2 directly binds to the promoter regions of Ikaros and FOXO3. The deletion of Egr2 and 3 in lymphocytes led to the downregulation of Ikaros, Aiolos and FOXO3 expression. The impaired expression was found to be associated with proliferative disorder and the development of T and B cell lymphoma. Secondly Egr2 strongly inhibits STAT3 transcriptional activity by regulating SOCS3, which is a known inhibitor of STAT3. The breakdown of this regulation could be an important mechanism in lymphomagenesis. A model is proposed which defines Egr2 and Egr3 as the backbone of important tumour suppressor genes that control cell fate decision and regulates homeostasis in the lymphoid system. Thus, our results suggest that Egr2 and 3 are important regulators of lymphocyte function by their involvement in multiple cell signalling pathways, which could potentially be key genes for future cancer therapy

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated

Investigating disease associated immune signatures in Diffuse Large B-cell Lymphoma.

PhD Theses MedicalDiffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS) is the most frequent subtype of lymphoma with approximately 4,800 new cases per year in the UK. Although it is a curable disease with standard immunochemotherapy, up to one third of patients are primary refractory or relapse after a period of remission. Until recently the prognosis for these patients was extremely poor. The recent approval of chimeric antigen receptor T (CAR-T) cell therapy has significantly improved the outlook for this group, however over half of the patients treated will progress and many others will not be suitable due to rapid disease progression. There are currently a multitude of new agents in development which hold much promise and will likely improve the outlook further for the highest risk patients. However, there remain several unmet needs including improved translation of biological insights to directly benefit patient care. Recent studies have focused on the genomic landscape in DLBCL, with new subgroups proposed based on the presence of recurrent and potentially actionable mutations, including many which facilitate escape from immune detection. In addition to molecular signals from the malignant lymphoma cells, there are reproducible signals from the nonmalignant compartment in both the tissue and peripheral blood microenvironment, with relevance to disease biology. Considerable variation is seen in immune cell composition and function between individuals in both health and disease, but this has not been well characterised in DLBCL. We focused predominantly on the peripheral blood immune compartment in this work, confirming the presence of a relative monocytosis and lymphopenia in DLBCL and their relevance to survival. We present a detailed description of the immune landscape in DLBCL, and document disease and outcome associated immune signatures. We identify mechanisms to account for these variations, widespread cytokine dysregulation and multiple bases of immune dysfunction. We also establish monocytes as the main peripheral blood source of cytokine production in DLBCL. Finally, we establish a pipeline for detailed characterisation of the tissue immune microenvironment using imaging mass cytometry

Immune profiling of the tumour microenvironment in prostate cancer

Prostate cancer is the most common cancer among men in the UK and is characterised by large biological and clinical heterogeneity. There is an urgent need for better-personalised patient stratification, for example in accurately identifying patients with regional lymph node metastasis. Nodal involvement negatively impacts on patient survival outcomes and the current pre-operative staging tools to determine the need for extended pelvic lymph node dissection at time of radical prostatectomy are far from precise. The primary tumour immune microenvironment influences tumour immune editing and therefore disease progression. The primary aim of this research was to investigate the in situ phenotype of prostate cancer tumour infiltrating immune cells and determine their potential as biomarkers for regional lymph node invovlement and further explore possible underlying mechanisms for their distribution. The discovery tissue microarray comprised of index lesions from 94 patients undergoing radical prostatectomy and pelvic node dissection (50 with and 44 without histologic evidence of pelvic nodal disease respectively, referred to as LN+ and LN- thereafter). Two multiplex immunofluorescence panels were optimised to comprehensively characterise the immune microenvironment: (1) The macrophage and B cell panel includes CD68, CD163, CD20, AE1/3 (PanCK) and DAPI and (2) The T lymphocytic panel assays for CD4, CD8, FoxP3, PD-1, AE1/3 and DAPI. The macrophage (CD68, CD163+), T (CD8+, CD4+) and B (CD20+) cell immune cell subpopulations within the malignant epithelium and associated stroma were measured and correlated to the nodal status. Stromal infiltration by M1-like macrophages (CD68+CD163-) (p=0.047), CD8 effector (CD8+FoxP3-PD-1-) (p=0.008) and CD4 effector (CD4+FoxP3-PD-1-) T cells (p=0.0003, Mann Whitney test) were lower in LN+ patients. Stromal CD4 effector immune cell density remained a statistically significant independent predictor of lymph node spread in multivariate regression analysis (OR= 0.15, p=0.004). Additionally, in an independent validation cohort of 184 radical prostatectomy specimens, stromal CD4 effector immune cell density predicted the presence of nodal metastasis (OR=0.26, p=0.0004). Addition of stromal CD4 effector T cell density to currently used clinicopathological factors, namely T stage, PSA level, Gleason score and percentage of tumour positive cores, improved the predictive accuracy of current nomograms (from 63.5% to 76.8%, p<0.0001). Tumour infiltrating immune cells did not however correlate with common molecular alterations of prostate cancer such as ERG overexpression and PTEN deletion. Transcriptomic analysis (by HTG EdgeSeq) of the tumour microenvironment was performed to assay 1,041 host immune response related genes. Surprisingly, I did not observe significant differences in the expression levels of adhesion molecules or chemokines (common regulators of immune cell migration) between LN+ and LN- cases. Instead, there was significant upregulation (FC>1.5, adj p value <0.05) of extracellular matrix components (collagen I, collagen III, fibronectin 1) in LN+ tumours, suggesting increased extracellular matrix fibrosis to be associated with reduced T lymphocytic infiltration and tumour immune evasion. Increased collagen III and fibronectin 1 protein expression were confirmed in LN+ patients. Collagen I had increased density score (by second generation harmonic), but not overall abundance, in LN+ patients, eluding to a disorganised stroma with increased cross-linking and elongated fibres. B7-H3 is a newly discovered member of the B7 family of immune checkpoint molecules with both immune and non-immune functions. I investigated the relationship of B7-H3 to the tumour microenvironment as well as its non-immune functions in prostate cancer. Contrast to PD-1, high B7-H3 expression correlated with worse clinicopathological patient features: higher T stage (p<0.0001), perineural invasion (p=0.01) and lymph node spread (p=0.0006). Furthermore, there was significant decrease in migration and invasion in vitro following suppressed B7-H3 expression in multiple human prostate cancer cell lines. RNA sequencing identified extracellular space chemotactic cytokines and their receptors to be highly downregulated genes in PC3M cells with B7-H3 knocked out. Future experiments will investigate the mechanistic downstream pathways of this phenotype and further evaluate the role of B7-H3 in metastasis in vivo. Data presented in this thesis reveal differences in the immune infiltrates, particularly CD4 effector (CD4+FoxP3-PD-1-) T cells between LN+ and LN- patients. Prospective clinical studies are needed to test the predictive value of stromal CD4 effector T cell density in diagnostic prostatic biopsies for regional nodal disease. The role of increased extracellular matrix components in determining tumour immune infiltrates also warrants additional research