Digital Spatial Profiling identifies distinct patterns of immuno-oncology-related gene expression within oropharyngeal tumours in relation to HPV and p16 status.

Background: The incidence of oropharyngeal cancer (OPC) is increasing, due mainly to a rise in Human Papilloma Virus (HPV)-mediated disease. HPV-mediated OPC has significantly better prognosis compared with HPV-negative OPC, stimulating interest in treatment de-intensification approaches to reduce long-term sequelae. Routine clinical testing frequently utilises immunohistochemistry to detect upregulation of p16 as a surrogate marker of HPV-mediation. However, this does not detect discordant p16-/HPV+ cases and incorrectly assigns p16+/HPV- cases, which, given their inferior prognosis compared to p16+/HPV+, may have important clinical implications. The biology underlying poorer prognosis of p16/HPV discordant OPC requires exploration. Methods: GeoMx digital spatial profiling was used to compare the expression patterns of selected immuno-oncology-related genes/gene families (n=73) within the tumour and stromal compartments of formalin-fixed, paraffin-embedded OPC tumour tissues (n=12) representing the three subgroups, p16+/HPV+, p16+/HPV- and p16-/HPV-. Results: Keratin (multi KRT) and HIF1A, a key regulator of hypoxia adaptation, were upregulated in both p16+/HPV- and p16-/HPV- tumours relative to p16+/HPV+. Several genes associated with tumour cell proliferation and survival (CCND1, AKT1 and CD44) were more highly expressed in p16-/HPV- tumours relative to p16+/HPV+. Conversely, multiple genes with potential roles in anti-tumour immune responses (immune cell recruitment/trafficking, antigen processing and presentation), such as CXCL9, CXCL10, ITGB2, PSMB10, CD74, HLA-DRB and B2M, were more highly expressed in the tumour and stromal compartments of p16+/HPV+ OPC versus p16-/HPV- and p16+/HPV-. CXCL9 was the only gene showing significant differential expression between p16+/HPV- and p16-/HPV- tumours being upregulated within the stromal compartment of the former. Conclusions: In terms of immune-oncology-related gene expression, discordant p16+/HPV- OPCs are much more closely aligned with p16-/HPV-OPCs and quite distinct from p16+/HPV+ tumours. This is consistent with previously described prognostic patterns (p16+/HPV+ >> p16+/HPV- > p16-/HPV-) and underlines the need for dual p16 and HPV testing to guide clinical decision making.

Heightened Epstein-Barr virus immunity and potential cross-reactivities in multiple sclerosis.

BACKGROUND: Epstein-Barr virus (EBV) is a likely prerequisite for multiple sclerosis (MS) but the underlying mechanisms are unknown. We investigated antibody and T cell responses to EBV in persons with MS (pwMS), healthy EBV-seropositive controls (HC) and post-infectious mononucleosis (POST-IM) individuals up to 6 months after disease resolution. The ability of EBV-specific T cell responses to target antigens from the central nervous system (CNS) was also investigated. METHODS: Untreated persons with relapsing-remitting MS, POST-IM individuals and HC were, as far as possible, matched for gender, age and HLA-DRB1*15:01. EBV load was determined by qPCR, and IgG responses to key EBV antigens were determined by ELISA, immunofluorescence and Western blot, and tetanus toxoid antibody responses by multiplex bead array. EBV-specific T cell responses were determined ex vivo by intracellular cytokine staining (ICS) and cross-reactivity of in vitro-expanded responses probed against 9 novel Modified Vaccinia Ankara (MVA) viruses expressing candidate CNS autoantigens. RESULTS: EBV load in peripheral blood mononuclear cells (PBMC) was unchanged in pwMS compared to HC. Serologically, while tetanus toxoid responses were unchanged between groups, IgG responses to EBNA1 and virus capsid antigen (VCA) were significantly elevated (EBNA1 p = 0.0079, VCA p = 0.0298) but, importantly, IgG responses to EBNA2 and the EBNA3 family antigens were also more frequently detected in pwMS (EBNA2 p = 0.042 and EBNA3 p = 0.005). In ex vivo assays, T cell responses to autologous EBV-transformed B cells and to EBNA1 were largely unchanged numerically, but significantly increased IL-2 production was observed in response to certain stimuli in pwMS. EBV-specific polyclonal T cell lines from both MS and HC showed high levels of autoantigen recognition by ICS, and several neuronal proteins emerged as common targets including MOG, MBP, PLP and MOBP. DISCUSSION: Elevated serum EBV-specific antibody responses in the MS group were found to extend beyond EBNA1, suggesting a larger dysregulation of EBV-specific antibody responses than previously recognised. Differences in T cell responses to EBV were more difficult to discern, however stimulating EBV-expanded polyclonal T cell lines with 9 candidate CNS autoantigens revealed a high level of autoreactivity and indicate a far-reaching ability of the virus-induced T cell compartment to damage the CNS.

Circulating tumour DNA detects somatic variants contributing to spatial and temporal intra-tumoural heterogeneity in head and neck squamous cell carcinoma.

Background: As circulating tumour DNA (ctDNA) liquid biopsy analysis is increasingly incorporated into modern oncological practice, establishing the impact of genomic intra-tumoural heterogeneity (ITH) upon data output is paramount. Despite advances in other cancer types the evidence base in head and neck squamous cell carcinoma (HNSCC) remains poor. We sought to investigate the utility of ctDNA to detect ITH in HNSCC. Methods: In a pilot cohort of 9 treatment-naïve HNSCC patients, DNA from two intra-tumoural sites (core and margin) was whole-exome sequenced. A 9-gene panel was designed to perform targeted sequencing on pre-treatment plasma cell-free DNA and selected post-treatment samples. Results: Rates of genomic ITH among the 9 patients was high. COSMIC variants from 19 TCGA HNSCC genes demonstrated an 86.9% heterogeneity rate (present in one tumour sub-site only). Across all patients, cell-free DNA (ctDNA) identified 12.9% (range 7.5-19.8%) of tumour-specific variants, of which 55.6% were specific to a single tumour sub-site only. CtDNA identified 79.0% (range: 55.6-90.9%) of high-frequency variants (tumour VAF>5%). Analysis of ctDNA in serial post-treatment blood samples in patients who suffered recurrence demonstrated dynamic changes in both tumour-specific and acquired variants that predicted recurrence ahead of clinical detection. Conclusion: We demonstrate that a ctDNA liquid biopsy identified spatial genomic ITH in HNSCC and reliably detected high-frequency driver mutations. Serial sampling allowed post-treatment surveillance and early identification of treatment failure.

Developing and Validating a Multivariable Prognostic-Predictive Classifier for Treatment Escalation of Oropharyngeal Squamous Cell Carcinoma: The PREDICTR-OPC Study.

PURPOSE: While there are several prognostic classifiers, to date, there are no validated predictive models that inform treatment selection for oropharyngeal squamous cell carcinoma (OPSCC).Our aim was to develop clinical and/or biomarker predictive models for patient outcome and treatment escalation for OPSCC. EXPERIMENTAL DESIGN: We retrospectively collated clinical data and samples from a consecutive cohort of OPSCC cases treated with curative intent at ten secondary care centers in United Kingdom and Poland between 1999 and 2012. We constructed tissue microarrays, which were stained and scored for 10 biomarkers. We then undertook multivariable regression of eight clinical parameters and 10 biomarkers on a development cohort of 600 patients. Models were validated on an independent, retrospectively collected, 385-patient cohort. RESULTS: A total of 985 subjects (median follow-up 5.03 years, range: 4.73-5.21 years) were included. The final biomarker classifier, comprising p16 and survivin immunohistochemistry, high-risk human papillomavirus (HPV) DNA in situ hybridization, and tumor-infiltrating lymphocytes, predicted benefit from combined surgery + adjuvant chemo/radiotherapy over primary chemoradiotherapy in the high-risk group [3-year overall survival (OS) 63.1% vs. 41.1%, respectively, HR = 0.32; 95% confidence interval (CI), 0.16-0.65; P = 0.002], but not in the low-risk group (HR = 0.4; 95% CI, 0.14-1.24; P = 0.114). On further adjustment by propensity scores, the adjusted HR in the high-risk group was 0.34, 95% CI = 0.17-0.67, P = 0.002, and in the low-risk group HR was 0.5, 95% CI = 0.1-2.38, P = 0.384. The concordance index was 0.73. CONCLUSIONS: We have developed a prognostic classifier, which also appears to demonstrate moderate predictive ability. External validation in a prospective setting is now underway to confirm this and prepare for clinical adoption.

FBXL7 Body Hypomethylation Is Frequent in Tumors from the Digestive and Respiratory Tracts and Is Associated with Risk-Factor Exposure.

Squamous cell carcinoma is the main histological tumor type in the upper aerodigestive tract (UADT), including the esophagus (ESCC) and the head and neck sites, as well as the oral cavity (OCSCC), larynx (LSCC) and oropharynx (OPSCC). These tumors are induced by alcohol and tobacco exposure, with the exception of a subgroup of OPSCC linked to human papillomavirus (HPV) infection. Few genes are frequently mutated in UADT tumors, pointing to other molecular mechanisms being involved during carcinogenesis. The F-box and leucine-rich repeat protein 7 (FBXL7) is a potential tumor-suppressing gene, one that is frequently hypermethylated in pancreatic cancer and where the encoded protein promotes the degradation of AURKA, BIRC5 and c-SRC. Thus, the aim of this study was to evaluate the methylation and expression profile of FBXL7 in the UADT and the gene's association with the clinical, etiological and pathological characteristics of patients, as well as the expression of its degradation targets. Here we show that the FBXL7 gene's body is hypomethylated in the UADT, independently of histology, but not in virus-associated tumors. FBXL7 body methylation and gene expression levels were correlated in the ESCC, LSCC, OCSCC and OPSCC. Immunohistochemistry analysis showed that FBXL7 protein levels are not correlated with the levels of its degradation targets, AURKA and BIRC5, in the UADT. The high discriminatory potential of FBXL7 body hypomethylation between non-tumor and tumor tissues makes it a promising biomarker.

Immediate Sample Fixation Increases Circulating Tumour Cell (CTC) Capture and Preserves Phenotype in Head and Neck Squamous Cell Carcinoma: Towards a Standardised Approach to Microfluidic CTC Biomarker Discovery.

INTRODUCTION: Research demonstrates strong evidence that circulating tumour cells (CTCs) can provide diagnostic and/or prognostic biomarkers in head and neck squamous cell carcinoma (HNSCC) and a potential tool for therapeutic stratification. However, the question still remains as to the optimum method of CTC enrichment and how this can be translated into clinical practice. We aimed to evaluate the Parsortix microfluidic device for CTC enrichment and characterisation in HNSCC, seeking to optimise a sample collection and processing protocol that preserves CTC integrity and phenotype. METHOD: Spiking experiments of the FaDu and SCC040 HNSCC cell lines were used to determine the Parsortix capture rate of rare "CTC-like" cells. Capture rates of cancer cells spiked into EDTA blood collections tubes (BCTs) were compared to the Transfix fixative BCT and Cytodelics whole blood freezing protocol. The Lexogen Quantseq library preparation was used to profile gene expression of unfixed cells before and after microfluidic enrichment and enriched cell line spiked Transfix blood samples. An antibody panel was optimised to enable immunofluorescence microscopy CTC detection in HNSCC patient Transfix blood samples, using epithelial (EpCAM) and mesenchymal (N-cadherin) CTC markers. RESULTS: Across a spiked cell concentration range of 9-129 cells/mL, Parsortix demonstrated a mean cell capture rate of 53.5% for unfixed cells, with no significant relationship between spiked cell concentration and capture rate. Samples preserved in Transfix BCTs demonstrated significantly increased capture rates at 0 h (time to processing) compared to EDTA BCTs (65.3% vs. 51.0%). Capture rates in Transfix BCTs were maintained at 24 h and 72 h timepoints, but dropped significantly in EDTA BCTs. Gene expression profiling revealed that microfluidic enrichment of unfixed cell lines caused downregulation of RNA processing/binding gene pathways and upregulation of genes involved in cell injury, apoptosis and oxidative stress. RNA was successfully extracted and sequenced from Transfix preserved cells enriched using Parsortix, demonstrating epithelial specific transcripts from spiked cells. In a proof-of-concept cohort of four patients with advanced HNSCC, CTCs were successfully identified and visualised with epithelial and epithelial-mesenchymal phenotypes. CONCLUSION: We have optimised a protocol for detection of CTCs in HNSCC with the Parsortix microfluidic device, using Transfix BCTs. We report a significant benefit, both in terms of cell capture rates and preserving cell phenotype, for using a fixative BCT- particularly if samples are stored before processing. In the design of large cohort multi-site clinical trials, such data are of paramount importance.

Lack of predictive tools for conventional and targeted cancer therapy: Barriers to biomarker development and clinical translation.

Predictive tools, utilising biomarkers, aim to objectively assessthe potentialresponse toa particular clinical intervention in order to direct treatment.Conventional cancer therapy remains poorly served by predictive biomarkers, despite being the mainstay of treatment for most patients. In contrast, targeted therapy benefits from a clearly defined protein target for potential biomarker assessment. We discuss potential data sources of predictive biomarkers for conventional and targeted therapy, including patient clinical data andmulti-omicbiomarkers (genomic, transcriptomic and protein expression).Key examples, either clinically adopted or demonstrating promise for clinical translation, are highlighted. Following this, we provide an outline of potential barriers to predictive biomarker development; broadly discussing themes of approaches to translational research and study/trial design, and the impact of cellular and molecular tumor heterogeneity. Future avenues of research are also highlighted.

Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Head and Neck Cancer.

PURPOSE: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxia-immune classifier with potential application in patient prognostication and prediction of response to targeted therapy. EXPERIMENTAL DESIGN: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n = 275) and validated using two independent cohorts (n = 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining. RESULTS: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and survival profiles: hypoxialow/immunehigh, hypoxiahigh/immunelow, and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P = 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxialow/immunehigh and hypoxiahigh/immunelow tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3+ T cells (r = -0.5464; P = 0.0377), further corroborating the transcription-based classification. CONCLUSIONS: We developed and validated a hypoxia-immune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination.

Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three "first wave" proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501), as well as subdominant responses through common class I alleles (e.g. B7 and C*0304). Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that "first wave" antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design.

The immunology of Epstein-Barr virus-induced disease.

Epstein-Barr virus (EBV) is usually acquired silently early in life and carried thereafter as an asymptomatic infection of the B lymphoid system. However, many circumstances disturb the delicate EBV-host balance and cause the virus to display its pathogenic potential. Thus, primary infection in adolescence can manifest as infectious mononucleosis (IM), as a fatal illness that magnifies the immunopathology of IM in boys with the X-linked lymphoproliferative disease trait, and as a chronic active disease leading to life-threatening hemophagocytosis in rare cases of T or natural killer (NK) cell infection. Patients with primary immunodeficiencies affecting the NK and/or T cell systems, as well as immunosuppressed transplant recipients, handle EBV infections poorly, and many are at increased risk of virus-driven B-lymphoproliferative disease. By contrast, a range of other EBV-positive malignancies of lymphoid or epithelial origin arise in individuals with seemingly intact immune systems through mechanisms that remain to be understood.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

T cell detection of a B-cell tropic virus infection: newly-synthesised versus mature viral proteins as antigen sources for CD4 and CD8 epitope display.

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8+ and CD4+ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the B-lymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8+ and CD4+ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36-48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4+ T cells.

Cyclical expression of EBV latent membrane protein 1 in EBV-transformed B cells underpins heterogeneity of epitope presentation and CD8+ T cell recognition.

CD8(+) T cells specific for EBV latent cycle epitopes can be reactivated in vitro by stimulating with the autologous EBV-transformed B lymphoblastoid cell line (LCL). The resultant CD8(+) clones kill epitope peptide-loaded targets, but frequently do not kill or show only low levels of lysis of the unmanipulated LCL in 5-h cytotoxicity assays. However, they reproducibly show clear LCL recognition in cytokine (IFN-gamma) release assays and inhibit LCL outgrowth in long-term coculture assays. We show that this growth inhibition is not mediated by cytokines, but by slow killing detectable in extended cytotoxicity assays. The paradoxical earlier findings reflect the fact that cytokine assays are more sensitive indicators of Ag-specific recognition in situations in which the target population is heterogeneous at the single-cell level in terms of epitope display. Such heterogeneity exists within LCLs with, at any one time, subpopulations showing large differences in sensitivity to T cell detection. These differences are not cell cycle related, but correlate with differing levels of EBV latent membrane protein (LMP)1 expression at the single-cell level. In this study, LMP1 is not itself a CD8(+) T cell target, but its expression enhances Ag-processing capacity and HLA class I expression. We propose that LMP1 levels fluctuate cyclically in individual cells and, over time, all cells within a LCL pass through a LMP1(high) T cell-detectable phase.

CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1.

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I-restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.