Using Blood and Stool Samples From Patients With Blood Cancer and/or Haemophagocytic Lymphohistiocytosis to Study the Influence of Host Microbial Factors on Toxicity and Survival

Introduction Immuno-therapy in the form of bispecific antibodies (BiSp) and chimeric antigen receptor T-cell (CAR-T) therapy have resulted in a paradigm shift in treatment for both aggressive and indolent B-cell lymhomas such as diffuse large B-cell lymphom and (DLBCL) and follicular lymphoma (FL). Immunotherapy utilises the patient's own immune system to help fight their diseases. Despite transformative results, CAR-T continues to have limitations in terms of both efficacy and adverse events, with a toxicity profile characterised by T-cell proliferation and over-activation primarily in the form of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) with frequent intensive care admissions. CRS is a non-antigen-specific toxicity that occurs as a result of high-level immune activation, while the underlying mechanism driving ICANS is not fully understood, but is thought to relate to the release of inflammatory cytokines increasing vascular permeability, endothelial activation and leading blood-brain barrier breakdown. Similar toxicities are seen with BiSp although mitigation in the form of step up dosing and corticosteroid prophylaxis means these are somewhat less than that seen with CAR-T.

Haemophagocytic Lymphohistiocytosis (HLH) is a rare and often lethal hyper-inflammatory syndrome induced by aberrantly activated macrophages and cytotoxic T-cells. Patients develop fever, cytopenias and haemophagocytosis which may rapidly develop into multi-organ failure, akin to CRS. In general, infants tend to have inherited T- and NK-cell defects impairing cytotoxic function, younger adults have underlying acute viral infections and autoimmunity while older adults are those most likely to suffer from underlying cancer and have extremely poor outcomes

Over the past decade, a body of literature has emphasised interplay between the gut microbiota (GM), the immune system and the gut-brain axis. Compositional and functional changes in the GM (gut dysbiosis) may result in expansion of pro-inflammatory pathobionts with potential to enter the blood circulation, triggering cytokine signalling, as well as loss of normally residing microbiota or general loss of diversity. Host-gut microbiota interactions are mediated by small molecule metabolites, and dysbiosis may lead to alterations in innate and adaptive immunity, including regulatory T-cell generation and NK-cell function. Recent studies suggest dysbiosis to be associated with inferior outcomes and greater toxicity following CAR-T cell therapy. A retrospective study examining reported broad spectrum antibiotic therapy prior to CAR-T to be associated with worse survival and increased neurotoxicity, with the presence of Clostridia species associated with complete response. A further study found significant correlations between baseline Bifi dobacterium longum abundance and survival, and developed an algorithm predicting responders based on Bacteroides, Ruminococcus, Eubacterium, and Akkermansia abundance. These studies suggest the GM is intertwined with outcomes following CAR T-cell therapy but the mechanism and pathophysiology are not yet known. To date, there is no data regarding the GM and BiSp responses or immune related toxicities, or outcomes following a diagnosis of HLH.

In order to understand the effects of both the GM and the host immune system on blood cancer in general as well as outcomes and toxicities of immunotherapies in blood cancer and HLH, the investigators plan to prospectively collect stool and blood samples from patients undergoing treatment at multiple time-points. These samples will be used to characterise changes in GM and faecal metabolites, aiming to identify novel inflammatory pathogenic pathways within the GM and associated metabolites which contribute to CRS, ICANS and HLH and survival which are amenable to therapeutic targeting. For patients with follicular lymphoma, the investigators will assess if the GM affects the risk of disease progression and the need for systemic immuno-chemotherapy.

The ongoing collection of stool and blood samples from people with blood cancer or diagnosed with HLH will occur in a single centre, Nottingham University Hospitals NHS Trust. Samples will be used to study the effects of the host GM on toxicity, progression and survival. Stool and serum samples are planned to undergo shotgun metagenomics (microbiome profiling) and untargeted metabolomics. Multi-omics integration analysis will be conducted, including taxonomy, functional and metabolite analysis. Any remaining samples will, given patient permission, be kept for future ethically approved related studies, including potential downstream investigations such as organ-on-chip (gut, liver, brain), stool and serum mRNA analysis (RNA-seq) to identify downregulated targets, stool and blood non-coding RNA analyses, proteomics, epigenomics and glycomics as well as in vivo zebrafish and/or other animal studies. Stool and peripheral blood mononuclear cells can be co-cultured in primary cell lines or organ-on-chips (EMULATE) to mimic disease and to evaluate therapies. These studies may be carried out at the University of Nottingham, or be transferred to other researchers within and outside of the UK and other commercial institutions.