Understanding the pathogenesis of Epstein-Barr virus-associated complications arising in patients treated with immune checkpoint regulators

Immune checkpoint regulators (iCR) are a revolutionary new class of treatment which manipulate the immune system to fight cancer. Whilst iCR regimens have yielded improved outcomes for patients with cancer, they are associated with a number of autoimmune complications termed 'immune-related adverse events' (irAE). One of the commonest irAE to affect patients is immune related colitis (irColitis), which in approximately 1-5% of patients, will result in severe inflammation of the bowel and perforation. Colonic perforation is a serious and life threatening emergency which has potential long term implications for patients. It is known that bacteria can modulate the effects and complications of immune checkpoint therapy. Nothing is known about the effect of viruses on outcome following iCR.

In a small retrospective study of diagnostic tissue specimens, we found a proportion of iCR treated patients with irCol showed multiple ulcerations along the course of their gastrointestinal tract. On further investigation, these ulcerations contained immune cells infected with the Epstein-Barr virus (EBV). This entity is called EBV mucocutaneous ulcer (EBVMCU). Critically, the 4 of 13 cases which showed EBVMCU were all associated with severe inflammation and perforation.

The findings suggest the either iCR, or the anti-immune drugs used to treat colitis, affects the way the immune system control EBV, which is latently present in 95% of the population. This is the first ever example of a link between viruses and complications related to iCR treatment. As new iCR drugs are developed, it is possible that such interactions will become ever more important to how iCRs are used clinically.

The aim of this study is to:
1. Characterise the clinical course and pathological features of iCR associated EBVMCU.
2. Study the changes in the immune system in blood and tissue that occur upon commencement if iCR therapy, development of irColitis.

Plan
Part 1:
We will characterise the features of EBVMCU that occur in patients treated on iCR therapy. We will examine surplus diagnostic tissue from patients who have suffered bowel perforated following irColitis. We will perform the following tests:
i. EBV: Test for the presence of EBV.
ii. Genes: we will see if variations in the CTLA-4 gene, the target of some iCR, effect EBVMCU development in iCR.
iii. We will examine the cellular composition and the immune cells of the EBVMCU lesions.
This will enable us to establish the incidenece of EBVMCU in irColitis and provide insight into how these lesions develop.
Part 2:
We will follow 50 patients started on iCR therapy and measure a the effect iCR has on the immune system, specifically, the control of EBV. This will provide new information on the effects of iCRs on human viruses and the immune responses that is essential for controlling them.

The study will provide a valuable insight into the regulation of EBV immunity and could have clinical consequences as to how irAE are managed.

Background: Immune checkpoint regulators (iCR) have revolutionised the management of some cancer types, but are associated with immune related adverse events including immune related colitis (irColitis). Whilst we know the bacterial microbiome mediates response and complication in iCR therapy, little is known about the effects of the viriome. EBV is a ubiquitous viral pathogen present in 95% of the population; we have recently made the first link between this virus and complications of iCR. In a series of 13 patients suffering immune related colitis following ipilimumab, all four colonic perforations were associated with the recently recognised EBV positive mucocutaneous ulcer (EBVMCU). The presence of EBVMCU in this context is unexpected, but treatment related immunosuppression and specific CTLA-4 mediated effects are implicated.

Purpose of the study:
1. Characterise the clinicopathological features of EBVMCU in irColitis.
2. Examine the effects of iCR on EBV specific immunity.

Plan of investigation: The study will comprise two components:
1. A multicentre study to characterise the clinicopathological features of EBVMCU in irColitis.
We will retrospectively examine the surplus diagnostic FFPE material of all resection specimens (n~40) and a subset of biopsy specimens (n=50). We will perform the following experiments:
i. Effects of CTLA-4 polymorphisms on EBVMCU development in irColitis
ii. Immune cell composition determined by VECTRA multiplex immunofluorescence
iii. T/B-cell clonality to determine if there are clonal expansion within EBVMCU
We will also seek to identify any colectomy specimens prospectively, with a view to examine lesional EBV specific immunity.

2. Prospectively examine the effects of iCR therapy on EBV immune surveillance in 50 patients.
We will prospectively examine changes in systemic EBV viral load and EBV specifiv T-cell immunity following iCR therapy prospectively in a cohort of 50 patients.

Immune checkpoint inhibitors are rapidly becoming the 'fourth modality' of cancer treatment, and are being used to treat a widening range of cancers eliciting profound clinical benefits for some patients. These agents are, unfortunately also associated with autoimmune complications which can be life threatening. As new immune checkpoint inhibitors are developed, and as their usage increases, it is vital that we understand how best to deploy these new drugs, maximising benefit while reducing harm., A key factor influencing treatment benefit and side effects is the microbiome, bacteria and viruses that colonise human beings. As yet, while certain bacteria haven been shown to influence these drugs, no viral agents have been investigated in detail.

I have recently shown, for the first time, that Epstein-Barr virus is linked to an important complication of checkpoint therapy. This study builds on this foundation, seeking ti understand more about the interactions between these powerful immune modifying drugs and a key component of the human virome. As such, it provides several impactful outcomes. First, it may allow drug treatment to be improved, benefiting patients in the short and long term. Second, it has the potential to guide the deployment of novel immune modifying drugs being developed to treat cancer. Third, it has the potential to reveal new insights into the human immune system and EBV, a key pathogen that infects 95% of people yet is also linked to 200,000 cases of cancer annually.