Early Detection of Immunotherapy-mediated Toxicity

Rationale: The number of tumor types and settings in which immune checkpoint inhibitors
(immunotherapy) is standard treatment is rapidly expanding. However, toxicity is a frequent
adverse event (irAE) often necessitating high-dose of immunosuppressant treatments such as
corticosteroids and sometimes even requiring permanent discontinuation of checkpoint
inhibitors. Early detection of immunotherapy-mediated toxicity and early initiation of
immunosuppressant treatment might reduce the disease burden from immunotherapy, in
addition to reducing the total dose of glucocorticoids and other immunosuppressants needed for
clinical management. More importantly, it might result in a lower discontinuation rate of
treatment due to severe toxicity.
Objective: We hypothesize that during an irAE (immune-related adverse event), as a result of
inflammation and damage to the specific organ, organ-derived DNA will be detectable in blood
of patients. The aim of this project is to investigate whether the presence of cell-free DNA
originating from the organ towards which immunotherapy-induced toxicity is directed, can be
detected using epigenetic profiling of cell-free DNA.
Study design: Organ specific methylation patterns in cell-free DNA will be derived from 1)
paired blood samples collected from patients during an episode of immunotherapy-mediated
toxicity and in absence of immunotherapy-mediated toxicity and 2) samples from patients
without checkpoint inhibitor treatment but with organ confined auto-inflammatory diseases.
Optionally, feces will be collected at the same time points to investigate inflammation
biomarkers during an episode of immunotherapy-mediated colitis. In addition, blood will be
drawn for investigation of other biomarkers of inflammation.
Study population: Adult patients planned to receive or receiving immune checkpoint inhibitors
as anti-cancer treatment and adult patients with auto-inflammatory diseases directed to a
specific organ
Main study parameters/endpoints: The primary study endpoint is the detection of organspecific
methylation patterns in cell-free DNA during an irAE. Secondary endpoints are the
levels of other biomarkers of inflammation during immunotherapy-mediated toxicity and the
comparison organ-specific methylation profiles in blood with other biomarkers of inflammation.

We hypothesize that during an irAE, as a result of inflammation and damage to the specific organ,
organ-derived DNA will be detectable in blood of patients. The aim of this project is to investigate
whether the presence of cell-free DNA originating from the organ towards which immunotherapyinduced
toxicity is directed, can be detected using epigenetic profiling of cell-free DNA. If this
hypothesis is confirmed, epigenomic profiling of cell-free DNA should further be explored to test
Figure 2. Overview of MeD-seq technology. Active genes display gene body methylation (1), whereas inactive
genes show promoter and enhancer methylation. LpnPI digests methylated templates in 32 bp fragments (3)
surrounding the LpnPI containing reads and alignment to genome (7). Shown are gene tracks of MeD-seq readcounts
displaying differential DNA methylation of the HOXB locus in liver and ovary
NL77494.078.21 Early Detection of Immunotherapy-mediated Toxicity EDIT study
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Version number 1.0, date: 18-05-2021
whether it can serve as an early detection marker for ieAEs, i.e. before the patient experiences
symptoms or when the patient only experiences mild symptoms.

start immunotherapy, during immunotherapy, during irAE, after irAE