Liquid biopsies for CNS tumors

Brain tumors are a heterogeneous group of tumors that constantly evolve,
generally recur and often progress to more malignant subtypes. Patients often
have a short life expectancy. Current diagnostic criteria an clinical
management rely on the tissue profiling obtained by invasive neuro-surgical
procedures.
Gross total tumor resection is associated with a better survival and better
quality of life in patients with brain tumors. However, in many patients gross
total resection is not safely possible. In these patients, a biopsy of the
tumor has to be taken with the sole reason to obtain tissue for diagnosis. No
patient benefit is to be expected from the biopsy. A biopsy comes with a
significant risk of neurological deterioration (even death).

Following the initial diagnosis, patients are currently monitored by magnetic
resonance imaging (MRI) to determine residual tumor mass, examine growth speed
and screen for signs of malignant dedifferentiation (i.e. presence of regions
with contrast enhancement). A major limitation of MRI is the limited ability to
distinguish therapy-induced necrosis (*pseudo-progression*) from genuine
tumoral burden or to identify minimal tumoral burden. New advanced MRI
techniques and nuclear imaging techniques cannot distinguish pseudo-progression
form genuine tumoral burden with a sufficient diagnostic accuracy.

Liquid biopsies may bypass the need for surgery but its use in brain tumor
patients has historically been challenging. Recent data has demonstrated that
circulating tumor DNA (ctDNA) can reliably be detected in plasma of brain tumor
patients using high throughput methylation profiling. Clinical implementation
however requires an additional increase in detection sensitivity. Med-Seq is a
novel methylation technique that we hypothesize to be more sensitive than
currently employed techniques to detect ctDNA for brain tumor diagnostics. This
project will explore the use of this technique and its suitability for tumor
diagnosis and monitoring.

The overall goal of this project is to bring the promising non-invasive *liquid
biopsy* technique from the lab to clinical practice in brain tumor patients. We
therefore aim to identify and characterize ctDNA in blood from brain tumor
patients using Med-Seq, and assess its diagnostic value. Our research question
is: *Can we identify ctDNA from brain tumor patients using Med-Seq and use it
for tumor diagnosis and disease monitoring?*.

As the concept of liquid biopsies in glioblastoma has exclusively focused on
the detection of tumor cells or tumor DNA in the peripheral blood with limited
results, we here also assess a new angles to solve this problem. Interestingly,
the value of immunophenotyping of lymphocytes in the peripheral blood to
monitor glioblastoma disease progression has never been assessed. In previous
work, we identified a significant increase in T cells in recurrent glioma
compared to their initial counterpart. Remarkably, these T cells are in
majority CD3+ but not CD8+, probably suggesting an immunosuppressive phenotype.
These T cells are recruited from the peripheral blood. We hypothesize that upon
glioblastoma progression, peripheral blood T cell counts decrease and T cell
phenotype becomes more tumorsuppressive as compared to the stable disease
status.

The overall aim of this project is to improve glioblastoma disease monitoring
with T cells as peripheral blood biomarker.

Med-Seq will be used to identify ctDNA in plasma of patients with a brain
tumor. Genome-wide DNA methylation profiling will be done by sequencing
fragments generated by a methylation sensitive restriction enzyme. All data
will be combined with data from other tumor types and normal tissues, and
bio-informatical analysis (principle component analysis/tSNE/UMAP, logistic
regression, Random Forest) will be used to predict tumor presence and type.

To assess the phenotype of T cells in the tumor in comparison to peripheral
blood, we will use spectral flow cytometry. A pre-existing 38 marker-panel
dedicated to T cells in the brain will be applied towards 12 fresh glioblastoma
samples that will be transferred from the operating room directly to the lab,
and on peripheral blood mononuclear cells (PBMCs) derived on the day of surgery
from the same patient. We will isolate tumor DNA and RNA, perform multiplex
immunofluorescence stainings on the tumor to inform on spatial distribution of
immune effector cells, and annotate clinical data including age, sex and
ethnicity.
T cells in tumor and peripheral blood will be compared. We anticipate to find
major differences, but hypothesize to also find intriguing similarities, like T
cells in the peripheral blood with a brain residency associated phenotype.
Prospective analysis of peripheral blood T cells and relation with tumor
progression
To find a change in peripheral T cell count and phenotype in the course of the
disease, we will apply a 13-panel flow cytometry on PBMCs on prespecified time
points during the course of the disease, until tumor progression is confirmed
on MRI. Thirty glioblastoma patients (first 12 included in previous step) will
be included. Patients will be treated and followed-up as per clinical protocol.
Clinical and imaging data will be collected.

This is a pilot study to test the technique. We will draw 30ml of blood from 30
brain tumor patients. Their data will be compared to the already available data
of healthy controls.

Blood will be drawn at timepoints where blood drawing is already indicated for
medical reasons, except for one moment (day of surgery), when an extra blood
drawn will be scheduled. 30ml extra blood will not cause burden or risk for the
patient