Liquid Biopsy in Neuro-oncology in the St Antonius Hospital

Dissemination of cancer to the central nervous system (CNS) is a feared
complication of a cancer. These metastases are localized in the brain
parenchyma or leptomeninges including the cerebrospinal fluid - so called
leptomeningeal metastases.
Brain metastases (BM) and leptomeningeal metastases (LM) may also co-occur. The
highest propensity for CNS metastases are with small cell and non-small cell
lung carcinoma, breast carcinoma and melanoma. Brain metastases incidence rates
are reported between 9%-26%, and leptomeningeal incidence rates between 0,8- 8.%
Progression of disease in the CNS might occur despite good response of systemic
disease to systemic cancer treatment - so called 'neuro-systemic dissociation'.
An example of this is CNS progression in female patients with HER-2 positive
breast carcinoma treated with anti- HER directed therapies.
Lack of response of CNS metastases is often explained by insufficient drug
penetration in the CNS compartment due to the Blood-Brain Barrier.
However CNS progression also occurs despite penetration of drugs in the CNS
compartment. A likely explanation for this 'immunity' of CNS metastases is
discordance of the metastases at a molecular level, as compared to the primary
tumour. In solid material of brain metastases clinically informative new DNA
mutations were detectable in approximately 50% of patients, not detectable in
the primary tumour. These additional DNA mutations could either offer a
molecular explanation for treatment resistance or offer new targetable
mutations for the CNS metastases.
Translating this insight into clinical practice is hampered by the fact that
solid material of brain metastases is not available in most patients with CNS
metastases.
In the underlying study we will further explore the additional diagnostic and
predictive value of so-called Liquid biopsy for patients who present with
suspected CNS metastases in the Sint Antonius Hospital. The principle of liquid
biopsy is that tumours shed parts in bodily fluids - for example as circulating
tumour DNA (ct-DNA)- that can be isolated and analysed. The main advantage of
liquid biopsy is that it is largely non-invasive- in contrast to traditional
biopsy. Furthermore, it can better track tumours and mutations over a duration
of time, and it may also be used to validate the efficiency of cancer
treatment. There is increasing interest in tumour mutation detection (e.g.
EGFR, BRAF, ALK) and treatment-resistance related mutations (e.g. T790M when
using EGFR inhibitors, PIK3CA mutations when using anti-HER directed therapies)
in liquid biopsies. Detection of ctDNA in blood in metastasized cancer has been
extensively studied and is increasingly used in clinical practice for example
in breast cancer and non-small cell lung cancer.
The use of ctDNA detection in CSF is currently being explored in patients with
leptomeningeal metastases and/ or brain metastases. Recent data suggest that
ctDNA can also be isolated from CSF from patients which appear on MRI as
*isolated* brain metastases without leptomeningeal involvement. Detection of
tumour specific mutations in CSF may lead to a better understanding of therapy
resistance of CNS metastases and improved future targeted treatment for CNS
disease. Small series of LM and BM confirm that both digital droplet (dd-) PCR
and Next Generation Sequencing (NGS) are applicable in CSF and can thereby
identify respectively single or multiple cancer-driver- and resistance
mutations.
In previous years liquid biopsy techniques have been developed at the
Department of Clinical Chemistry in the SAZ with financial support of the Sint
Antonius Innovation fund.

Implementation of liquid biopsy by NGS in cell-free circulating tumor DNA in
CSF and / or blood, in patients clinically suspected of leptomeningeal
metastases and / or brain metastases

Demonstrate that the detection of additional tumor mutations in CSF-ct DNA,
compared to the blood or (if available) primary tumor, has additional value in
explaining therapy resistance and demonstrating targeted mutations in patients
with CNS metastases of solid tumors

laboratory experimental, prospective cohort

A venipuncture is performed after the planned lumbar puncture. The tubes with
extra cerebrospinal fluid and blood are handed over to the trial coordinator of
the Laboratory for Clinical Chemistry.
Next generation sequencing (NGS) is performed with the Oncomine Pancancer
cell-free assay in both CSF and blood, if available NGS is also performed on
solid material of the primary tumor Particularly for the secondary research
objectives, a limited number of patient and clinical parameters are obtained.
These mainly concern the primary tumor, cancer treatment and course of disease.
The liquid biopsy findings are compared with routine parameters with which the
diagnosis of leptomeningeal metastases has been made, namely routine CSF
parameters and results of neuro-imaging. The patient and clinical data are
stored encrypted in Redcap
Presentation of the data is mainly descriptive - namely, the percentage of
samples in which NGS analysis is successful, and the number of additional
clinically relevant mutations in CSF, relative to blood and solid material
Statistic analysis such as multivariate survival analysis will take place for
the secondary outcome measures
All material (CSF and blood) and data will be retained until September 1, 2030
(5 years after completion of the study on September 1, 2025) and then destroyed.
During this storage period, related residual material can still be examined -
for example for specific new mutations or proteins - if new developments occur
in this research area
In principle, all tests will take place in the SAZ. However, for possible
confirmation of NGS test results, encoded material (CSF and blood), without
patient data, can be sent to the NKI / AVL.

The burden of sampling includes the lumbar puncture and veni-puncture. There is
no extra burden due to participation in this study except for the
veni-puncture- as the lumbar puncture was already clinically indicated. The
amount of CSF drawn is approx. 20 ml, of which 10 ml is used for ct DNA
analysis, the amount of blood drawn approx. 60 ml. The risk of participation
in this trial is therefore negligible. Patients also have no personal benefit
as the investigation of new laboratory techniques does not influence current
standard clinical care.