Exploring the Feasibility of Cerebrospinal Fluid (CSF) Liquid Biopsy in Patients With Metastatic Solid Tumours and Leptomeningeal Disease (Cohort A), Parenchymal Brain Metastases (Cohort B), or No Evidence of Central Nervous System (CNS) Metastases (Cohort C): A Pilot Study
Phase: NA
Conditions: Solid Tumor Malignancies, Brain Metastasases, Leptomeningeal Disease (LMD)
Sponsor: Sunnybrook Health Sciences Centre

 Nov 2025 
 
 
Exploring the Feasibility of Cerebrospinal fluid (CSF) Liquid Biopsy in Patients with 
Metastatic Solid Tumours and Leptomeningeal Disease (Cohort A), Parenchymal Brain 
Metastases (Cohort B), or No Evidence of Central Nervous System (CNS) Metastases 
(Cohort C): A Pilot Study 
 
STUDY SYNOPSIS  
 
Research Questions: 
1. Among patients with metastatic solid tumours in each of three pre-defined cohorts (A, 
B, C), what proportion of patients have a positive cytology, a positive circulating 
tumour DNA (ctDNA), and/or positive circulating tumour cells (CTC) in 
cerebrospinal fluid (CSF) samples? 
2. Among the patients enrolled in the study, how do ctDNA and CTC results obtained 
from cerebral spinal fluid (CSF) and plasma compare in each of three cohorts (A, B, 
C)? 
3. Is it feasible to perform proteomic analysis of CSF among patients with metastatic 
solid tumours who have at least one positive CSF biomarker (cytology and/or ctDNA 
and/or CTCs) in each of three cohorts (A, B, C)? 
 
Population: 
Inclusion Criteria:  
1. Age > 18. 
2. Diagnosed with a metastatic solid tumour in one of the following scenarios: 
a. Patients in Cohort A will have leptomeningeal metastatic disease (LMD) with 
or without parenchymal brain metastases.  
b. Patients in Cohort B will have parenchymal brain metastases but no evidence 
of LMD.  
c. Patients in Cohort C will have metastatic solid tumours no CNS metastases 
(i.e. no LMD nor brain metastases).  
3. Patient is suitable for lumbar puncture and/or has an Ommaya reservoir that is 
accessible for CSF collection. 
4. Patient is eligible at any time point in their treatment whether they have started 
treatment for LMD  
5. Patients who previously enrolled in the study but for whom CSF biomarkers were 
negative can re-enroll later (e.g. upon progression of disease in the CNS). 
Exclusion Criteria: 
1. Inability to understand and not willing to sign a written informed consent document 
(language barrier not excluded; patients can have translator). 
 
Intervention: One-time CSF collective via lumbar puncture or Ommaya reservoir and 
collection of concurrent plasma of peripheral blood (liquid biopsy). 
  
Comparison: Not applicable. 
 

 Nov 2025 
 
 
 
End points: 
1. Primary endpoint: Proportion of eligible patients with positive CSF cytology, positive 
ctDNA, and positive CTCs. 
2. Secondary endpoint: Concordance of mutations identified on CSF ctDNA with 
plasma ctDNA. 
3. 3. Exploratory endpoint: Feasibility of conducting proteomic analysis of CSF.  
Study Design: 
Prospective single centre feasibility trial conducted at the Sunnybrook Odette Cancer Centre 
(SOCC).  
• 
Cohort A: 20 patients with any malignant solid tumour with LMD either suspected on 
imaging and/or confirmed via CSF cytology will be recruited. 
• 
Cohort B: 20 patients with any malignant solid tumour with parenchymal brain 
metastases will be recruited.  
• Cohort C: 20 patients with metastatic solid tumours and no evidence of LMD or 
parenchymal brain metastases (control group) will be recruited. 
 
 
Time Target: 
Patients will be recruited over a 12-month time period.  
 
1. OBJECTIVES  
 
Primary Objectives 
To estimate the proportion of patients with solid tumours and CNS metastases (either 
meeting criteria for Cohort A or B), or no CNS metastases (Cohort C) who have 
positive CSF cytology, positive ctDNA, and/or positive CTCs 
 
Secondary Objectives  
1. The concordance of mutations identified based on ctDNA profiling in the CSF 
and blood plasma in each of three cohorts (A, B, C). 
2. To explore the feasibility of proteomic analysis using CSF samples in each of 
three cohorts (A, B, C). 
 
 
 
 
 
 
 
 
 
 
 

 Nov 2025 
 
 
2. BACKGROUND 
 
Leptomeningeal metastatic disease (LMD) is the spread of malignant cells into the pia and 
arachnoid mater of the brain and spinal cord, affecting approximately 5-8% of all solid 
tumour types1. It is a rare complication that is associated with a high symptom burden and 
poor prognosis, with median survival ranging from 6-8 weeks without treatment and 3-6 
months with treatment1,2. Common solid tumours that result in LMD include breast, lung, 
gastro-intestinal (GI), melanoma, and primary central nervous system (CNS) cancers1,3. 
Resulting from the infiltration of cancer cells into the CNS, LMD can present with various 
symptoms such as headache, seizures, visual disturbances, weakness, and confusion1. The 
diagnosis and monitoring of LMD remain challenging due to the nonspecific clinical 
symptoms and false negative rates for MRI imaging and cerebrospinal fluid (CSF) analysis1.  
 
Current research states that CSF analysis is the gold standard for diagnosing LMD, however 
circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA) are new alternative 
diagnostic methods that have not been thoroughly evaluated to-date1. In this study, CSF 
samples collected via lumbar puncture (LP) or an Ommaya reservoir, along with blood 
plasma, will be obtained from patients with solid tumours who have diagnosed or suspected 
LMD. These samples will be analyzed for biomarkers to explore their association with 
clinical outcomes and potential therapeutic targets, ultimately guiding more effective 
treatment strategies.  
 
In addition, there is an unmet need to identify biomarkers in the CSF among patients with and 
without parenchymal brain metastases, even with the absences of LMD. If our exploratory 
study is successful, we may identify a novel mechanism to understand targetable mutations of 
CNS metastases through relatively less invasive mechanisms than craniotomy to surgically 
resect brain metastases. 
 
 
3. STUDY DESIGN  
 
 
Cohort A 
20 patients with any malignant solid tumour with LMD either suspected on imaging and/or 
confirmed via CSF cytology will be recruited with or without brain metastases.  
 
Cohort B 
20 patients with any malignant solid tumour and parenchymal brain metastases without 
evidence of LMD will be recruited. 
 
Cohort C 
20 patients with any malignant solid tumour and no evidence of CNS involvement (no LMD 
or parenchymal brain metastases) will be recruited as a control group. 
 

 Nov 2025 
 
 
 
Figure 1: Study Schema 
 
 
4. ENROLLMENT PROCEDURES 
 
Patient Enrollment  
Patients will be screened for eligibility to participate in the study. The study team will inform 
the treating oncologist regarding their eligibility prior to their medical appointment. After the 
study is introduced to eligible patients by their oncologist, the study will be discussed in more 
detail by the study team to obtain informed consent. 
 
5. PLANNED DATA COLLECTION  
 
Planned CSF and Blood Plasma Collection:  
CSF collection: Using standard techniques, CSF samples of 15-20 cc will be collected via 
lumbar puncture (LP) or an Ommaya reservoir. For LP’s, the use of a 20-gauge spinal needle 
and 2% lidocaine anaesthesia at L3/L4 or L4/L5 intervertebral disc space, with the patient in 
the lateral decubitus position4. For ventricular reservoirs such as the Ommaya, a 25-gauge 
butterfly needle will be used without anaesthesia, and the first 2 mL of CSF collected will be 
discarded4. Due to the fast clearance of tumour cells after sampling, analysis must be 
performed rapidly by a cytologist4,5.  
 
Cohort A 
Adult patients with any 
metastatic solid 
tumour and with LMD 
either suspected on 
imaging and/or 
confirmed via CSF 
cytology (with or 
without concurrent 
brain metastases) 
Cohort B 
Adult patients with any 
metastatic solid tumour 
and parenchymal brain 
metastases 
Cohort C (Control) 
Adult patients with any 
metastatic solid tumour 
with no evidence of 
LMD or parenchymal 
brain metastases 
Collection of CSF via 
lumbar puncture or 
Ommaya reservoir 
Cytology, ctDNA, and 
CTC proteomic 
analysis of CSF and 
blood plasma 

 Nov 2025 
 
 
Blood plasma collection: Blood samples will be drawn into Streck Tubes. Two 10 mL vials 
of blood will be drawn per participant, gently mixed, and started upright at 4℃ until 
processing6. Processing should occur within four hours of collection6. Samples will be 
centrifuged at 1500 g for 10 minutes at 4℃ using a sealed swinging bucket rotor to separate 
the plasma6. Plasma will then be carefully extracted with a pipette set to 900 μL into 1.8 mL 
aliquots in 2 mL labelled microcentrifuge tubes and placed on ice6. A second high-speed 
centrifugation at 10,000 rpm will be used to remove residual cellular debris6. The buffy coat 
layer will be isolated and stored separately, and any remaining plasma will be used to 
resuspend the cellular pellet6. Compatible plasma samples will be pooled, aliquoted, and 
stored at -80℃, alongside the buffy coat and pellet6. All samples will be clearly labelled and 
handled under appropriate biosafety and waste disposal protocols6. 
 
 
6. BIOMARKER 
 
Biomarker Plan  
This study involves the assessment of several biomarkers (cytology, ctDNA, CTCs, 
proteomics) in the blood and CSF of participants.  These analyses will be performed at 
Sunnybrook and through collaborations with external centres/companies once appropriate 
material and data transfer agreements are obtained. 
 
 
7. STATISTICAL ANALYSIS 
 
Planned Cytological Analysis: 
CSF and blood plasma samples will undergo proteomic and cytological analysis using the 
genomic equipment at the Sunnybrook Research Institute (SRI). While the specific platform 
has yet to be finalized, technologies such as Next Generation Sequencing (NGS), the QX200 
Droplet Digital PCR System, and ANGLE plc’s Parsortix® system are under consideration 
for their suitability in cytological and proteomic applications6.  
 
Planned Statistical Analysis:  
For the first objective, binary data will be analyzed to determine the proportion of positive 
CSF samples from the total number of samples collected. These proportions will then be 
stratified by tumour type for comparison. The second objective is comparing ctDNA 
detection in CSF and blood plasma samples. This will involve observing the unique and 
shared mutations across sample types, and ultimately summarizing findings in a concordance 
table. From the table, test performance metrics such as sensitivity and specificity will also be 
derived. The third objective evaluates the feasibility of proteomic analysis and will be 
measured by the proportion of CSF samples that yield sufficient protein concentrations. The 
mean number of proteins detected per samples and any commonalities will be reported. All 
statistical analysis will be conducted using Excel and R. Given the exploratory nature of the 
study, graphical illustrations and plots will be emphasized over hypothesis testing with p-
value. 

 Nov 2025 
 
 
References  
1. Zhu JW, Shum M, Qazi MA, et al. Cerebral spinal fluid analyses and therapeutic 
implications for leptomeningeal metastatic disease. Journal of Neuro-Oncology. 
2024;172(1):31-40. doi:10.1007/s11060-024-04902-0. 
2. Sener U, Wilcox JA, Boire AA. Leptomeningeal disease: Current approaches and 
Future Directions. Current Neurology and Neuroscience Reports. 2025;25(1). 
doi:10.1007/s11910-025-01412-y. 
3. Nayar G, Ejikeme T, Chongsathidkiet P, et al. Leptomeningeal disease: Current 
diagnostic and therapeutic strategies. Oncotarget. August 16, 2017. Accessed April 
25, 2025. https://www.oncotarget.com/article/20272/text/. 
4. Glantz MJ, Cole BF, Glantz LK, et al. Cerebrospinal fluid cytology in patients with 
cancer. Cancer. 1998;82(4):733-739. doi:10.1002/(SICI)1097-
0142(19980215)82:4<733::AID-CNCR19>3.0.CO;2-Y 
5. Morganti S, Parsons HA, Lin NU, Grinshpun A. Liquid biopsy for brain metastases 
and leptomeningeal disease in patients with breast cancer. NPJ Breast Cancer. 
2023;9(1):43. doi: 10.1038/s41523-023-00550-1. 
6. Using a Liquid Biopsy to Monitor Metastatic Breast Cancer Treatment (BEACON): 
Blood Processing Standard Operating Procedure. Sunnybrook Odette Cancer Centre; 
2023. Unpublished report.