Determining the sensitivity and specificity of circulating tumor cells and cytology in cerebrospinal fluid of patients clinically suspected for leptomeningeal metastases

Leptomeningeal metastases (LM), also known as meningeal carcinomatosis or
neoplastic meningitis, is a diffuse dissemination of tumor cells into the
cerebrospinal fluid (CSF) and leptomeninges.[1] Up to 8% of all patients with
cancer develop LM. [2] The highest incidence of LM is seen in patients with
(lobular) breast carcinoma (10%), small cell and non-small cell lung carcinoma
(10%) and melanoma (5%).[3] Due to a spread of tumor cells in the CSF, LM is
characterized by multifocal symptomatology (cerebral, cranial nerve and/or
spinal nerve dysfunction). Gadolinium enhanced MRI of the symptomatic location
of the nervous system is the radiological method of choice when LM is
clinically suspected. In patients with a metastasized tumor, clinical signs of
LM and contrast enhancement of either the leptomeninges, pia mater/cortex or
cranial or spinal nerves on MRI, the diagnosis LM can be made. The sensitivity
of MRI with gadolineum for LM is 75% and the specificity 77%. [4] If MRI does
not show equivocal abnormalities, CSF cytology needs to be performed. In 55% of
patients with LM from solid tumors, malignant cells are found during the first
CSF examination. The sensitivity raises to 80-90% after the second CSF
sampling, as determined in the pre-MRI era.[5] The volume of sampled CSF
determines partly the sensitivity of CSF cytology. If possible, 10 ml CSF needs
to be taken and the material must be processed as quickly as possible. Clinical
chemical analysis of the CSF (leukocytes, lactate dehydrogenase, total protein,
glucose) is aberrant in 90% of patients with LM.[5] An abnormal clinical
chemical analysis of the CSF does not prove LM, but can also occur in other
neurological diseases, including (infectious) meningitis.

Recently, Patel et al (2011) described the detection of breast cancer cells in
the CSF using the Cell Search System (Veridex). [6] Using this method, the CSF
is enriched immuno-magnetically for the epithelial cell adhesion molecule
(EpCAM). Next nuclear staining with 4 ',6-diamidino-2-phenylindole (DAPI) and
immunofluorescent detection with cytokeratin and CD45 is performed in 5
patients with leptomeningeal metastases from breast cancer and approximately
104 circulating tumor cells (CTCs) in 7,5 ml CSF were found, using this
method. There seemed to be an association between the number of CTCs and
response to intrathecal administered chemotherapy in this small group of
patients.

ALK, BRAF. EGFR, MEK and PARP inhibitors are oral targeted therapies in the
treatment of breast cancer, lung cancer and melanoma. Since LM is common in
these forms of cancer, gaining knowledge about the CNS distribution of these
drugs is important. Clinical trials of vemurafenib, dabrafenib, trametinib and
olaparib show encouraging results with respect to systemic metastases. However,
they did not yet fully evaluate the distribution of these agents to the CSF.
By quantification of tyrosine kinase inhibitors (e.g. crizotinib,vemurafenib,
dabrafenib, erlotinib, gefitinib, afatinib, dacomitinib, trametinib and
olaparib) in CSF and in plasma the penetration of these agents through the
blood-CSF barrier can be investigated.

In the future, the determination of CTCs and ctDNA in the CSF could be a new
quantitative method for the anti-tumor response assessment of systemic or
intrathecal therapy (as opposed to CSF cytology, which is subjective and not a
quantitative method). If the method shows greater sensitivity than CSF cytology
and can reliably measure single tumor cells, the sensitivity of CSF examination
in patients with a clinical suspicion of LM will increase. Possibly, this
method can also be used to detect micrometastases in the CSF in patients
without neurological symptoms, but with a high risk of CNS metastases

We will investigate a maximum of 100 patients, being treated at the NKI-AVL and
Slotervaart Hospital for an Epcam positive tumor (breast cancer, lung cancer,
gastrointestinal cancer, melanoma) and undergo a diagnostic lumbar puncture
because of a clinical suspicion of LM.
Written informed consent will be obtained from patients prior to study start.
From every patient a sample of 1 x 2 ml of CSF for chemistry (the rest material
will be used for determining ctDNA), 1 x 5 ml of CSF for cytology and
EPCAM/MCSP staining (in case cytology shows positive result) and 1 x of 5 ml of
CSF for CTC enumeration will be taken by the neurologist or neurology resident
(in total 12 ml CSF per sampling). From patients that are treated with tyrosine
kinase inhibitors (e.g. crizotinib,vemurafenib, dabrafenib, erlotinib,
gefitinib, afatinib, dacomitinib, trametinib and/or olaparib) 2 ml extra CSF
for drug quantification will be taken (in total 14 ml CSF per sampling). One
standard 8ml tube for chemistry and three extra 8 ml samples of whole blood for
CTC assay will be drawn by blood sampling technitians (bloedafname)/OIO has
also an NKI- AVL Statement of competence for venipuncture
(bekwaamheidsverklaring voor venapunctie) - in total 32 ml blood per sampling),
to determine the amount of CTCs in blood. one standard tube of blood for
chemistry will be drawn. From patients that are treated with tyrosine kinase
inhibitors (e.g. crizotinib,vemurafenib, dabrafenib, erlotinib, gefitinib,
afatinib, dacomitinib, trametinib and/or olaparib) 4 ml extra blood for drug
quantification will be taken (in total 36 ml blood per sampling). If more
lumbar punctures would be clinically indicated, extra sampling as stated above
will be performed. If available, archived primary tumor tissue will be obtained
for EPCAM staining.

The CTC sampling of the CSF will be done by during a planned diagnostic lumbar
puncture for cytology and chemical analysis of the CSF. CTC blood sampling will
be done during standard blood sampling, directly before or after the lumbar
puncture. 1 extra tube of CSF and 3 extra tubes of blood samples will be drawn
for purpuses of this study. From patients treated with tyrosin kinase
inhibitors (e.g. vemurafenib, dabrafenib, trametinib, crizotinib, erlotinib,
gefitinib, afatinib, dacomitinib, and olaparib) 2 another 2 ml CSF and 4 ml
blood will be taken.

When the patient visits the NKI-AVL for blood and CSF sampling, the following
medical information will be collected: gender and date of birth, detailed
diagnosis based on existing pathology reports, current stage and status of
disease, treatment history and current treatment, co-medication, neurological
signs and symptoms and Karnofsky performance score. After sampling and data
registration, on day 3 the nurse practitioners will call patients to collect
information about possible adverse events from collecting extra material. If no
related adverse events occur the participation is finished.