This document discusses the identification and quantification of malignant cells (CSFTCs) in cerebrospinal fluid (CSF) as a diagnostic method for leptomeningeal metastases. Current diagnostic methods like cytological assessment of CSF and MRI scans lack sensitivity and specificity. The document outlines that the CellSearch technology can detect and quantify tumoral epithelial cells (CSFTCs) in CSF from breast, lung, and other cancer patients. Sequential analysis of CSFTCs levels can be used to evaluate responses to treatments and CSFTCs may provide insights into metastatic properties different from blood CTCs. This technique opens up opportunities for clinical evaluation and monitoring of therapies, as well as basic research into the characteristics of cells that metastasize

1. Identification and quantification of malignant cells
(CSFTCs) in cerebrospinal fluid :
GC Faure, Q Tu (Nancytomique- Nancy),
E Le Rhun (Lille)
We declare not having a conflict of interest
Diagnosis of leptomeningeal
metastases (LM) in patients with solid
tumors (breast, lung, prostate...) and
melanomas remains difficult.
Usual diagnostic methods of
cytomorphological assessment of
cerebro-spinal fluid (CSF) and
gadolinium enhanced MRI lack both
specificity and sensitivity.

2. Plan
• Leptomeningeal Metastases (LM)
• The biological fluid CSF
• Diagnostic challenges Gold Standard
• Method
• Results
• Openings

3. LM or meningeal carcinomatosis
Epidemiology... Prognosis
 3 to 5% of cancer patients, incidence up to 9.6% (J Clin
Oncol 2004;22:2865)
 Up to 19% of autopsied patients with cancer and
neurological symtoms (Glass, 1979)
– Breast cancer (5%), lung (11%), melanoma (20%)
 Increasing incidence
– Better survival of cancer patients
+ New molecules for systemic disease have bad meningeal
diffusion Kodack DP et al. PNAS 2012, 109, E3119
 Very Bad prognosis (4 weeks to 6 months) and bad
quality of life
– But promise of new intrathecal drugs (MTX,
trastuzumab...) and trials (Chamberlain)

4. Cerebrospinal fluid: CSF
• Volume 150mL
• Production #500mL per day, (3.7x)
• Choroid plexuses
• Lumbar puncture Berlin
– Heinrich Ireneus Quincke
– Berl klin Wochenschr 1891;28:929 +965

5. LM: Diagnostic challenge
Gold standard
Dux et al, J Neurol Sci, 1994; 121; 74-78
 CSF volume
– 3.5mL: 68% positivity
– 10.5mL: 97% positivity
 Time interval between sampling and analysis
 Cell viability 30 mns 50%; 60 mns 20%; 90 mns 10%
 Good sensitivity requires
 First LP 40%
 Second LP 80%, Third LP to reach 90-95%
 No reliable quantification
– Response at 50% threshold

6. Methods
• CellSave® tubes allowing multicentric
studies
• CellSearch® Veridex
– Cell Tracks AutoPrep
– Analyzer II... Multispectral imaging
• CellTracks® CTCs and CMC kits
– EpCAM enrichment, KT label, CD45
• Validation on image galeries
CellTracks®
AutoPrep®
System
CellTracks®
Analyzer II
Cancer type
Breast Lung Melanoma
Tumorcells/5mLLCR
0,1
1
10
100
1000
10000
100000

7. LM and « CSFTCs » in Breast
Cancer (BMC Clinical Pathology 2012)
• Detection
• Quantification: 1 to 103-4
• Characterization
– Her2-Neu
Specificity: no contaminating ependymal cells
Sensitivity: detection and quantification in all
established LM patients studied compared to
cytology
High homogeneity (and reproducibility) of
images
High purity compared to blood samples

8. Sequential analysis of CSFTCs
confirms repetability of numerations
with two subgroups (high > 700/mL vs low)
BMC Clin Pathol 2012
Sample number
1st 2nd 3rd 4th 5th
Tumoralcells/5mLLCR
0,1
1
10
100
1000
10000
100000
DM CTC
WA CTC
CJ CTC
DMB CTC
VT CTC
PV CTC
BE CTC
CS CTC
HE CMC
ST CMC
DC CTC

9. Melanoma CSFMCs
Medical Oncology 2013;
• CMC kit (J&J, VERIDEX)
– CD146 capture, MEL14 , CD45
• Four patients 9 points
• Good reproducibility during follow-up
• Cell morphology of melanoma cells in CSF far
better than in blood

10. LUNG Cancer
CSFTCs + CSFTMs
Cytomorphology: cell galleries
allow to differentiate SCLC, NSCLC
(adenocarcinoma, squamous carcinoma)
and detect apoptosis, autophagy...
• Numerous CTMs in some NSCLC patients
up to 80%, in sequential study

11. CSFTCs: a new frontier!
• Tumoral (epithelial) cells can be detected
and quantified in CSF with the CellSearch®
technology (CSFTCs)
• Their numbers can be sequentially followed-
up in breast, lung and other cancers
– allowing to evaluate the efficacy of
treatments (intrathecal and/or systemic)
• Tumoral cell population in CSF might be
different from blood CTCs, allowing further
studies of metastatic properties
• CSFMCs can also be detected and quantified in
CSF

12. Clinical, Basic, Therapeutic
openings
• Evaluation of systemic and intrathecal therapies in
clinical settings
• Comparison with other CTCs tecnologies (Wuhan CTC
chip, … ScreenCell, ImageStreamX AMNIS flow, EPIC on
slide)
• Early Clinical detection of brain and meningeal metas!
• Characteristics of cells going through the BBB (HER-2,
EGF-R, etc..)... understanding metastasis
• Enrichment/isolation for further cell and molecular
biology studies. ... (Parrot project: lung and prostate)
• New therapy targets?