CSFTCs can be detected, quantified and

1. Tumoral Cells in CSF (CSFTCs):
E.Le Rhun (Lille) GC Faure (Nancy)
Nancytomique CHU Nancy
Diagnosis of leptomeningeal metastases (LM) in
patients with solid tumors (breast, lung, ...)
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. After CTCs.......... CSFTCs
• A new acronym
– CSF:volume 150 mL vs blood (4,5L)
– Another biological fluid
– From choroid plexuses to pathology
• A new gold standard for carcinoma
meningitis or leptomeningeal
metastasis definition
• A new frontier for cancer research
– Metastatic processus
– New therapeutic approaches?

3. 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

4. Leptomeningeal
Metastasis
• Clinic
– Very sick: seizures, severe headaches,
blurry vision, mental status changes,
inability to walk or perform everyday
tasks... completely incapacitated
• Diagnosis
– Imaging (MRI)
• Meningeal enhancement
– Cytology
– Biomarkers? Molecular, Cellular

5. LM in Media,
and Internet 3/2013
• Huffington Post
• People: Valerie Harper
Harper, famous as the spunky best friend
Rhoda Morgenstern on The Mary
Tyler Moore Show, told People she
was stunned after receiving her
diagnosis, but realized she could
help spread awareness for the rare
condition. « I think there's
an opportunity to help
people! »

6. CSFTCs: a new frontier in
Cancer?
- L. Nayak, M. Fleisher, R. Gonzalez-Espinoza et al. (MSK, NY)
Immunomagnetic platform technology (IMPT) for the diagnosis of
leptomeningeal metastasis in solid tumors (LMST) 2010 ASCO
Poster Discussion Session, Abstract Number: 2032.
Neurology 2013, small heterogeneous series of 15 LM
- Patel et al Hershey (Oncotarget 2011 Oct;2(10):752-60)
Spiking in normal blood
- Burns TF, Wolff AC (Johns Hopkins, Baltimore) Cell Cycle. 2012
Jan 15;11(2):203-4. Epub 2012 Jan 15. Detection of
circulating tumor cells in the cerebrospinal fluid: a new
frontier.

7. LM: 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)

8. Epidemiology of LM
• Probably underestimated, but
• 100 000 to 170 000 patients with cancer each year
in the USA develop CNS metastases (Clin Canc Res
2007, 13; 1648) (J Clin Oncol 2004;22:2865)
– Breast (5 %)
– Prostate (less)
– Lung (20%)
– Melanoma (7%)
– Renal (6%)
– Colorectal (2%)
• With major quality of life consequences

9. Quality of life
• Literature is poor
• Support Care Cancer 2011;19: 467-
473 (Lung cancer)
• Complaints: pain, fatigue, dyspnea
• Symptoms
– related to brain tumors: consciousness
deterioration, headache, cranial nerve
palsy, delirium
– Carcinomatous meningitis: headache,
cranial nerve palsy, epilepsy, nausea +
vomiting

10. LM: 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

11. Methods
• >80 Samples from Lille and Nancy
– Volume 5mL
– Up to 4 days delay between sampling
and study...
– Lumbar and ventricular punctures
• Cytology (on 7.5 to 10mL) and biochemistry
according to classical diagnostic procedures
• Cellsearch® technology (CTCs kit;
CMCs kit)

12. Patients
• Established or suspected LM from
primitive cancers
– Breast (45), Lung (15), Prostate and Lung
(1), Melanoma (5), Ovary (1)
– Cytologically defined (50%) and MRI+
– Patients included in DEPOSEIN clinical
research protocol (14)
– Patients sampled for diagnostic and follow-
up procedures, at time of intrathecal
treatment
• Control patients sampled in context
of other neurological disorders

13. CSFTCS Breast (BMC Clinical
Pathology)
CSFTCs Lung

14. STA Ch 2: LCR mélanome CMC
New Developments
CSF vs BLOOD
• Preservation: CSF paradox in Cell Save
tubes!
• Morphology, numbers and cell biology
characteristics
– Similarities: Breast
– Discrepancies: Lung (+CTMs), Melanoma

15. Main Results
 Specificity: no contaminating ependymal cells
in controls
 Sensitivity: Detection and quantification in all
established LM patients studied
– Initial point of follow-up
• From 1 to >10000 cells
– Sequential study in 9 patients from
Deposein with #30 assays
 High homogeneity (and reproducibility) of
images in patients according to primitive cancer
types
 High purity compared to blood samples
 Presence of CTM in lung cancer

16. CSFTCs and CSFMCs numbers
With the CellSearch® Veridex
Cancer type
Breast Lung Melanoma
Tumorcells/5mLLCR
0,1
1
10
100
1000
10000
100000

17. 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

18. Melanoma Leptomeningeal
metastasis: current status
• CMCs are not easy to detect in
bloodwith Cell Search technology
• Meningitis is underdiagnosed with
severe prognosis
– L Harstad et al: Neuro Oncol 2008; 10:
1010-8 MD Anderson

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

20. LUNG Cancer
CSFTCs, CSFTMs
• Patients 15, samples 18
• Numerous CSFTCs
– Sequential follow-up (3)
– Cell galleries allow to differentiate
SCLC (1), NSCLC adenocarcinoma
(4+), NSCLC squamous carc. (3)....
– Aspects of apoptosis, autophagy...
• Numerous CTMs in some NSCLC
patients up to 80%

21. LUNG Carcinoma meningitis
• CSFTCs --->
• <---CSFTMs

22. 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

23. Research (1)
Clinical
• Validation of sensitivity and
specificity CSFTCs and CSFMCs is
underway
– Sensitivity and reliability of the method for
detection of rare events invites to use it
earlier in clinical evolution of metastatic
cancers to detect infraclinic LM
– CTM-like in the CSF are detectable
and can be quantified with the
CellSearch technology. Are they
prognostically significant?

24. Research (2)
Understanding cancer biology
Tumour Cell characteristics
HER-2, EGF-R, etc
Fi Melanoma CTCs are expressing
HER-2
• Are CTM-like agregates in the
CSF the metastatic ones?
Nature Cell Biology 15 [3] (février 3): 317 324.
doi:10.1038/ncb2681.

25. Research (3)
Metastases through the BBB
How do cells migrate
preferentially to the brain
and leptomeninges?
• Breast: Dario Marchetti
In epithelial cell adhesion molecule (EpCAM)–
negative CTCs, ... identified a potential
signature of brain metastasis comprising
“brain metastasis selected markers
(BMSMs)” HER2+/EGFR+/HPSE+/Notch1+
Others?

26. Research (4) drug screening...
for new therapy targets
• In clinically established LM, CSFTCs are not rare
events, and
– Cells available from CSF of LM patients for
further studies will help detecting new
molecules for systemic or local treatment
– Sensitivity and reliability of the method for
detection of rare events invites to use it
earlier in clinical evolution of metastatic
cancers (breast, lung, melanoma...)
• Not only to detect infraclinic LM in
order
• ...to try local treatments as soon as
possible and evaluate their efficacy

27. Participants:
• Centre Oscar Lambret (Lille)
• CHU (Nancy)
– Pôle Laboratoires (Immunologie)
– Pôle Neurologie (L Taillandier, Internes:
Marie, Maud?, Basile...)
• Université Lorraine: SIGRETO (F Plenat),
CRAN CNRS UMR 7039 (D Wolff)
• Hôpital Zhongnan (ZHOU Yunfeng, TU
Jiancheng, XIONG Bin Wuhan University
• NENO Network
– Amiens, Besançon, Colmar, Reims,
Strasbourg... Luxembourg, Liège...

28. Acknowledgments
EA 4369 RHEM UMR CNRS 7039
• GC Faure
• M de Carvalho
• MC Béné (Nantes)
• Wuhan PhD students (Chen Min, Cai
Huili, Tu Qian)
• Laboratoire d'Immunologie, CHU Nancy,
Pôle Laboratoires et Faculté de Médecine,
Université Lorraine