Presentation at the

1. CTCs and CSFTCs in metastatic
Lung Cancer
• GC Faure, Q Tu, H Cai, M de
Carvalho, C Kohler Immunologie
• C Clément-Duchêne, Y Martinet
Pneumologie
• L Taillandier et al., Neurologie
• E Le Rhun, COL, Lille

2. CTCs... CSFTCs... ETCS?
• CTCs: A dream come true?
– 1869, Thomas Ashworth
– 2013, A new cell
biomarker
• MRD a challenge in
oncohaematology:
– Blood, Bone marrow
• MRD in solid oncology:
– Prognosis
– Personalized Medicine
– From DTCs to CTCs

3. CTCs in patients with prostate cancer.
Danila D C et al. Clin Cancer Res 2011;17:3903-3912
©2011 by American Association for Cancer Research

4. So many CTCs approaches
post 3rd
World CTC USA Boston
• Immunomagnetic (Cell Search, MagSweep..)
• Cellular Size filtration (ISET, Screencell...)
• Density gradient separation (Rarecell)
• Dielectrophoresis (DepArray, Silicon Biosystem)
• Microchip microfluidics (GEDI, Harvard SLS,
CTC-Chips... )
• Multispectral imaging (on slide EPIC, flow
AMNIS)
• Functional properties CAM (VITATEX)
• In vivo sampling (Gilupi)

5. Microfluidics
many approaches NIH funded
• +/- dielectrophoresis
• S Stott, X Zhang

6. CTC-CHIPS
not enough clinical data yet
• From LA to Wuhan
• HR Tseng... B Xiong
• 'nanovelcro'

7. EPIC (Scripps Research
Institute, P Kuhn Lab, La Jolla)
• From 2009, lung case
• Concept of fluid
biopsy and HD-CTCs
• CECs...

8. FOR INTERNAL AND EXTERNAL USEMKG-1866, Rev. 1 8
CellSave Tube
CellSearch™
Circulating Tumor Cell Kit
Circulating Tumor Cell Control Kit
CellSearch™ System
VERIDEX, Immunicon, J&J
CellTracks®
AutoPrep®
System
MagNest®
CellTracks®
Analyzer II

9. FOR INTERNAL AND EXTERNAL USEMKG-1866, Rev. 1 9
Circulating Tumor Cell
Y
Anti-EpCAM
Ferrofluid
EpCAM
Nucleus
DAPI
Y
Anti-
CK-PE
CK
Immunomagnetic Labeling and
Immunofluorescent Identification
of Cells
Plus anti-CD45 do distinguish contaminant mononuclear cells
Enrichment of cells of interest with
PROFILE Kit

10. Literature on CTCs
PubMed 3/2013
• Breast (274)
• Prostate (112)
• Digestive (ColoRectal +...) (66)
• Lung (<85, #45)
• Others
– Melanoma (16)
%ProbabilityofSurvival
Time from Baseline Blood Draw (Months)
0 2 4 6 8 10 12 14 16 18 22 24 26 28 30
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
20
Logrank
p < 0.0001
21.7 Months
11.5
Months
Cox Hazard Ratio = 3.3
chi-square = 34.48
(p-value < 0.0001)
CTC / 7.5mL Median OS in
at Baseline N (%) Months (95% C.I.)
<5 CTC 94 (43%) 21.7 (21.3 to ------)
>5 CTC 125 (57%) 11.5 ( 9.3 to 13.7)

11. Clinical research on
CTCs in Lung Cancer (1)
• After initial molecular biology
techniques
• Microchip (Haber Nature, 2007)
• Cell Search
– Wu C et al (Beijing) J Thorac Oncol 2009
Jan;4(1):30-6.
– Okumura Y et al Pulmonary venous blood Ann
Thorac Surg 2009 Jun;87(6):1669-75.
– Tanaka F et al Clin Canc Res 2009 Nov
15;15(22):6980-6.)

12. Clinical research
on CTCs in Lung Cancer (2)
• Filtration methods (ISET)
– Vona G... P Paterlini-Brechot Amer J Pathol 2000,
156, 57-63
– Hofman V et al, Clin Cancer Res. 2011 Feb 15;17(4):827-
35
– Hou JM...Dive C Am J Pathol. 2011 Mar;178(3):989-96;
– Hou JM et al J Clin Oncol. 2012 Feb 10;30(5):525-32.

13. Comparison
CellSearch vs ISET
Farace F Br J Cancer. 2011 Sep 6;105(6):847-
53.

14. CTMs: Circulating Tumour
Microemboli
Hou JM,.. Dive C J Clin Oncol 2012
Cho EH,.. Kuhn P Phys Biol. 2012
Feb;9(1):016001.

15. SCLC
Cell Search
Hou JM et al Am J Pathol 2009 Aug;175(2):808-16;
Hou JM et al J Clin Oncol. 2011 Apr 20;29(12):1556-63
Naito T et al J Thorac Oncol. 2012 Mar;7(3):512-9
Hiltermann N et al Ann Oncol. 2012 Nov;23(11):2937-42
Boshuizen R al J Thorac Dis. 2012 Oct;4(5):456-8 PK

16. CTCs in SCLC and NSCLC
• pharmacodynamic biomarker, in vivo
and ex vivo
• prognosis

17. Metaanalysis
Metaanalysis of circulating tumor cells
as a prognostic marker in lung cancer
Ma XL, Xiao ZL, Liu L, Liu XX, Nie W, Li P, Chen
NY, Wei YQ.
Asian Pac J Cancer VOLUME 13, 2012 Issue Number 4 , 1137-1144
12 + 15 articles, 2615 patients
The hazard ratio (HR) for OS predicted by pro-treatment
CTCs was 2.61 [1.82, 3.74], while the HR for PFS was
2.37 [1.41, 3.99].
The HR for OS predicted by post-treatment CTCs was
4.19 [2.92, 6.00], while the HR for PFS was 4.97 [3.05,
8.11].

18. CERONC/CIRCUBRONCH projects
NSCLC preliminary results
• Patients inclusion 31
– Sequential study 19

19. After CTCs............ CSFTC
• A new acronym
– CSF:volume 150 mL vs blood (4,5L)
– Another biological fluid
– From choroid plexuses
• A new gold standard for carcinoma
meningitis definition
• A new frontier for cancer research
– Metastatic processus
– Therapeutic approaches

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

21. 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
- Patel et al Hershey (Oncotarget 2011 Oct;2(10):752-60.)
- 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.

22. Leptomeningeal Metastasis
• Clinique
– 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

23. Media...
• Huffington Post (03/06/2013)
Valerie Harper
• People
– I think there's an opportunity to help
people!

24. 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
 Very Bad prognosis (4 weeks to 6 months) and bad
quality of life
– But promise of new intrathecal drugs (MTX,
trastuzumab...) and trials (Chamberlain)

25. 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

26. 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

27. CSFTCS Breast (BMC Clinical
Pathology)
CSFTCs Lung

28. First 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

29. Sequential analysis of CSFTCs
confirms repetability of numerations
with two subgroups (high > 700/mL vs low)
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

30. Melanoma Leptomeningeal
metastasis
• CMCs are not so easy to detect in
blood
• Meningitis is underdiagnosed with
severe prognosis
• L Harstad et al: Neuro Oncol 2008; 10: 1010-8
MD Anderson

31. Melanoma CSFMCs
Medical Oncology (in press)
• CMC kit
• Four patients
• 9 points
• Reproducibility

32. LUNG Cancer
CSFTCs, CSFTMs
• Samples (13)
• Numerous CSFTCs
– One sequential follow-up (3)
– Cell galleries might differentiate
SCLC (1), adenocarcinoma (2),
NSCLC (3)....
– Aspects of apoptosis, autophagy...
• Numerous CTMs in some NSCLC
patients up to 80%

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

34. 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 is more homogeneous
and somehow different from blood CTCs,
allowing further studies of metastatic properties
• CSFMCs can also be detected and quantified in
CSF

35. 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...

36. 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