La présentation sur Nancytomique faite lors de la réunion scientifique sur le Laboratoire sans murs organisée avec le consulat de France dans le cadre de la Filière Médicale Francophone Nancy-Wuhan à la Faculté de Médecine de Wuhan et à l'Hôpital Zhongnan.

1. NANCYTOMIQUE
• A unique platform dedicated to clinical and
translational research
• Focusing on rare cell events
– New cellular biomarkers for personalized
medicine
• In Oncology
• In Cardiovascular and immunological
diseases

2. Equipments,
Methods and Skills
• Technical and Medical competences
– Flow cytometry, cell imaging, immunology,
hématology, cell biology
– Cancerology, Cardiovascular, Internal medicine
• 3 complementary Innovative Devices
– Multiparametric Flow cytometer (3 lasers, 10 colours)
Navios (Beckman Coulter)
– Cell Search VERIDEX
– ImageStream X AMNIS
• Making available for Clinical Research
– Multiparametric immunophenotyping
– Immunomagnetic enrichment
– Multispectral imaging on slide and in flow

3. MANPOWER
• GC FAURE, (MC Béné), M de CARVALHO
• Engineer
• PhD Students
– CHEN Min
– CAI Huili
– TU Qian
• M2 Research
– De Kayser Manon
– WU Xanglei
• Open to other labs in Nancy (CRAN CNRS
UMR7039, Fdération de Recherche CNRS)
– DING Haixia
– WANG YingYing
– MASTRONICOLA R, ZUILY S

4. Filière médicale francophone
法国医疗领域
depuis la classe 2001/2
在医学院总理事会的摩泽尔总理事会的摩泽尔

5. TOPICS
• Onco-Haematology:
– Minimal Residual disease in ALL, AML, CLL
– Myeloma phenotyping and thrombosis (MCB + VLC)
• Solid Oncology
– CTCs (Minimal Residual disease: a one century
challenge
– CSFTCs and ETCs
• Immunology: transplantation, tumour
– Discrete cells (Tregs, dendritic cells),
– Cytokine signatures of cell subpopulations
• Cardiovascular diseases: CECs, pECs,
microparticles
– Vasculitis and Angiogenesis

6. ONCO-HEMATOLOGY
CHEN M, CAI H
• From extensive and optimal
immunophenotyping in
– ALL/AML
– CLL
• To Minimal residual disease (MRD) and
Peripheral Blood Blast decrease
• To Evaluation of potential New Therapies
(CHEN M)
– Thèse UL BIOSE April 8, 2013: Stimulation
of CLL cells through TLR-7 and TLR-9
– Chen M et al Leuk & Lymphoma
2012;53:739-742

7. Multiparametric flow cytometry
MC Béné, on behalf of GEIL, GTLLF, GOELAMS
Normal Bone marrow
Minimal Residual Disease
Rare events: detection and quantification
in « bermudes »
CD34 CD45
CD13 SSC

8. CLL: Innate immune receptors
on B CLL cells, and beyond…
• B-CLL cells express TLR-7 and over-
express TLR-9
• Extensive study of 40 CLL B-cells
stimulated
– ODN M362(CpG-C) ligand of TLR-9
– Imiquimod R837, ligand of TLR-7
• Multiparametric flow cytometry for
apoptosis, proliferation,
immunophenotypic modulation (CD20,
CD52) , cytokines (IL-6, IL-8) production,
signaling pathways (AKT, STAT, MAPK)
• Imiquimod and ODN could be used as an
adjuvant immunotherapy strategy for
patients with CLL
D
0
D
1
D
2
D
3
0
20
40
60
80
100
* *
*
*
*
*
Time (n=8)
S
%ofcellslabelled
positivelybyTUNEL
D
1
D
2
D
3
D
1
D
2
D
3
D
1
D
2
D
3
D
1
D
2
D
3
D
1
D
2
D
3
D
1
D
2
D
3
D
1
D
2
D
3
D
1
D
2
D
3
0
1
2
3
4
*
*
*
*
*
*
*
*
*
**
*
*
S
Time (n=11)
RelativeMFI

9. Solid ONCOLOGY
TU Q
• CirculatingTumor Cells (CTCs)
– STIC DHOS METABREAST (Institut Curie)
– Cancéropole Grand Est (Inserm Strasbourg)
• CerebroSpinalFluidTCs (Breast, Lung)
– DEPOSEIN BMC Clin Pathology 2012
• CSFMelanomaCells
– Medical Oncology 2013
• World CTC USA 2012, World CTC Berlin 2013, Lung
Cancer 2013 Luxbembourg: Invited speaker,
• Cytology 2013 Prize Junior Cytopathologist
• EffusionTCs
– Tumor Cells quantification in peripheral blood and ascitic fluid
from metastatic esophageal cancer submitted
%ProbabilityofSurvival
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Time from Baseline (Months)
0 5 10 15 20 30 35 40 45 5025
Logrank
p < 0.0001
Cox Hazards Ratio = 2.4
chi-square = 19.54
(p-value < 0.0001)
21.9 Months
10.9
Months
CTC / 7.5mL Median OS in
at Baseline N (%) Months (95% C.I.)
<5 CTC 89 (50%) 21.9 (20.1 to 28.6)
>5 CTC 88 (50%) 10.9 ( 7.0 to 15.2)

10. 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

11. 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

12. 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

13. 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

14. Immunology
CHEN M, M de Carvalho, X WU
Discrete cells
Tregs and Dendritic cell subpopulations
Cytokine signatures according to tumoral antigenic load
Immunomonitoring for
Infectious diseases (HIV, tuberculosis)
Transplantation follow-up
Chen M et al Clin exp Immunol 2011;163:104-12 Reduced levels of both
circulating CD4+CD25+CD127(low/neg) and CD4+CD8(neg) invariant natural killer regulatory T
cells in stable heart tranplant recipients
Chen M et al 2nd ECI Berlin 2009. B1 cells expressing CXCR3 in cardiac
allograft rejection
Tumor immunity (Prostate, Colic and ENT Cancer)
M2 Project WU X

15. Phenotypic markers and Cytokine signatures
Functional diversity associated with
• cell migration to systemic and mucosal lymphoid
tissues
• proliferation, cytotoxicity
•antigenic load
Pantaleo et al. Nature Reviews Immunology 6, 417–42

16. Cardiovascular diseases
CAI H, TU Q, DING H
• From Endothelial Progenitors to CECs, and
microparticles
– Number, Morphology, Apoptosis?
• Angiogenesis disorders
– STB and Rendu-Osler telangiectasia
• Inflammation
– Vasculitis: SAPL, Wegener
– Heart infarction
• Myeloma and thrombosis (VLC)
• Glioblastoma and exosomes (BF)

17. CECs: Preliminary results
• Number >20/mL
• Morphology
• Clusters (Wegener)
• Multinucleated cells?
(Rendu Osler)
• Apoptosis (myeloma)
• Association with
mononuclear cells
(Wegener)

18. Collaborations and projects
• France
– Nancy (EA4369) CRAN/UMR7039
• Anatomopathologie CAV, Méd Interne,
Méd Vasculaire, Urologie, Neuro-
oncologie, Pneumologie, MIMOS...
– Cancéropole Grand Est (Prostate, Colorectal)
– Lille (Centre Oscar Lambret-CSFTCs,
CMCs/PHRC)
– Paris Institut CURIE (STIC DHOS
METABREAST)
• International
– Estonia (TARTU, GORCE Parrot project)
– China (WUHAN...)
– USA EPIC: Fluid biopsy and CECs
– ScreenCell