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

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

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

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

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

ONCO-HEMATOLOGYCHEN 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

Multiparametric flow cytometryMC Béné, on behalf of GEIL, GTLLF, GOELAMSNormal Bone marrowMinimal Residual DiseaseRare events: detection and quantification in « bermudes »

CD34 CD45

CD13 SSC

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

D0 D1 D2 D30

20

40

60

80

100

* **

* *

*

Time (n=8)

S

% o

f cel

ls l

abel

led

posi

tive

ly b

y T

UN

EL

D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D30

1

2

3

4

*

*

*

*

*

*

*

*

***

*

*

S

Time (n=11)

Rel

ativ

e M

FI

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

%P

rob

ab

ilit

y o

f S

urv

iva

l

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

Logrankp < 0.0001

Cox Hazards Ratio = 2.4chi-square = 19.54(p-value < 0.0001)

21.9 Months

10.9Months

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)

LM and « CSFTCs » in Breast Cancer (BMC Clinical Pathology 2012)

• Detection• Quantification: 1 to 103-4

• Characterization– Her2-Neu

Specificity: no contaminating ependymal cellsSensitivity: detection and quantification in all established

LM patients studied compared to cytology

High homogeneity (and reproducibility) of images

High purity compared to blood samples

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

Tu

mora

l ce

lls

/ 5 m

L L

CR

0,1

1

10

100

1000

10000

100000

DM CTCWA CTCCJ CTCDMB CTCVT CTCPV CTCBE CTCCS CTCHE CMCST CMCDC CTC

Melanoma CSFMCsMedical 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

LUNG CancerCSFTCs + 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

ImmunologyCHEN M, M de Carvalho, X WU Discrete cellsTregs 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

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

Cardiovascular diseasesCAI 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)

CECs: Preliminary results

• Number >20/mL • Morphology• Clusters (Wegener)• Multinucleated cells?

(Rendu Osler)• Apoptosis (myeloma)• Association with

mononuclear cells (Wegener)

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