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

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

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

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)

Microfluidicsmany approaches NIH funded

• +/- dielectrophoresis• S Stott, X Zhang

CTC-CHIPSnot enough clinical data yet

• From LA to Wuhan• HR Tseng... B Xiong• 'nanovelcro'

EPIC (Scripps Research Institute, P Kuhn Lab, La Jolla)

• From 2009, lung case

• Concept of fluid biopsy and HD-CTCs

• CECs...

FOR INTERNAL AND EXTERNAL USEMKG-1866, Rev. 1 8

CellSave Tube

CellSearch Circulating Tumor Cell Kit

Circulating Tumor Cell Control Kit

CellSearch SystemVERIDEX, Immunicon, J&J

CellTracks AutoPrep System

MagNest

CellTracks Analyzer II

FOR INTERNAL AND EXTERNAL USEMKG-1866, Rev. 1 9

Circulating Tumor CellY

Anti-EpCAMFerrofluid

EpCAM

NucleusDAPI

YAnti-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

Literature on CTCs PubMed 3/2013

• Breast (274)• Prostate (112)• Digestive (ColoRectal +...) (66)• Lung (<85, #45)• Others

– Melanoma (16)%

Pro

babi

lity

of S

urvi

val

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

Logrankp < 0.0001

21.7 Months

11.5Months

Cox Hazard Ratio = 3.3chi-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)

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.)

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.

ComparisonCellSearch vs ISET

Farace F Br J Cancer. 2011 Sep 6;105(6):847-53.

CTMs: Circulating Tumour Microemboli

Hou JM,.. Dive C J Clin Oncol 2012 Cho EH,.. Kuhn P Phys Biol. 2012

Feb;9(1):016001.

SCLCCell 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

CTCs in SCLC and NSCLC

• pharmacodynamic biomarker, in vivo and ex vivo

• prognosis

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 patientsThe 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].

CERONC/CIRCUBRONCH projectsNSCLC preliminary results

• Patients inclusion 31 – Sequential study 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

CSF

• Volume 150mL• Production #500mL per day, (3.7x)• Choroid plexuses • Lumbar puncture Berlin

– Heinrich Ireneus Quincke Berl klin Wochenschr 1891;28:929 +965

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.

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

Media...

• Huffington Post (03/06/2013) Valerie Harper

• People– I think there's an opportunity to help

people!

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)

LM: Gold standardDux 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

STA Ch 2: LCR mélanome CMCNew DevelopmentsCSF vs BLOOD

• Preservation: CSF paradox in Cell Save tubes!

• Morphology, numbers and cell biology characteristics

– Similarities: Breast– Discrepancies: Lung (+CTMs), Melanoma

CSFTCS Breast (BMC Clinical Pathology)

CSFTCs Lung

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

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

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 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

Melanoma CSFMCsMedical Oncology (in press)

• CMC kit• Four patients• 9 points• Reproducibility

LUNG CancerCSFTCs, 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%

LUNG Carcinoma meningitis

• CSFTCs --->

• <---CSFTMs

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

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...

AcknowledgmentsEA 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