world ctc berlin 2013

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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.

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CSFTCs can be detected, quantified and

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Page 1: World CTC Berlin 2013

Detection and quantification of 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.

Page 2: World CTC Berlin 2013

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?

Page 3: World CTC Berlin 2013

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

Page 4: World CTC Berlin 2013

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

Page 5: World CTC Berlin 2013

LM in Media, and Internet 3/2013

• Huffington Post• People: Valerie HarperHarper, 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! »

Page 6: World CTC Berlin 2013

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.

Page 7: World CTC Berlin 2013

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)

Page 8: World CTC Berlin 2013

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

Page 9: World CTC Berlin 2013

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

Page 10: World CTC Berlin 2013

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

Page 11: World CTC Berlin 2013

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)

Page 12: World CTC Berlin 2013

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

Page 13: World CTC Berlin 2013

CSFTCS Breast (BMC Clinical Pathology)

CSFTCs Lung

Page 14: World CTC Berlin 2013

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

Page 15: World CTC Berlin 2013

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

Page 16: World CTC Berlin 2013

CSFTCs and CSFMCs numbers With the CellSearch® Veridex

Cancer type

Breast Lung Melanoma

Tu

mor

cell

s /

5m

L L

CR

0,1

1

10

100

1000

10000

100000

Page 17: World CTC Berlin 2013

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

Page 18: World CTC Berlin 2013

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

Page 19: World CTC Berlin 2013

Melanoma CSFMCsMedical 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

Page 20: World CTC Berlin 2013

LUNG CancerCSFTCs, 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%

Page 21: World CTC Berlin 2013

LUNG Carcinoma meningitis

• CSFTCs --->

• <---CSFTMs

Page 22: World CTC Berlin 2013

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

Page 23: World CTC Berlin 2013

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?

Page 24: World CTC Berlin 2013

Research (2)Understanding cancer biology

Tumour Cell characteristicsHER-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.

Page 25: World CTC Berlin 2013

Research (3)Metastases through the BBB

How do cells migrate preferentially to the brain and leptomeninges?

• Breast: Dario MarchettiIn 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?

Page 26: World CTC Berlin 2013

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

Page 27: World CTC Berlin 2013

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...

Page 28: World CTC Berlin 2013

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

Page 29: World CTC Berlin 2013