Identification and quantification of malignant cells (CSFTCs) in cerebrospinal fluid : GC Faure, Q Tu (Nancytomique- Nancy),

E Le Rhun (Lille)We declare not having a conflict of interest

Diagnosis of leptomeningeal metastases (LM) in patients with solid tumors (breast, lung, prostate...) and melanomas remains difficult.

Usual diagnostic methods of cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium enhanced MRI lack both specificity and sensitivity.

Plan

• Leptomeningeal Metastases (LM)• The biological fluid CSF• Diagnostic challenges Gold Standard• Method • Results• Openings

LM or meningeal carcinomatosisEpidemiology... 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)

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

LM: Diagnostic challengeGold 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

Methods

• CellSave® tubes allowing multicentric studies

• CellSearch® Veridex– Cell Tracks AutoPrep– Analyzer II... Multispectral imaging

• CellTracks® CTCs and CMC kits– EpCAM enrichment, KT label, CD45

• Validation on image galeries

CellTracks AutoPrep System

CellTracks Analyzer II

Cancer type

Breast Lung Melanoma

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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

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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

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

Clinical, Basic, Therapeuticopenings

• Evaluation of systemic and intrathecal therapies in clinical settings

• Comparison with other CTCs tecnologies (Wuhan CTC chip, … ScreenCell, ImageStreamX AMNIS flow, EPIC on slide)

• Early Clinical detection of brain and meningeal metas!

• Characteristics of cells going through the BBB (HER-2, EGF-R, etc..)... understanding metastasis

• Enrichment/isolation for further cell and molecular biology studies. ... (Parrot project: lung and prostate)

• New therapy targets?