Tumoral Cells in CSF (CSFTCs):E.Le Rhun (Lille) GC Faure (Nancy)Nancytomique CHU NancyDiagnosis of leptomeningeal metastases (LM) inpatients with solid tumors (breast, lung, ...)and melanomas remains difficult.Usual diagnostic methods of cytomorphologicalassessment of cerebro-spinal fluid (CSF) andgadolinium enhanced MRI lack both specificityand sensitivity.
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 carcinomameningitis or leptomeningealmetastasis definition• A new frontier for cancer research– Metastatic processus– New therapeutic approaches?
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
LeptomeningealMetastasis• Clinic– Very sick: seizures, severe headaches,blurry vision, mental status changes,inability to walk or perform everydaytasks... completely incapacitated• Diagnosis– Imaging (MRI)• Meningeal enhancement– Cytology– Biomarkers? Molecular, Cellular
LM in Media,and Internet 3/2013• Huffington Post• People: Valerie HarperHarper, famous as the spunky best friendRhoda Morgenstern on The MaryTyler Moore Show, told People shewas stunned after receiving herdiagnosis, but realized she couldhelp spread awareness for the rarecondition. « I think theresan opportunity to helppeople! »
CSFTCs: a new frontier inCancer?- L. Nayak, M. Fleisher, R. Gonzalez-Espinoza et al. (MSK, NY)Immunomagnetic platform technology (IMPT) for the diagnosis ofleptomeningeal metastasis in solid tumors (LMST) 2010 ASCOPoster 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. 2012Jan 15;11(2):203-4. Epub 2012 Jan 15. Detection ofcirculating tumor cells in the cerebrospinal fluid: a newfrontier.
LM: Epidemiology... Prognosis 3 to 5% of cancer patients, incidence up to 9.6% (J ClinOncol 2004;22:2865) Up to 19% of autopsied patients with cancer andneurological symtoms (Glass, 1979)– Breast cancer (5%), lung (11%), melanoma(20%) Increasing incidence– Better survival of cancer patients+ New molecules for systemic disease have bad meningealdiffusion Kodack DP et al. PNAS 2012, 109, E3119 Very Bad prognosis (4 weeks to 6 months) and badquality of life– But promise of new intrathecal drugs (MTX,trastuzumab...) and trials (Chamberlain)
Epidemiology of LM• Probably underestimated, but• 100 000 to 170 000 patients with cancer each yearin the USA develop CNS metastases (Clin Canc Res2007, 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
Quality of life• Literature is poor• Support Care Cancer 2011;19: 467-473 (Lung cancer)• Complaints: pain, fatigue, dyspnea• Symptoms– related to brain tumors: consciousnessdeterioration, headache, cranial nervepalsy, delirium– Carcinomatous meningitis: headache,cranial nerve palsy, epilepsy, nausea +vomiting
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
Methods• >80 Samples from Lille and Nancy– Volume 5mL– Up to 4 days delay between samplingand study...– Lumbar and ventricular punctures• Cytology (on 7.5 to 10mL) and biochemistryaccording to classical diagnostic procedures• Cellsearch® technology (CTCs kit;CMCs kit)
Patients• Established or suspected LM fromprimitive cancers– Breast (45), Lung (15), Prostate and Lung(1), Melanoma (5), Ovary (1)– Cytologically defined (50%) and MRI+– Patients included in DEPOSEIN clinicalresearch protocol (14)– Patients sampled for diagnostic and follow-up procedures, at time of intrathecaltreatment• Control patients sampled in contextof other neurological disorders
CSFTCS Breast (BMC ClinicalPathology)CSFTCs Lung
STA Ch 2: LCR mélanome CMCNew DevelopmentsCSF vs BLOOD• Preservation: CSF paradox in Cell Savetubes!• Morphology, numbers and cell biologycharacteristics– Similarities: Breast– Discrepancies: Lung (+CTMs), Melanoma
Main Results Specificity: no contaminating ependymal cellsin controls Sensitivity: Detection and quantification in allestablished LM patients studied– Initial point of follow-up• From 1 to >10000 cells– Sequential study in 9 patients fromDeposein with #30 assays High homogeneity (and reproducibility) ofimages in patients according to primitive cancertypes High purity compared to blood samples Presence of CTM in lung cancer
CSFTCs and CSFMCs numbersWith the CellSearch® VeridexCancer typeBreast Lung MelanomaTumorcells/5mLLCR0,1110100100010000100000
Sequential analysis of CSFTCsconfirms repetability of numerationswith two subgroups (high > 700/mL vs low)BMC Clin Pathol 2012Sample number1st 2nd 3rd 4th 5thTumoralcells/5mLLCR0,1110100100010000100000DM CTCWA CTCCJ CTCDMB CTCVT CTCPV CTCBE CTCCS CTCHE CMCST CMCDC CTC
Melanoma Leptomeningealmetastasis: current status• CMCs are not easy to detect inbloodwith Cell Search technology• Meningitis is underdiagnosed withsevere prognosis– L Harstad et al: Neuro Oncol 2008; 10:1010-8 MD Anderson
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 farbetter than in blood
LUNG CancerCSFTCs, CSFTMs• Patients 15, samples 18• Numerous CSFTCs– Sequential follow-up (3)– Cell galleries allow to differentiateSCLC (1), NSCLC adenocarcinoma(4+), NSCLC squamous carc. (3)....– Aspects of apoptosis, autophagy...• Numerous CTMs in some NSCLCpatients up to 80%
CSFTCs: a new frontier!• Tumoral (epithelial) cells can be detectedand 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 oftreatments (intrathecal and/or systemic)• Tumoral cell population in CSF might bedifferent from blood CTCs, allowing furtherstudies of metastatic properties• CSFMCs can also be detected and quantified inCSF
Research (1)Clinical• Validation of sensitivity andspecificity CSFTCs and CSFMCs isunderway– Sensitivity and reliability of the method fordetection of rare events invites to use itearlier in clinical evolution of metastaticcancers to detect infraclinic LM– CTM-like in the CSF are detectableand can be quantified with theCellSearch technology. Are theyprognostically significant?
Research (2)Understanding cancer biologyTumour Cell characteristicsHER-2, EGF-R, etcFi Melanoma CTCs are expressingHER-2• Are CTM-like agregates in theCSF the metastatic ones?Nature Cell Biology 15  (février 3): 317 324.doi:10.1038/ncb2681.
Research (3)Metastases through the BBBHow do cells migratepreferentially to the brainand leptomeninges?• Breast: Dario MarchettiIn epithelial cell adhesion molecule (EpCAM)–negative CTCs, ... identified a potentialsignature of brain metastasis comprising“brain metastasis selected markers(BMSMs)” HER2+/EGFR+/HPSE+/Notch1+Others?
Research (4) drug screening...for new therapy targets• In clinically established LM, CSFTCs are not rareevents, and– Cells available from CSF of LM patients forfurther studies will help detecting newmolecules for systemic or local treatment– Sensitivity and reliability of the method fordetection of rare events invites to use itearlier in clinical evolution of metastaticcancers (breast, lung, melanoma...)• Not only to detect infraclinic LM inorder• ...to try local treatments as soon aspossible and evaluate their efficacy
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, TUJiancheng, 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, CaiHuili, Tu Qian)• Laboratoire dImmunologie, CHU Nancy,Pôle Laboratoires et Faculté de Médecine,Université Lorraine