Single cell genetic analysis helps validating cytopathological identification of CTCs in patients with Clear Cells Renal Carcinoma
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1 Single cell genetic analysis helps validating cytopathological identification of CTCs in patients with Clear Cells Renal Carcinoma Patrizia Paterlini Bréchot, MD, Ph.D. Professor of Cell Biology/Oncology University Paris Descartes/INSERM
2 Disclosure Inventor/coinventor of ISET patents Founder and CSO of Rarecells I do not receive conpensation from Rarecells nor from any other company I have a triple task: Teaching (University Paris Descartes) Developing and implementing new tests (Hôpital Necker) Research activity (INSERM Unit)
3 Overview Purpose Technical note on ISET Published results New workflows Molecular analyses of CRC Molecular analysis of CTC from Kidney cancer
4 Toward a diagnostic use of CTC in predictive oncology CS-CTC= EpCam and CK positive, CD45 negative FDA clearence: clinical validity but clinical utility not demonstrated or demonstrated in M+ patients Sensitivity and specificity problems Reproducible data does not mean clinically useful data Underevaluation of CTC heterogeneity Confounding results for molecular analyses and theranostic application Diagnosis of CTC: diagnosis of tumor and clinical utility for patients
5 Tumor cells with epithelial markers in tumors: minority 34 PAC 20 (59%) tumors with pos. cells 5-70% range of pos cells 27 HCC 5 (18%) tumors with pos. cells 1-50% range of pos. cells HCC and PAC derived cell lines 95-99% pos. Cells A. Sabile et al Am. J. Clin. Pathol. 112: , 1999 Epithelial to Mesenchymal transition + tumor heterogeneity Why Epithelial markers are used to isolate CTC?
6 Diagnosis of CTC in Oncology Tumor cells from Cervix Tumor cells from Ascitis Tumor cells from Urine Tumor cells from Blood
7 CTC- Circulating Tumor Cells Inflammation increases TC diffusion Epithelial- to Mesenchymal Transition (EMT) Early TC diffusion at In situ carcinoma stage Apoptotic CTC Circulating Tumor Cell (CTC) Circulating Tumor Microembolus (CTM) EMT essential for TC invasion Stem character Invasion Stem CTCs concentrated 100 times in blood versus tumor (animal model) Tumor cell Proliferation Angiogenesis Mesenchymal to Epithelial Transition (MET) Paterlini Brechot, Cancer letters 2007, updated Circulating Tumor cells are released into the blood system long before the development of metastasis, Klein CA, Nature Reviews, 2009 Rhim A et al, Cell, 2012 Tumor cells heterogeneity in tumor tissue Gerlinger et al, NEJM, 2012
8 ISET Isolation by Size of Tumor/Trophoblastic cells Leukocytes are the smallest cells in the body Patented combination of parameters allowing very sensitive and rapid isolation of CTC and CFTC Blood treatment within 5-6 h after collection Storage for molecular analyses: 7 years Open system allowing all types of analysis on CTCs Vona et al, Am J Pathol, 20008
9 ISET is diagnostic and more sensitive than CellSearch independent studies in vivo: % of positive patients ISET (size+ cytopathology) versus CellSearch (EpCAM +, CK+, CD45-) Type of cancer Number of patients Reference ISET % of patients with CTC Non-Small Cell Lung cancer 210 (non-metastatic and metastatic) Hofman V et al, Int J of Cancer (metastatic) Farace F et al, Br J of Cancer, (IIIA - IV) Krebs M et al, J. Thoracic Onc Prostate cancer 20 (metastatic) Farace F et al, Br J of Cancer, 2011 Breast cancer 20 (metastatic) Farace F et al, Br J of Cancer, 2011 Pancreatic cancer 54 (non-metastatic and metastatic) Khoja L et al, Br J of Cancer, 2011 CellSearch % of patients with CEpC (cut-off) 50% 39% ( 1) 21% ( 2) 100% 45% ( 1) 25% ( 2) 15% ( 5) 80% 23% ( 2) 100% 90 % ( 1) 60 % ( 5) 85% 75% ( 1) 40% ( 5) 93% 40% ( 1) ISET detects CTCs in higher numbers and in more patients CTM (Circulating Tumor Microemboli) are detected by ISET only
10 ISET - RARECELLS ISET Enrichment/detection principle: Size, cytopathology 2 x 10 ml of blood ISET isolates all types of CTC (all types of cancer) and all types of CRC CTC Pores Cytopathological analysis is diagnostic in Oncology ISET allows the diagnosis of CTC Work of process: Rapid, easy to use, efficient Fixed cells 15 minutes Blood to be processed within 3 hours after collection Scientific results: Sensitivity, Diagnostic specificity Fresh cells CYTOPATHOLOGICAL DEFINITION OF CTC Nuclear size equal or larger than 16 microns, Irregularity of the nulear countour Irregularity of the chromatin Presence of a visible cytoplasm Circulating normal epithelial cells Circulating Tumor cells Vimentine + CTC ISET is diagnostic for CTC Unparalleled sensitivity (1/10 ml) Identifies the most malignant CTCs (expressing Vimentin) CTC microdissection Spot Lysis Identifies Circulating Tumor Microemboli (CTM) ISET allows molecular analysis of CTCs High nuclear to cytoplasmic ratio (>0.8) Low cost, filters and cells storable; multiplex tests Aspiration controlled by a pump allows to optimize filtration of all types of blood samples
11 SPECIFICITY Heterogeneity of circulating rare cells Tumor Atypical Normal Hofman et al, Clinical Cancer Research, 2011
12 Preoperative CTC detection using the ISET method for patients with lung cancer is a new prognostic biomarker 208 patients 49% CNHC 36% CTC Hofman Clinical Cancer Research 2011
13 Diagnostic value of ICC for the detection of BRAF V600E mutation on CTC isolated by ISET from metastatic melanoma patients Hofman V et al, J Invest Dermatology, 2012 Patient 1 BRAF V600E mutation searched in 98 tumour tissues and CTCs by pyrosequencing (T) and by ICC using the VE1 antibody (T, CTC). 87/98 (89%) patients with CTCs Patient 2 53/98 (54%) patients with BRAF V600E mutation detected by pyrosequencing in T Consistent results (T, CTC) 8/87 (8%) patients had CTCs positive by ICC but no mutation detected on tumour tissues. The ICC detection of BRAF V600E mutation on CTCs isolated by ISET is a very sensitive and specific method
14 EML4-ALK-gene rearrangement is consistently found in CTC/CTM isolated by ISET and in the tumorous tissue Ilie M et al, Annals of Oncology, 2012 CTCs isolated by ISET from 87 patients with lung adenocarcinoma ALK break-apart FISH (Abbott) anti-alk antibody (5A4 clone) Blindly on CTCs and corresponding tumor tissues Five patients positive for ALK-gene rearrangement and strong ALK ICC in CTCs and T tissue. 82 patients negative for both ALK FISH and ALK ICC in CTCs and T tissue
15 18 ALK-positive patients 14 ALK-negative patients 100 % consistant result (T, CTC) 100% of patients with detectable CTCs by ISET Representative example of vimentin/cytokeratins/ CD45/DAPI immunofluorescent staining of ALKrearranged CTCs in a ALK-positive patient Single cell analysis: ALKrearranged CTCs expressed a mesenchymal phenotype contrasting with heterogenous epithelial and mesenchymal marker expressions in tumors
16 CTC challenges Without decreasing the sensitivity and keeping cells with intact morphology CTC isolated unfixed for NGS, RNA analysis, culture, injecton into mice CTC collection on a slide: high throughput immuno and FISH analysis CTC stabilization and long storage for days
17 ISET* by Rarecells & CellCelector by ALS: powerful enrichment & selection of circulating rare cells Blood drawing Filtration Sample preparation Cell selection and picking Single-cell genetic characterization BF 15 µm CK Drawing a 10 ml-blood sample Dilution with Rarecells buffer for unfixed cells Filtration by Rarecells Device (3 min) Bright field or Immune Labeling : ex: EpCAM or Cytokeratin or Vimentin, etc.. Very Fast scanning, cell detection, selection and picking with CellCelector - WGA, genotyping, aneuploidy detection, mutation panel detection - Similar workflow to PGD * ISET: Isolation by SizE of Tumor/Trophoblastic Cells
18 Sensitivity of isolation unfixed CRC Number of micropipetted cells in 1 ml of blood Number of cells detected (A549) Recovery (A549) % 1 100% 1 100% 1 100% 1 100% 1 100% 1 100% % % 4 80% 5 100% 4 80% % 3 60% 5 100% 9 90% 8 80% % % 8 80% Overall recovery 91% Unpublished data 18
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21 Whole genome amplification methods PEP MDA RepliG (Qiagen) Genomiphi (GE) GenomePlex (Sigma) PicoPlex (Rubicon genomics) MALBAC (Yikon Genomics) Time 8 h (o/n) 2 h /8 h ~ 4 h 2 h 4 h Cost per reaction 2,6 5,24 (Qiagen) 3,88 (GE) 12,7 28 $ 34,3 Yield ng range? 10 µg / 40 µg from 10 ng purified gdna 5-10 µg (fresh single cell) 2-5 µg (fresh single cell) 3 µg (fresh single cell) Proportional Better than MDA Genome coverage in HTS (fresh single cell) 72% of the genome at 1x coverage (25x seq depth) (Zong et al, 2012) 6% of the genome at 0,2x coverage (Navin et al, 2011) unpublished 85 to 93 % of the genome at 1x coverage (25x seq depth) (Zong et al, 2012)
22 Feasibility of single cell NGS by whole exome sequencing (Illumina) Sample type Unfixed (Fresh) Single cell number of reads number of mapped reads Percentage of coverage Average sequencing depth % 27 Fixed single cell % 22 Fixed and microdissected single cell % 20 Genomic DNA % 23 In collaboration with 22
23 CTC transfer on slides Step 1 Blood Collection 10 ml Step 2- Filtration with Rarecells System (15 min) Blood to be processed within 5 hours of collection 10 min Patient Dilution with Rarecells Buffer Filtration by Rarecells Device Step 3- Transfer on slide Step 4 Staining and cytopathology leucocyte A549 Ongoing: sensitivity validation (1 tumor cell per ml of blood)
24 VHL mutation analysis in CTCs from patients with Clear Cells Renal Carcinoma 30 patients with CCRC 22 men et 8 women Mean age 68,5 Two patients with metastasis Tested by ISET before surgery Mutation analysis: T-CTC- gdna CTM from CCRC patients VHL gene Identified in 1993 by Latif et al. Tumor suppressor gene Position: 3p Kb 3 exons 2 ARNm de 4,5 et 4,7 Kb, two protein isoforms More than 500 mutations
25 Résultats moléculaires VHL mutations found EXON CODON MUTATION TYPE OF MUTATION CODON INITIAL CODON MUTE CpG PROTEIN CHANGE 9 c.27g>t Tr GAC TAC Oui D9Y 18 c.53c>a Tr GCA GAA Non A18E 61 c.183c>g Tr CCC CCG Non P61P 65 c.194c>a Tr TCG TAG Non S65X 69 c delcg Fr CGC - - E69X 88 c.263g>a Ts TGG TAG Non W88X 92 c.275dela Fr GAC - - D92X 100 c.299delc Fr ACG - - T100X 109 c.327delc Fr ATC - - H109X 116 c.346c>g Tr CTT GTT Non L116V 118 c.353t>c Ts CTC CCC Non L118P 140 c.418delc Fr CTC - - L140X 145 c.435g>t Tr CAG CAT - Q145H 158 c.472delc Fr CTG - - L158X 163 c.486delc Fr TGC - - L163X 176 c.526a>t Tr AGG TGG Non R176W 183 c.548c>a Tr TCG TAG Non S183X 207 c.620c>t Ts GCA GTA Non A207V Tr : Transversion; Ts : Transition; Fr : Frameshift
26 Résultats moléculaires Exemple of transversion : Mutation c.27g>t, codon 9 / Exon 1 (1 patient) CTC Tumor Genomic DNA
27 Résultats moléculaires Exemple of a transversion found in 2 patients Mutation c.263g>a, codon 88 / Exon 1 CTC Tumor Genomic DNA CTC from patient CGN CTC from patient GRA
28 VHL mutation analysis can help cytopathological diagnosis of CTCs in patients with Clear Cells Renal Carcinoma Blind mutation analysis of VHL gene in T, leucocytes and CTC isolated by ISET Cytoapthology 30 patients with CCRC 25 with VHL mutation in tumor 20 patients with CTC 205 single cell analysis : 64 CTC VHL gene exon 2: T insertion in position 171 Mutation: GGTGTGGTCTCTTTAA (WT: GGTGTGGCTCTTTAA) VHL mutations: 57/64 CTC 7 VHL-neg CTC from tumors neg for VHL Ben Njima B et al, manuscript in preparation
29 Acknowledgements Ms. Katia Hormigos Dr Sophie Laget INSERM Unit 807 Dr Thierry Capiod Dr Basma Ben Jima Dr Ingrid Pfifer Pr Patrizia Paterlini Bréchot Collaborations Pr Paul Hofman, Patology Dpt, Nice Dr. Marius Ilie, Patology DptNice Pr. Nicole Brousse, Patology Dpt, Paris Pr. Khaled Ben Romdhane, Tunis Pr. Arnaud Mejean, Urology Dpt, Paris THANKS FOR YOUR ATTENTION!
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