CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
Localized lung cancer
COPD Screening
Metastatic lung cancer ALK liquid biopsy
Technical Insights into Highly Sensitive Isolation and Molecular Characterization of circulating tumor cells for early
detection of tumor invasion.
Patrizia Paterlini Bréchot, MD, Ph.D. Professor of Cellular and Molecular Biology University Paris Descartes– Paris
Lucile Broncy, Msc, Ph.D. student (bourse CIFRE)
University Paris Descartes– Paris
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
Circulating Tumor Cells, more than a biomarker. Actor of invasion
ü Epithelial Tumor cells ü ≠ Normal Epithelial cells ü Mesenchymal Tumor cells ü Hybrid epithelial- mesenchymal Tumor cells ü Tumor stem cells ≠ Normal stem cells ü Circulating Tumor Microemboli (CTM)
Heterogeneous
ü Few per ml, mixed with ü 10 millions WBC, 5 billion RBC
Very rare
2
Tumor
metastasis
blood
Disease progression
Diagnostic Screening Treatment efficacy Prognostic Theranostic
Potential for reducing cancer mortality
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
ISET® TECHNOLOGY: OPEN SYSTEN: Isolation by Size of Tumor cells (all types) Blood collection
Dilution with the « ISET® Buffer »
Sample Incubation
Blood treatment with the Rarecells® Device
Filtration (-) (10 min.) (5 min.) ISET® Filters
CCC are isolated by the ISET® system on the ISET®
Filter
(5 min.)
• Cytopathological diagnosis • CCC counting
è Achieve Cytopathological diagnosis Isolated Circulating Cancer Cells (CCC)
• Circulating Cancer Cells can be characterized by immunolabelling, FISH, and Next Generation Sequencing (NGS)
è CCC characterization
• Molecular analyses targeted to tumor cells after laser microdissection (CGH, DNA mutation, RT-PCR, NGS)
è Perform Theranostic Analyses
Enriched circulating rare cells including tumor cells
FIXED
+ Possible storage and shipment of filters
• Collection of 10ml of blood from the patients • Leukocytes are the smallest cells in the body
Vona et al Am J Pathol, 2000 Paterlini Bréchot, Cancer Letters 2007 Paterlini Bréchot, Cancer Microenvironment 2014
Patented combination of parameters allowing very sensitive and rapid isolation of CTC
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
Circulating Cancer Cells (CCC) are tumor cells isolated without loss
and undamaged : diagnosed by cytopathology
Tumor cells EMT+ are lost by CellSearch
Normal CTC by CellSearch
Atypical CTC by CellSearch
Hofman et al, Clinical Cancer Research, 2011 Paterlini Bréchot, Cancer Microenvironment, 2014
!
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
Hofman Clinical Cancer Research 2011
Preoperative CCC detection using the ISET method is a prognostic biomarker for patients with lung cancer and uveal melanoma
208 patients 208 lung cancer patients 49% CNHC 36% CCC
• 31 uveal melanoma patients (29 non-metastatic, 2 metastatic) :
– 17/31 with CCC/CCM – 8/31 with CCM
• 17 control with benign disease: – 0/17 with CCC/CCM
Mazzini et al, Cancers, 2014
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
An independently funded, 6-year-long monocentric study, published in 2014, demonstrated that ISET® was able to make a very early diagnosis of lung cancer.
Project Air: Based on these results, a multicenter study with ISET® of 600 patients funded by independent Public National and Regional sources has started in November 2015 to confirm the results.
Project Detector: Another multicenter study using ISET® will start in December 2016 focusing on screening of tobacco-induced cancers. This independent study is part of the “Priorité Cancers Tabac” national research plan and has received significant financing from INCa (Institut national du cancer)
Major Clinical Trials M
ultic
ente
r Stu
dies
• 254 patients including 168 patients with COPD followed by ISET and low dose CT-scan screening
– 5 of 168 presented CCC detected by ISET – Per 10 mL of blood, 19 to 67 isolated CCC and 1 to
3 CCM, Vimentin expression • In these 5 patients,
– CT-scan detected a lung nodule 1 to 4 years after CCC detection by ISET leading to surgical resection and early diagnosis of Lung Cancer (stage 1A)
– Follow up of the 5 patients at 18 months after surgery: no tumor recurrence, no CCC.
Mon
ocen
tric
Stu
dy
Ilie et al. Plos One 2014
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
Lower Limit Of Detection (LLOD): 1 Tumor cell /10 ml of blood; sensitivity at LLOD: 100%
n=2 n=13
n=9
n=30
n=6 n=6
y = 0.9991x R2 = 0.96263
1
10
100
1000
1 10 100 1000 Num
ber o
f A54
9 ce
lls d
etec
ted
by IS
ET®
(log
sca
le)
Number of spiked A549 cells in 10 mL of blood or equivalent (log scale)
y = 1.0128x R! = 0.99514
0
20
40
60
80
100
120
0 20 40 60 80 100 120
Num
ber o
f HeL
a ce
lls d
etec
ted
by IS
ET®
Number of spiked HeLa cells in 1 mL of blood
Laget et al, Plos One, 2017
Capture of individual cells by micropipetting
Incubation with Rarecells® Buffer
Fixed cancer cells on the filter
Sens
itivi
ty te
sts
ISET®
Filtration
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
Culture of tumor cells and cytoskeleton analysis after live isolation from blood
Laget et al, Plos One, 2017
Blood Collection
Incubation with Rarecells® Live Cells Buffer (Ref 540208) Li
ve C
ells
CTC culture and functional assays
CTC-derived xenografts
RNA / Transcriptomic DNA / NGS analyses
ISET®
Filtration
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
Targeted and global Next Generation Sequencing results obtained on single cancer cells isolated by ISET
Ion AmpliSeq Hotspot Cancer Panel V2: 2800 theranostic mutations in 50 genes K-RAS, PIK3CA and SMO: non-silent mutations found in single HCT116 cells
No mutations detected in ABL1, APC, FGFR3 and STK11 genes : these specific variants are not in the list of mutations of the panel
KRAS G13D mutant allele PIK3CA H1047R SMO V404M
Sample mutant allele
frequency mutant allele
coverage amplicon coverage
mutant allele
frequency
mutant allele
coverage amplicon coverage
mutant allele
frequency
mutant allele
coverage amplicon coverage
Single HCT116 (H1) 38% 37 97 73% 710 979 55% 1099 2000 Single HCT116 (H2) 100% 11 11 46% 453 981 12% 245 1996 Single HCT116 (H3) 100% 29 29 32% 312 986 67% 1346 2000
Pooled data 3 HCT116 56% 77 137 50% 1475 2946 45% 2690 5996
WGA-Amplified HCT116 DNA 42% 59 140 46% 99 213 53% 1064 1998
Unanmplified bulk HCT116 DNA 51% 995 1958 51% 1010 1996 50% 1009 2000
Single leukocyte (L1) 0% 0 13 0% 0 280 0% 0 1992 Single leukocyte (L2) 0% 0 52 0% 0 524 0% 0 1983 Single leukocyte (L3) ND 0 0 0% 0 498 0% 0 1986 Bulk DNA blood donor 0% 0 1942 0% 0 1996 0% 0 1998
Laget et al, Plos One, 2017
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
ü ISET sensitivity is 1 tumor cell per 10 mL of blood
ü Live tumor cells isolated by ISET can be cultured for drug sensitivity and immunomodulation assays
ü ISET allows single tumor cells genetic characterization for theranostic purposes
ü Ongoing developments for single tumor cell transcriptomic characterization to identify the organ of origin
Developments concerning CCC isolation for early detection of invasive cancer
CONFIDENTIAL – NOT FOR REDISTRIBUTION Patrizia Paterlini Bréchot, MD, Ph.D.
Acknowledgements
THANKS FOR YOUR ATTENTION! [email protected]
Rarecells Diagnostics • Ms. Katia Hormigos • Dr Sophie Laget • Lucile Broncy INSERM / Paris Descartes University • Lucile Broncy • Dr Basma Ben Jima • Dr Thierry Capiod • Dr Fatima Ben Mohamed • Pr Patrizia Paterlini Bréchot
Scientific collaborators • Dr Stephen Jackson and Dahlia Dhingra from Thermofisher Scientific • Dr Filip Van Nieuwerburgh and Lieselot Deleye from Ghent University