Download Technical Insights into Highly Sensitive Isolation

Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
Technical Insights into Highly Sensitive Isolation and
Molecular Characterization of circulating tumor cells for early
detection of tumor invasion.
COPD
Screening
Localized lung
cancer
Metastatic lung cancer
ALK liquid biopsy
Patrizia Paterlini Bréchot, MD, Ph.D.
Lucile Broncy, Msc,
Professor of Cellular and Molecular
Biology
University Paris Descartes– Paris
Ph.D. student (bourse CIFRE)
University Paris Descartes– Paris
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
Circulating Tumor Cells, more than a biomarker. Actor of invasion
Very rare
ü  Few per ml, mixed with
ü  10 millions WBC, 5 billion RBC
Tumor
Heterogeneous
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)
blood
metastasis
Disease progression
Screening
Diagnostic
Prognostic
Potential for reducing cancer mortality
2
Theranostic
Treatment
efficacy
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
ISET® TECHNOLOGY: OPEN SYSTEN: Isolation by Size of Tumor cells (all types)
(-)
Blood collection
Sample Incubation
(10 min.)
Filtration
(5 min.)
ISET® Filters
(5 min.)
• Collection of
10ml of blood
from the
patients
• Leukocytes are
the smallest cells
in the body
Dilution
with the
« ISET®
Buffer »
Blood
treatment
with the
Rarecells®
Device
CCC are isolated by the
ISET® system on the ISET®
Filter
Patented combination of parameters allowing very sensitive and rapid isolation of CTC
è Achieve Cytopathological diagnosis
Isolated Circulating Cancer Cells (CCC)
•  Cytopathological diagnosis
•  CCC counting
è CCC characterization
•  Circulating Cancer Cells can be characterized by
immunolabelling, FISH, and Next Generation
Sequencing (NGS)
è Perform Theranostic Analyses
•  Molecular analyses targeted to tumor cells after
laser microdissection (CGH, DNA mutation, RTPCR, NGS)
Enriched circulating rare
cells including tumor cells
FIXED
+ Possible storage and
shipment of filters
Vona et al Am J Pathol, 2000
Paterlini Bréchot, Cancer Letters 2007
Paterlini Bréchot, Cancer Microenvironment 2014
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
Circulating Cancer Cells (CCC)
are tumor cells isolated without loss
and undamaged : diagnosed by cytopathology
Tumor cells
EMT+ are lost by CellSearch
Atypical
CTC by CellSearch
Normal
CTC by CellSearch
Hofman et al, Clinical Cancer Research, 2011
Paterlini Bréchot, Cancer Microenvironment, 2014
!
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
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
Hofman Clinical Cancer
Research 2011
• 
• 
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
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
Multicenter Studies
Monocentric Study
Major Clinical Trials
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.
• 
• 
Ilie et al. Plos One 2014
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.
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)
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
Lower Limit Of Detection (LLOD): 1 Tumor cell /10 ml of blood;
sensitivity at LLOD: 100%
Sensitivity tests
Capture of
individual cells by
micropipetting
Incubation with
Rarecells® Buffer
ISET®
Filtration
Number of HeLa cells detected by ISET®
Number of A549 cells detected by ISET®
(log scale)
n=2
n=13
1000
y = 0.9991x
R2 = 0.96263
n=9
100
n=30
n=6
n=6
10
1
1
10
100
Fixed cancer cells on
the filter
120
y = 1.0128x
R! = 0.99514
100
80
60
40
20
0
0
1000
20
40
60
80
100
Number of spiked HeLa cells in 1 mL of blood
Number of spiked A549 cells in 10 mL of blood or equivalent
(log scale)
Laget et al, Plos One, 2017
120
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
Culture of tumor cells and cytoskeleton analysis after live isolation from blood
Live Cells
CTC culture and functional
assays
Blood
Collection
Incubation with
Rarecells® Live Cells
Buffer
(Ref 540208)
ISET®
Filtration
CTC-derived xenografts
RNA / Transcriptomic DNA /
NGS analyses
Laget et al, Plos One, 2017
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
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
KRAS G13D mutant allele
Sample
mutant allele
frequency
PIK3CA H1047R
mutant
allele
mutant allele amplicon
coverage coverage frequency
SMO V404M
mutant
mutant
allele
allele
amplicon
coverage coverage frequency
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)
Pooled data 3
HCT116
100%
29
29
32%
312
986
67%
1346
2000
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
No mutations detected in ABL1, APC, FGFR3 and STK11 genes :
these specific variants are not in the list of mutations of the panel
Laget et al, Plos One, 2017
mutant
allele
amplicon
coverage coverage
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
Developments concerning CCC isolation for early detection of
invasive cancer
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
Patrizia Paterlini Bréchot, MD, Ph.D.
CONFIDENTIAL – NOT FOR REDISTRIBUTION
Acknowledgements
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
THANKS FOR YOUR ATTENTION!
[email protected]
Scientific collaborators
•  Dr Stephen Jackson and Dahlia Dhingra from Thermofisher Scientific
•  Dr Filip Van Nieuwerburgh and Lieselot Deleye from Ghent University