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! patriziapaterlini@gmail.com

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