Circulating Tumor DNA Clinical Application -...

Role of liquid biopsies and circulating tumor DNA

Pierre Laurent-Puig

European Georges Pompidou Hospital

Paris Descartes University

DISCLOSURE

AMGEN

ASTRAZENECA

BIOCARTIS

BOERINGHER INGELHEIM

INTEGRAGEN

MERCK SERONO

RAINDANCE TECHNOLOGIES NOT yet BIORAD

ROCHE

SANOFI

Detection of circulating tumor DNA• By searching for the genetic or epigenetic alterations which

characterize the DNA coming from tumor cells

• Tumor specific mutations

• Tumor specific structural changes (i.e. rearrangement)

• Copy Number Variations

• MicroRNA

• Methylated DNA

Zonta et al. Advances in Clinical Chemistry, Volume 70, 2015, Pages 197-246

Why detection of ctDNA is challenging ?

• Circulating tumor DNA is mixed with circulating normal DNA

• The detection of circulating tumor DNA and its validation as a biomarker of cancer have long been limited due to inefficient methods of detection

Zonta et al. Advances in Clinical Chemistry, Volume 70, 2015, Pages 197-246

0.001

0.01

0.1

1

colon cancer

16%

0.02

Alle

le fr

eq

ue

ncy m

ea

su

red

by N

GS

223 mutations from 293 plasma samples

0.001

0.01

0.1

1

pancreatic cancer


26%

0.02

Alle

le fr

eq

ue

ncy m

ea

su

red

by N

GS

0.001

0.01

0.1

1

lung cancer


45%

0.02

Alle

le fr

eq

ue

ncy m

ea

su

red

by N

GS

Adapted from Pecuchet N, … Laurent-Puig in Clin Chem 2016

Proportion of advanced cancer patients with lessthan 2% of ctDNA in plasma

Massively digital PCR in emulsion

Droplet-based digital PCR can overcome PCR-based procedure limitations.

See Williams, R. et al. Nature Methods. 2006.

Digital Droplet PCR

100 µm

Pekin D. et al., Quantitative and sensitive detection of rare mutations using droplet-based microfluidics, Lab on Chip, 2011; 11: 2156-66.

Taly V. et al., Detecting biomarkers with microdroplet technology,Trends in Molecular Medicine, 2012;18:405-16.

Sensitivity demonstrated for 1 mutant sequence in

~200,000 wild type sequences

Quantitative procedure and relatively simple to set up

48%45.1% 54.8%

➢Patient follow up will benefit from unique assays allowing to screen as much patients as possible!

30 assays1see cBIoPortal2Laurent-puig, Taly et al., unpublished data

Somatic mutation detection for ctDNA monitoring ?The follow-up is linked to our ability to detect specific mutation(s) within the tumor

The example of colon cancer

Massively parallel sequencing Different methods have been used to improve sensitivity of NGS

CAPP seq, Safe SeqBPER methodCircle sequencing

All these methods tend to improve sensitivity of detection by differentiating true mutation to background noiseLess than 0.1%

First question : is liquid biopsy ready

for colorectal cancer screening ?

THE ANSWER IS NO BUT …..

Adapted from Garrigou S et al. Clin Chem 2016;62

Circulating Tumor DNA according to Tumor stage

SEPT9 methylation as a marker of circulating

tumor DNA 7941 asymptomatic individuals >50 years old

53 cases of colorectal cancers

Sensitivity of the SEPT9 test for CRC : 48.2% CI95% 32.4%-63.6%

Stage I 35%, Stage II 63%, Stage III 46%, Stage IV 77.4%

Specificity of the SEPT9 plasma detection 91.5% CI95% 89.7%-93.1%

Sensitivity for advanced adenoma was low 11.2%

Based on this study FDA approved the test in April 2016 with this labelling « The epi procolon test is

indicated to screen adults of either sex, 50 years or older, defined as average risk for crc, who have

been offered and have a history of not completing crc screening. The Epi proColon test is not

intended to replace colorectal cancer screening tests that are recommended by appropriate

guidelines »

Church TR et al. Gut 2014;63:315-325

Second question :

Is the liquid biopsy ready to search for the RAS

gene mutations?

KRAS detection : plasma versus tumor

Sensitivity Specificty and Accurracy

Authors Journal Detection Methods N Sensitivity Specificity Accurracy

Yen Clin Cancer Res

2009

Membrane arrays

KRAS codon 12 13 15 18

20 30

76 84% 95.3% 90%

Thierry Nature Med 2014 Intplex system

KRAS 12 & 13

7 frequent mutations

95 92% 98% 96%

Bettegowda Sci Transl Med

2014

SafeSeqS

KRAS 12 & 13

206 87.2% 99.9% 95%

Second question :


gene mutations?

THE ANSWER IS PROBABLY YES waiting for

prospective series in all RAS mutation

Second question :


gene mutations?

THE ANSWER IS PROBABLY YES waiting for

prospective series in all RAS mutation

So we do it

RASANC study

• Prospective biospecimen collection study in previously untreated advanced mCRC(NCT02502656)

• AGEO French collaborative study

• Plasma collected in Streck tubes

• Plasma NGS performed using established panel

Bachet et al. Submitted; ASCO 2017 Poster discussion

BPER METHOD

Presence of a mutation in ≥1 of the 22 tested

genes

Presence of ctDNA

RAS mutated RAS non mutated Bachet et al. Submitted; ASCO 2017 Poster discussion

Absence of a mutation in 1 of the 22 tested genes

Primary analysis

BPER METHOD


genes

Presence of ctDNA

RAS mutatedN=187 (45.4%)

RAS non mutatedN=225 (56.4%) Bachet et al. Submitted; ASCO 2017 Poster discussion


RAS mutation in plasma sample

Absence No.

Presence No.

Total No.

RA

Sm

utatio

n in

tu

mo

r

AbsenceNo.

167(41%)

3(1%)

170 (41%)

Presence No.

58 (14%)

184 (45%)

242 (59%)

Total No.225

(55%)187

(45%)412 (100%)

Primary analysis

BPER METHOD


genes

Presence of ctDNA


RAS non mutatedN=142(43%)



Secondary analysis

Digital PCR for detecting methylation universal

biomarkers WIF1 & NPY

No detection of methylation

Inconclusive resultsn=83 (20.1%)


Absence No.

Presence No.

Total No.

RA

Sm

utatio

n in

tu

mo

r

AbsenceNo.

128(39%)

3(1%)

131 (40%)

Presence No.

14 (4%)

184 (56%) 198 (60%)

Total No. 142(43%) 187 (45%) 329 (100%)

BPER METHOD


genes

Presence of ctDNA


RAS non mutatedN=225 (56.4%)



Secondary analysis

Digital PCR for detecting methylation universal

biomarkers WIF1 & NPY

No detection of methylation

Inconclusive resultsn=83 (20.1%)


Absence No.

Presence No.

Total No.

RA

Sm

utatio

n in

tu

mo

r

AbsenceNo.

128(39%)

3(1%)

131 (40%)

Presence No.

14 (4%)

184 (56%) 198 (60%)

Total No. 142(43%) 187 (45%) 329 (100%)

Bachet et al: RASANC study• Tumor DNA shed is

related to a range of clinical and biologic factors

• Thus, this is a tool that is most effective when used in the right patients


Third question: Is the liquid biopsy a

prognostic marker for colorectal cancer in early

and advanced stage?

Tie et al., Sci Transl Med 2016;8:346ra92Published by AAAS

Relapse free survival in stage II patients n=250 patients

The [ctDNA] is associated with tumor burden

Tie J, Ann Oncol 2015

53 patients receiving standard chemotherapy in first-line

mCRC patients (n=82)

First-line (n=68) or second-line (n=14) chemotherapy regimen

Mutation analysis in tumor

Identification of

targetable mutation* in the tumor

n=43 (52.4%)

No targetable mutation* identified in

the tumor

n=39 (47.6%)

Circulating tumor DNA detected at baseline

n=63 (76.8%)

Testing plasmatic DNA at baseline

for WIF1 and NPY hypermethylation

n=27/39 positive at baseline (69.2%)

Testing plasmatic DNA at baseline

for a targetable mutation

n= 36/43 positive at baseline (83.7%)

Alternative strategy based on the

hypermethylation detection of WIF1 or

NPY

Figure 1

Garlan et al. Clin Can Res 2017

[ctDNA] at baseline


55% 36% 9%

Decreased ctDNA ≤0.1ng/mL

N=40Decreased ctDNA but >0.1ng/mL

N=26

Increased ctDNA

N=7

0 C1or20 C1or2 0 C1or2

[ctDNA] variation during the follow-up is a prognostic indicator of PFS and 0S


55% 36% 9%

Decreased ctDNA ≤0.1ng/mL

N=40Decreased ctDNA but >0.1ng/mL

N=26

Increased ctDNA

N=7

0 C1or20 C1or2 0 C1or2



[ctDNA] variation during the follow-up is a prognostic indicator of PFS

A B


Garlan et al. Clin Cancer Res in revision


A B

Conclusions Improvement of methods for detecting ctDNA allows them to be

used in a clinical setting

The presence of ctDNA in early stage cancer after surgery is an

indicator of early recurrence

Quantification of ctDNA is a probably a surrogate marker of tumor

burden and therefore a prognostic marker in advanced stage

Evolution of ctDNA concentration under treatment is likely an early

indicator of chemotherapy efficacy

Finally, ctDNA allows the identification of resistance mutation and

could be a decision-making tool

Oncologists, pathologists and biostaticians

Prof. O. BouchéDr. B. LandiProf. JF. EmileDr F. Bibaut

Thanks to...

Fundings- Supports

Institut National pour la recherche sur le Cancer (INCa)

Association pour la Recherche contre le Cancer (ARC)

Région Alsace

Université de Strasbourg

Centre National de la Recherche Scientifique (CNRS)

Institut National de la santé et la recherche médicale (INSERM)

Université Paris-Descartes

Hopital Européen Georges Pompidou (hEGP)

Agence Nationale de la recherche (ANR)

UMRS-1147/ UdS

V. TalyJB BachetL. BenhaimDr H. BlonsDr V. BoigeA. DidelotDr E. FabreF. GarlanS. GarrigouT. HorD. Le CorreDr P. NizardC. NormandDr N. Pecuchet

Dr D. PekinDr K. Perez TorallaDr Y. SkhiriDr A. ZaananDr E. Zonta

RainDance Technologies

Dr D. LinkDr B. HutchisonJ. OlsonS. KotsopoulosQ. ZhongI. Atochin

X. Li A. CornerC. MilburyM. Samuels

R. NiarraP. AucouturierG. ChatellierDr Y. Rozenholc

Circulating Tumor DNA Clinical Application -...

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