Liquid biopsy: the experience of real life case studies · Beatriz Bellosillo Servicio de Anatomía Patológica Hospital del Mar, Barcelona Liquid biopsy: the experience of real life

Beatriz Bellosillo

Servicio de Anatomía PatológicaHospital del Mar, Barcelona

Liquid biopsy: the experience of real life case studies

10th September 2018

• Introduction

• Experience in colorectal cancer research samples

• Experience in lung cáncer research samples

Agenda

Cell-Free Tumor DNA

Cell-Free Normal DNA

Circulating Tumor Cell

1%

100%

10%

0.1%

0.01% BEAMing/PCR digital

Real-Time PCR

Pyrosequencing

Sanger Sequencing

Detection Capability(mutant DNA/ total DNA)

BRAF NRAS

KIT

Papillary: BRAF

KRAS NRAS

BRAF

BRAF BRCA1

BRCA2

Metastatic melanoma

Metastatic thyroid

Metastatic serous low-grade ovarian

Metastatic colon

H. Davies et al. Nature 2002

EGFR KRAS

ALK ROS

RET BRAF

Lung cancer

Hospital del Mar experience: NGS and liquid biopsy

Junior 454 IonTorrent PGM IonChef IonTorrent S5

2011 2015 2016 2018

20152012 20162013

UMI basedNGS DNApanels

TNA basedNGS panels

For Research Use Only. Not for diagnostic procedures.

• Introduction


• Experience in lung cancer research samples

Agenda

OncomineTM Cell-free DNA Assays

Lab-created Report

Oncomine™ Knowledgebase

Reporter*

Template PrepcfDNA Isolation Sequencing

Applied Biosystems™ MagMAX™ cfDNA

Isolation Kit

Ion GeneStudio™ S5 Systems

Analysis

Variant caller in Torrent Suite , Ion

Reporter™ Software

Library Prep

Oncomine™ cfDNA Assays

Ion Chef™ Instrument

Single blood tube Refined variant data

Oncomine cfDNA Assays include library prep and analysis


KRAS/NRAS: G12/G13/Q61 BRAF: V600E PIK3CA: E545K, H1047R TP53: R175H R273H/C/LSMAD4: R361C/H Recurrent deleterious APC mutations (including p.R876*, p.R1114*, p.Q1378*, and p.R1450*)

Detect rare variants present down to 0.1% with Mean Sensitivity – 90% Mean Specificity – 98%

AKT1

BRAF

CTNNB1

EGFR

ERBB2

FBXW7

GNASKRASKRAS

MAP2K1

NRAS

PIK3CAMAD4

TP53

APC

Oncomine™ Colon cfDNA Assay

GENE LIST (14)

Heterogeneity and co-existence of mutations at progression of panitumumab based treatment

- At least one mutation was detected in 94% of pts (15/16)- Median mutations per sample was 2.5 (range 1 -13)

Vidal et al, ESMO 2017

Plasma Pre- Panitumumab Plasma Post-PanitumumabPt APC TP53 CTNNB1 KRAS NRAS BRAF PIK3CA EGFR MAP2K1 ERBB2 AKT1 GNAS SMAD4 FBXW7 APC TP53 CTNNB1 KRAS NRAS BRAF PIK3CA EGFR MAP2K1 ERBB2 AKT1 GNAS SMAD4 FBXW7

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

1 MUTATION IN THE SAME GENENO PLASMA AVAILABLE 2 MUTATIONS IN THE SAME GENE 3 MUTATIONS IN THE SAME GENE

- At least one mutation was detected in 94% of pts (15/16)- Median mutations per sample were 2.5 (range 1 -13)

- At least one mutation was detected in 80% of pts (8/10)- Median mutations per sample was 1.8 (range 1 - 4)

Heterogeneity and co-existence of mutations at progression of panitumumab based treatment

Vidal et al, ESMO 2017

• Introduction


• Experience in lung cancer research samples

Agenda

30.73%

1.068%

4.99%

16.253%

ND(< 0.02%)

0.295%0.948% 1.386%

0

2

4

6

8

10

12

14

16

18

27/08/2013 27/11/2013 12/11/2014 01/02/2015 04/03/2015

% m

uta

nt

alle

les

L858R T790M

TKI

EGFR L858R

(T790M Not detected)

Retrospective case study #1

Mutation %

EGFR p.L858R 37%

EGFR p.T790M Not detected

TP53 p.R273C 17%

Mutation %

EGFR p.L858R 21.%

EGFR p.T790M 1.9%

TP53 p.R273C 9.5%

Taus et al, CLC 2018

OncomineTM Cell-free DNA Assays


169 hotspot mutations like EGFR: T790M, L858R, Exon19 del, C797S KRAS: G12X, G13X, Q61XALK: 1151Tins, L1152R, C1156YBRAF: V600E

Detect rare variants present down to 0.1% with Mean Sensitivity – 90% Mean Specificity – 98%

ALK

BRAF

EGFR

ERBB2

KRAS

MAP2K1

MET

NRAS

PIK3CA

ROS1

TP53

Oncomine™ Lung cfDNA Assay

GENE LIST (11)

2

3

4

3

1T790M

T790M+TP53

T790M+OTHER

OTHER (TP53-RAS)UNKNOWN

N=13/22 cases

Mutational

profile by NGS

in NSCLC at

the time of

progression to

TKI

Oncomine Lung Cell-Free Total Nucleic Acid Research Assay

Detects SNVs, indels + FUSIONS!

Lung cfTNA

Assay

• 0.1% LOD

SNVs• CNV

• Structural

variation

Sequencing of sample on Ion S5

System

Analyze data in Ion

Reporter Cloud

software to generate test

report

cfTNAextractio

n

Single bloodtube

8

42

2

4

NO MUTATIONSFOUNDEGFRMUTATIONSKRASMUTATIONSFUSION DRIVERGENESOTHERMUTATIONS

N= 20


Hotspot GenesFull-

LengthGenes

Copy Number Genes

GeneFusions

Exon Skipping

Lung cfTNA

ALKBRAFEGFRERBB2KRASMAP2K1

- MET ALKRET

ROS1

MET

Lung cancer – case study #1Lung cancer

• Research sample from 69 year woman

• Diagnosed with lung adenocarcinoma

• No positive biomarker for targeted therapy (EGFR-ALK-ROS1)

• PDL1 expression : 0%

• Standard chemotherapy




• Clinical progression

NGS screening of liquid biopsy research sample

Lung cancer – case study #1






FISHprobe

Oncomine solid

tumor CE-IVD

assay




• Clinical progression

• Potential targeted therapy was identified, and results may guide treatment decisions in the future (alectinib)



Lung cancer – case study #2Lung cancer

• Research sample from 48 year old woman

• Diagnosed of lung adenocarcinoma

• EGFR mutated (deletion in exon 19 and S768I in exon 20)

• Treatment with TKI (Afatinib)

• 5 month post-diagnosis: decrease of afatinib due to toxicity

• 7 months later– slow progression

• Liquid biopsy : T790M not detected by ARMS real-time PCR, but positive for deletion in exon 19

Confirmation by digital PCR for T790M

Osimertinib identified as potential treatment path in the future


Challenges of ctDNA/TNA analysis

• Dilution of tumoral/mutated DNA with wild type DNA

• Limited amount of cfDNA/TNA

• Small fragmented DNA

• Increase in the amount of information requested

• Optimize pre-analytical phase

• Adequate sample size to ensure minimum amount of cfDNA/TNA

– For some patients – insufficient material

Important considerations

Bloodextrac on

TransportPlasma

separa on

DNA

extrac on




• Ensure high quality cfDNA


Bloodextrac on

TransportPlasma

separa on

DNA

extrac on

cfDNA

Contaminating

genomic DNA

22

cfDNA




• Ensure high quality cfDNA

• Participate in external quality controls and validation of technology


Bloodextrac on

TransportPlasma

separa on

DNA

extrac on

Current practice at Hospital del Mar for molecular testing

Pre-treatment

RxResponse

RxProgression

1º

Routine

Research

Negative molecular

marker

- Re-biopsy

- Repetition of liquid

biopsy

NGS NGS

Molecular data should be discussed in

multidisciplinary tumor boards

Take-home messages

• ctDNA is a complementary strategy to tissue for molecular

analysis that allows:

– Non-invasive analysis of tumor heterogeneity

– Dynamic biomarker monitoring

• NGS of ctDNA provides a broad molecular analysis: SNVs, CNVs,

fusions

• A comprehensive and dynamic monitoring of biomarkers is

mandatory for real-time precision medicine

• Standardization and validation of the technology is necessary to

ensure quality of the results

• Variant interpretation should be done in the potential clinical

context in multidisciplinary teams

Disclaimer

Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.

Edurne ArriolaClara MontagutAlvaro TausJoana Vidal Joan Albanell

Raquel LongarónLaura CamachoConchi FernándezLara PijuanAlba DalmasesGabriel Piquer

Servicio de Anatomía Patológica y Servicio de Oncología, Hospital del Mar

Thermo-Fisher

Chris AllenFrancesco Acquadro

Rosella Petraroli

Documents

Liquid biopsy: the experience of real life case studies · Beatriz Bellosillo Servicio de Anatomía Patológica Hospital del Mar, Barcelona Liquid biopsy: the experience of real life