Next generation sequencing in every future pathology laboratory, are we ready?

José Luis Costa(jcosta@ipatimup.pt)

Thermo Fisher Scientific symposium – ECP20199th Sepember 2019

Conflict of interests

• Lecture fees from Thermo Fisher Scientific

• Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration

Ipatimup – Porto - Portugal

Ipatimup• Founding member of i3S

• Leading cancer research institute in Portugal

• Founding partner of Porto Comprehensive Cancer Center

i3S

• Biggest research in health institute in Portugal (1250 researchers);

• Cancer, Neurosciences and Host-Pathogen interactions research lines

IPATIMUP diagnosticos - Mission

• Provide services in the areas of surgical pathology andcytopathology, genetic diagnosis and genetic identification andparentage, in order to improve Portuguese citizens life quality.

NP EN ISO 15189

NP EN ISO 17025

Laboratory accreditation

Annual test volume

Molecular diagnosis

• Tumor mutation screening – 4220 cases

• Genetic diagnosis – 2960 cases

• Pre-natal screening – 3260 cases

Pathology; 27733

Molecular diagnosis;

10430

Forensic genetics;

91

2018

International consultation

2011

Ion PGM

NGS clinical research timeline

Motivation: reduce turn-around-time of tests

For Research Use Only. Not for use in diagnostic procedures.

2011 2012 2013

Ion PGMIon Proton

Ion OT2

NGS clinical research timeline

Motivation: reduce turn-around-time of tests

For Research Use Only. Not for use in diagnostic procedures.

2011 2012 2013 2014 2015

Ion PGMIon Proton

Ion OT2 Ion Chef

Ion Reporter

Ion Chef

NGS clinical research timeline

Motivation: comprehensive biomarker characterization

For Research Use Only. Not for use in diagnostic procedures.

ROS1• Crizotinib 4

• Cabozantinib 2

• Ceritinib 2

• Lorlatinib 2

• DS-6051b 1

ALK• Crizotinib 4

• Alectinib 4

• Ceritinib 4

• Lorlatinib 2

• Brigatinib 2

EGFR sensitizing• Gefitinib 4

• Erlotinib 4

• Afatinib 4

• Osimertinib 4

• Necitumumab 4

• Rociletinib 3

KEY1 – Phase I 3 – Phase III2 – Phase II 4 - Approved

Lung cancer clinically relevant alterations

MEK1• Trametinib 2

• Selumetinib 3

• Cobimetinib 1

PIK3CA• LY3023414 2

• PQR 309 1

NTRK1• Entrectinib 2

• LOXO-101 2

• Cabozantinib 2

• DS-6051b 1

RET• Cabozantinib 2

• Alectinib 2

• Apatinib 2

• Vandetanib 2

• Ponatinib 2

• Lenvatinib 2

BRAF• Vemurafinib 2

• Dabrafenib 2

ROS1• Crizotinib 4

• Cabozantinib 2

• Ceritinib 2

• Lorlatinib 2

• DS-6051b 1

HER2• Transtuzumab emtansine 2

• Afatinib 2

• Dacomitinib 2

MET• Crizotinib 2

• Cabozantini 2

ALK• Crizotinib 4

• Alectinib 4

• Ceritinib 4

• Lorlatinib 2

• Brigatinib 2

EGFR sensitizing• Gefitinib 4

• Erlotinib 4

• Afatinib 4

• Osimertinib 4

• Necitumumab 4

• Rociletinib 3

KEY1 – Phase I 3 – Phase III2 – Phase II 4 - Approved

Lung cancer clinically relevant alterations

Comprehensive biomarker characterization

Comprehensive biomarker testing leads to better future patient care

For Research Use Only. Not for use in diagnostic procedures.

2011 2012 2013 2014 2015

Ion PGMIon Proton

Ion OT2 Ion Chef

Ion Reporter

Ion Chef

NGS clinical research timeline

Somatic testing moved to NGS

• Simultaneous identification of multiple genes better picture of tumor heterogeneity to enable future better clinical decision

• Single input of DNA/RNA

• Sensitive and quantitative detection of genomic aberrations

• Decrease sequencing costs per gene

Motivation: comprehensive biomarker characterization

For Research Use Only. Not for use in diagnostic procedures.

2011 2012 2013 2014 2015 2016

Ion PGMIon Proton

Ion OT2 Ion Chef

Ion Reporter

Ion Chef

NGS clinical research timeline

Motivation: more comprehensive and sensitive

For Research Use Only. Not for use in diagnostic procedures.

Tissue may not be available for all assays or situations

2011 2012 2013 2014 2015 2016 2017 2018 2019

Ion PGMIon Proton

Ion OT2 Ion Chef

Ion Reporter

Ion Chef Ion Chef

Ion S5xl Ion S5

NGS clinical research timeline

Motivation: more comprehensive and sensitive

For Research Use Only. Not for use in diagnostic procedures.

1 10 102 103 104 105 106 108107 >109

Kilobases Megabases Gigabases

COMPREHENSIVENESS

10%(1/10)

100%

0.1%(1/ 1000)

0.01%(1/10000)

1%(1/100)

0.001%(1/100000)

SEN

SITI

VIT

Y

NGS Targeted panels

NGSExome

NGSwhole genome

BEAMingdigital PCR

QPCR

Liquid Biopsysweet spot

• The capacity of dPCR to detect rare alleles combined with the ability to query the number of targets of a NGS targeted panel

Understand Peripheral monitoring - Liquid biopsy

Validated routine strategy

cfDNA isolation

NGSVariant

Discovery

dPCRvalidation

Plasmacollection

1. Plasma collection 2. cfDNA isolation

3. NGS variant identification

• Ion AmpliSeq Colon & LungCancer Research Panel

• Ion AmpliSeq RNA Fusion Lung Cancer Research Panel

• Oncomine cfDNA Lung assay

4. Digital PCR validation

QuantStudio 3D Digital PCR System

Ion S5xl System

BD Vacutainer PPT (K2EDTA)

MagMax cfDNA extraction kit

TaqMan Assays

ISO 15189 accreditationFor Research Use Only. Not for use in diagnostic procedures.

OncoNetwork multicentric study

Benchmarking at 0.1% AF

Genes (n) 11 62 73

SNVssensitivity 83.9% 67.3% 63.8%

PPV 99.1% 93.6% 92.1%

InDelssensitivity 83.9% 86.2% 67.8%

PPV 99.1% 100% 88.4%

OncoNetworkstudy

www.foundationmedicine.com www.guardanthealth.com

Confident detection of variants at 0.1% AF

Understand Peripheral monitoring - Liquid biopsy

Peripheral monitoring - Liquid biopsy

• Informative in different tumor models

• Informative for different future therapeutic strategies

• Allows real-time study of the disease

• Allows study of future clinical relapse anticipation

• Identification of resistance mechanism

Where we are now

Decreased time to diagnoses

0 2 4 6 8 10 12 14 16

2018

2014

Turnaroundtime

33%

Increased diagnostic yield

0 10 20 30 40 50 60 70 80

2018

2014

Unclassifiedcases

50%0 1000 2000 3000 4000 5000

2018

2016

2014

Tumormutationscreening

Increased number cases

53%

Either on FFPE or plasma samples

Both procedures accredited

For Research Use Only. Not for use in diagnostic procedures.

Technology challenging us

• Complexity of workflows

• Capacity/throughtput (batching)

• Data analysis

• Data interpretation

• Specialist equipment and data sorage requirements

Us challenging the technology

Ion Chef

Ion Reporter

Oncomine Knowledge Reporter

Ion chip series

Ion S5xl

• Ideally all-in-one system

• Tumor specific panels for routine testing• Pan cancer panel for specific questions

• Validated independently of type of biological specimen (FFPE, plasma, CSF...)• Validated for type of variants (SNV, CNV, Translocations, InDels)

Joana ReisSusana Neto

Joana MarquesFernando Schmitt

José Carlos Machado

Venceslau Hespanhol

Gabriela Fernandes

Fátima Carneiro

Conceição Souto Moura

nurses

Funding

Kelli BramletThomas Bittick

Elaine Wong-HoChris Allen

Rosella Petraroli

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