Next generation sequencing in every future pathology laboratory, are we ready?
José Luis Costa([email protected])
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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