liquid biopsies: monitoring patients with breast cancer ......shared mutations in cfdna and...
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Liquid biopsies: monitoring patients with breast
cancer and non-small cell lung cancer Jacqui Shaw, Director Leicester Precision Medicine Institute
email: [email protected]; [email protected]
https://www2.le.ac.uk/institutes/lpmi
https://twitter.com/lpmi_UK
Liquid biopsies
• cfDNA/ctDNA
• CTCs
• Tumour educated platelets
(TEPs)
• Exosomes and EVs
Developing blood tests for use in the clinic
ctDNA detects “sleeping” cancer
Guttery et al. Clin Chem 2015
9 of 54 patients had an ESR1 gene mutation in cfDNA
Liquid biopsy can help to guide treatment
Page et al. Clin Chem 2017
Shared mutations in cfDNA and individual CTCs
Shaw et al. Clin Can Res 2017
Individual CTCs
PIK3CA
p.H1047R
TP53
p.R175H
ctDNA
cfDNA
ESR1
p.E380Q
ESR1
p.Y537C
ESR1
p.E380Q
NGS
NGS
WT WT WTPIK3CA
p.H1047R
PIK3CA
p.H1047R
ESR1
p.E380Q
Based on a small fraction of cells from patients with high CTC counts, we see heterogeneity in mutations between matched CTCs and cfDNA
cfDNA Oncomine Panels
• Low limit of detection— variant detection down to 0.1% for SNV hotspots and indels
• Sample tolerance— flexible input amounts and tolerance of sample input variability to accommodate more samples - From 1 to 30 ng
• Optimized analysis – New variant caller module that removes PCR errors to help increase sensitivity and specificity
OncomineTM Lung cfDNA early access
• Horizon Multiplex cfDNA reference – 5%, 1%, 0.1%, 0% dilutions
• Consistent detection down to 0.1% VAF
TRAcking non small cell lung Cancer Evolution through therapy (Rx)
Liquid biopsy detects recurrence before scans
Abbosh et al. Nature. 2017
R
Detection of local recurrence
Local recurrence confirmed
Oncomine™ Lung cfDNA Assay >0.1% VAF 46 patients>150 hotspots ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53
Early breast cancer
• 26 early stage breast cancers, 40 patients with MBC and 92 healthy age matched female controls
• Applying a threshold of 1 or more driver mutations for detection of ctDNA (Cohen et al. Science 2018)
– 19 (73%) PCs, 29 (72.5 %) MBCs and 15 HCs (16%) were ctDNA positive
• Significant difference between ctDNA positive cancers and healthy controls
– (P < 0.0001) Shaw et al. AACR 2018
Oncomine™ Breast cfDNA Assay
AKT1
CCND1
EGFR
ERBB2
ERBB3
ESR1
FBXW7
FGFR1
KRAS
PIK3CA
SF3B1
TP53
550 S5 Prime Pan Cancer Study
550 chips 24 samples analysed with the Oncomine™ PanCancer cell-free assay (52-gene panel):
23/24 (5.8%) cfDNA samples were positive for ctDNA across 10 different genes:
• Hotspot genes (SNVs) and short indels: AKT1, ALK, AR, ARAF, BRAF, CHEK2, CTNNB1, DDR2,EGFR, ERBB2, ERBB3, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, RAF1, RET, ROS1, SF3B1, SMAD4, SMO
• Tumor suppressor genes: APC, FBXW7, PTEN, TP53
2009 2017
Surgery Nov 2009
Letrozole
Paclitaxel
April 2010
Tamoxifen
2013
March 2009
Anastrazole
1757
Nov
1732
Oct
1404 1512
May 16Jul 15
Developed ascites
Cap
Letrozole
Jan Aug Nov 15
1438
Blood sample
PIK3CA E545K1.57 2.35 2.21 2.71 1.24
KRAS G12D 1.1 1.1
0.14
Sample ID no of CTCs
Sample date Gene MAF% AA CHg
1404 68
NRAS 0.14 p.G12A
PIK3CA 1.57 p.E545K
01/07/2015 PIK3CA 0.56 p.H1047R
ESR1 0.14 p.D538G
TP53 0.17 p.G244D
1438 99 04/11/2015 PIK3CA 2.35 p.E545K
1512 67 06/05/2016 PIK3CA 2.21 p.E545K
1732 50 11/10/2017PIK3CA 2.71 p.E545K
KRAS 1.10 p.G12D
1757 91 15/11/2017PIK3CA 1.24 p.E545K
KRAS 1.06 p.G12D
0.56 PIK3CA H1047R
NRAS G12A
ESR1 D538G0.14
Pac
Developed pleural effusion
Stable disease
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Breakout Sensitivity, Standout Customization
• Low limit of detection-variant detection
down to 0.1%* enabled by unique molecular
tags and dual barcodes
• Customization in your hands-flexible panel
design customization available at an
accessible price
• Complete 2-day workflow-fast, targeted
NGS workflow from sample to data
New amplicon-based NGS library preparation technology that enables you to
design custom ultrahigh-sensitivity panels for your targets of interest
For Research Use Only. Not for use in diagnostic procedures.
Introducing Ion AmpliSeq HD Technology:
*For cell-free DNA samples
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Ion AmpliSeq HD Technology
Ultrahigh sensitivity to confidently detect
low frequency alleles
Flexible panel customization using the
Ion AmpliSeq™ Designer
Complete NGS workflow from sample to data in 2 days
For Research Use Only. Not for use in diagnostic procedures.
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Comparable Performance Between Ion AmpliSeq HD and Oncomine Cell-Free Panels
Figure 2. Ion AmpliSeq HD panel design targets over 50 genes with hotspots, CNV, fusions, tumor suppressor genes for cfDNA. 20 ng of cfDNA
input for each assay sequenced with Ion GeneStudio S5 system on Ion 540 Chip. PPV values based on Thermo Scientific™ Acrometrix™
Oncology Hotspot controls.
High specificity (PPV) through additional reductions in false positives
For Research Use Only. Not for use in diagnostic procedures.
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Ion AmpliSeq HD Panels: 2-Day NGS Workflow
Select targets Construct library Prepare template Run sequence Analyze data
Design a panel
at ampliseq.com• Ion AmpliSeq HD Library
Kit
• Ion AmpliSeq HD Dual
Barcode Kit
Ion Chef instrument* Ion GeneStudio S5 systems
• Torrent Suite Software
• Ion Reporter Software
• Oncomine Knowledgebase
Reporter
<2 hr hands-on time 45 min hands-on time 15 min hands-on time 15 min hands-on time 30 min hands-on time
Go from DNA to data in less than 2 days
For Research Use Only. Not for use in diagnostic procedures.
TAT: 1 hour 3 hours 13 hours 2.5 hours 15 hours
Sample
extraction
*Includes an overnight run on Ion Chef System
Acknowledgments
Karen PageDavid GutteryDaniel Fernadez-GarciaRobert HastingsJinli LuoBradley ToghillCaroline CowleyLindsay PrimroseJoan RileyLuke MartinsonMark OpenshawKevin Blighe
Mark RutherfordAlison GoodallJohn Le QuesneSamreen AhmedAnne Thomas
Leicester TRACERxteam
Charles CoombesJustin StebbingSimak AliAllison HillsStephanie MarchesseLaura WoodleyBrenda RosalesKelly GleasonKate GoddardAnna Boydell
Charles Swanton andTRACERx team
Special thanks to