Liquid biopsies: monitoring patients with breast cancer and non-small cell lung cancer Jacqui Shaw, Director Leicester Precision Medicine Institute email: js39@Leicester.ac.uk; LPMI@leicester.ac.uk 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 9 of 54 patients had an ESR1 gene mutation in cfDNA Guttery et al. Clin Chem 2015
Liquid biopsy can help to guide treatment Page et al. Clin Chem 2017
Shared mutations in cfDNA and individual CTCs PIK3CA ESR1 ctDNA p.H1047R p.E380Q NGS ESR1 TP53 cfDNA p.Y537C p.R175H NGS PIK3CA PIK3CA WT WT WT p.H1047R p.H1047R ESR1 ESR1 p.E380Q p.E380Q Individual CTCs Based on a small fraction of cells from patients with high CTC counts, we see heterogeneity in mutations between matched CTCs and cfDNA Shaw et al. Clin Can Res 2017
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
Oncomine TM 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
Detection of local recurrence R 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
Oncomine ™ Breast cfDNA Assay Early breast cancer AKT1 ERBB3 KRAS CCND1 ESR1 PIK3CA EGFR FBXW7 SF3B1 ERBB2 FGFR1 TP53 • 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
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
ESR1 D538G 0.14 0.56 PIK3CA H1047R NRAS G12A 0.14 KRAS G12D 1.1 1.1 1.57 2.35 PIK3CA E545K March Surgery 2.21 2.71 1.24 1404 1438 2009 Nov 2009 April 2010 1512 1732 1757 Jan Aug Oct Nov 2009 Jul 15 Nov 15 May 16 2017 2013 Letrozole Paclitaxel Tamoxifen Anastrazole Blood sample Stable disease Cap no of Letrozole Sample ID Sample date Gene MAF% AA CHg CTCs Pac NRAS 0.14 p.G12A PIK3CA 1.57 p.E545K 1404 68 01/07/2015 PIK3CA 0.56 p.H1047R ESR1 0.14 p.D538G TP53 0.17 p.G244D Developed pleural 1438 99 04/11/2015 PIK3CA 2.35 p.E545K effusion 67 1512 06/05/2016 PIK3CA 2.21 p.E545K PIK3CA 2.71 p.E545K Developed ascites 1732 50 11/10/2017 KRAS 1.10 p.G12D PIK3CA 1.24 p.E545K 1757 91 15/11/2017 KRAS 1.06 p.G12D
Breakout Sensitivity, Standout Customization Introducing Ion AmpliSeq HD Technology: New amplicon-based NGS library preparation technology that enables you to design custom ultrahigh-sensitivity panels for your targets of interest • 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 *For cell-free DNA samples For Research Use Only. Not for use in diagnostic procedures. 15
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. 16
Comparable Performance Between Ion AmpliSeq HD and Oncomine Cell-Free Panels High specificity (PPV) through additional reductions in false positives 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. For Research Use Only. Not for use in diagnostic procedures. 17
Ion AmpliSeq HD Panels: 2-Day NGS Workflow Go from DNA to data in less than 2 days Select targets Construct library Prepare template Run sequence Analyze data • Torrent Suite Software • Ion AmpliSeq HD Library Design a panel • Ion Reporter Software Ion Chef instrument* Ion GeneStudio S5 systems at ampliseq.com Kit • Oncomine Knowledgebase • Ion AmpliSeq HD Dual Reporter Barcode Kit <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 Sample extraction TAT: 1 hour 3 hours 13 hours 2.5 hours 15 hours For Research Use Only. Not for use in diagnostic procedures. *Includes an overnight run on Ion Chef System 18
Acknowledgments Karen Page Mark Rutherford Charles Coombes David Guttery Alison Goodall Justin Stebbing Daniel Fernadez-Garcia John Le Quesne Simak Ali Robert Hastings Samreen Ahmed Allison Hills Jinli Luo Anne Thomas Stephanie Marchesse Bradley Toghill Laura Woodley Caroline Cowley Leicester TRACERx Brenda Rosales Lindsay Primrose team Kelly Gleason Joan Riley Kate Goddard Anna Boydell Luke Martinson Mark Openshaw Kevin Blighe Charles Swanton and TRACERx team
Special thanks to
Recommend
More recommend
