Liquid biopsies: monitoring patients with breast cancer and - - PowerPoint PPT Presentation

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

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 WT

PIK3CA 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 16 Jul 15 Developed ascites

Cap

Letrozole Jan Aug Nov 15

1438

Blood sample

PIK3CA E545K

1.57 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 11/10/2017 PIK3CA 2.71 p.E545K KRAS 1.10 p.G12D 1757 91 15/11/2017 PIK3CA 1.24 p.E545K KRAS 1.06 p.G12D

0.56 PIK3CA H1047R

NRAS G12A ESR1 D538G

0.14

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 Page David Guttery Daniel Fernadez-Garcia Robert Hastings Jinli Luo Bradley Toghill Caroline Cowley Lindsay Primrose Joan Riley Luke Martinson Mark Openshaw Kevin Blighe Mark Rutherford Alison Goodall John Le Quesne Samreen Ahmed Anne Thomas Leicester TRACERx team Charles Coombes Justin Stebbing Simak Ali Allison Hills Stephanie Marchesse Laura Woodley Brenda Rosales Kelly Gleason Kate Goddard Anna Boydell Charles Swanton and TRACERx team

Special thanks to