Dr David Guttery Senior PDRA Dept. of Cancer Studies and CRUK Leicester Centre University of Leicester

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1 Dr David Guttery Senior PDRA Dept. of Cancer Studies and CRUK Leicester Centre University of Leicester

2 CFDNA/CTDNA Circulating-free AS A LIQUID DNA BIOPSY (cfdna) Tumour Biopsy Liquid Biopsy subclone 1 subclone 2 subclone 3 tumour DNA Healthy and tumour-derived cfdna Snapshot in space and time Invasive More representative of ITH? Minimally-invasive Longitudinal sampling

3 Robinson et al. Nat. Gen Toy et al. Nat. Gen. 2013

4 Can ESR1 mutations be detected in the blood? Are ESR1 mutations acquired during therapy?

5 CAN ESR1 MUTATIONS BE DETECTED IN THE BLOOD? 9 of 54 patients acquired a mutation in ESR1 Guttery et al. Clin Chem (2015)

6 ARE ESR1 MUTATIONS ACQUIRED DURING THERAPY? CTC count Letrozole, zolendronic acid Exemestane, everolimus RIP G3 IDC, DCIS, bone, axillary and pulmonary mets Guttery et al. (2015) Clin Chem Axillary progression Mets in skull, retina & jaw bone, radiotherapy

7 CONCURRENT DETECTION OF MUTATIONS AND CNV Gene TP53 ERBB2 KDELC2 PIK3CA MYC STK11 GATA3 CCND1 LMTK3 ESR1 FGFR1 NOMO2 PGR FGFR2 USP17L2 CNTNAP1 Page et al. under review

8 Can CNV be detected by targeted NGS? Can CNV be detected in the blood using targeted NGS? Is this clinically relevant?

9 CAN CNV BE DETECTED IN THE BLOOD? 42 baseline samples 9 healthy controls 50% had SNV/CNV ESR1 mutations correlated with poorer survival Page et al. under review

10 IS CNV CLINICALLY RELEVANT? Patient ID HER2 status of primary tumor Amplification Gene alteration in cfdna Mutation Disease status at time of blood sample 10 Positive ERBB2 - Progressing 40 Positive ERBB2 - Progressing 13 Positive ERBB2 and NOMO2 TP53 (p.i251s) Progressing 1 Positive ERBB2 and NOMO2 PIK3CA (p.h1047r) Progressing 20 Positive - - Progressing 2 Negative ERBB2 32 Negative ERBB2 and NOMO2 PIK3CA (p.h1047r); TP53 (p.r248q) PIK3CA (p.h1047r); TP53 (p. R248Q) Progressing Progressing 8 Negative ERBB2 and MYC** - Progressing 15 Positive - - Progressing 36 Positive - - Stable 5 Positive - - Stable 21 Positive - PIK3CA (p.h1047r) Stable 26 Negative* - NOMO2 (p.r418h) Stable *HER2 positive met biopsy, **detected in serial sample Page et al. under review

11 CTCS Yu et al. (2011) JCB

12 CTCS AND PROGNOSIS Cristofanilli et al. (2004) NEJM de Bono et al. (2008) Clin. Can. Res.

13 RECENT ADVANCES IN SINGLE CTC ANALYSIS Hodgkinson et al. (2014) Nat. Med. Polzer et al. (2014) EMBO Mol. Med.

14 RECENT DEVELOPMENTS IN CFDNA AND CTC SEQUENCING Strauss et al. (2016) Oncotarget

15 POOLED CTCS VS. CFDNA Guttery et al. (2015) Clin Chem

16 COMPARISON BETWEEN CFDNA AND SINGLE CTCS Shaw et al. (2016) Clin. Can. Res.

17 ANALYSIS OF SINGLE CTCS Blood draw 7.5 ml K2 EDTA tubes CTC isolation and staining CellSearch Single cell sorting DEParray TM Whole genome amplification Ampli1 TM WGA quality control Genome Integrity Index (GII) Targeted NGS Ampli1 and custom NGS panels

18 TARGETED NGS PANELS AcquiRes 4 genes, 30 amplicons ERBB2 (9) PIK3CA (6) ESR1 (7) TP53 (8) Ampli1 315 amplicons, 50 genes, 5 regions for CNV ABL1 FGFR3 PIK3CA (25) AKT1 FLT3 PTEN ALK GNA11 PTPN11 APC GNAS RB1 AR HNF1A RET ATM IDH1 SMAD4 BRAF IDH2 SMARCB1 CDH1 JAK3 SMO CDKN2A KDR SRC CSF1R KIT STK11 CTNNB1 KRAS TP53 (8) DDR2 MAP2K1 VHL EGFR MET 7q31 ERBB2 (4) MLH1 8p12 ERBB4 MPL 10q26 EZH2 MYC 11q13 FBXW7 NOTCH1 17q12 FGFR1 NRAS FGFR2 PDGFRA Jeselsohn et al. (2015) Nat. Rev. Clin. Oncol Bose et al. (2013) Cancer Discovery

19 CTC HETEROGENEITY MIRRORED IN MATCHING CFDNA Shaw et al. (2016) Clin. Can. Res.

20 SUMMARY Detected SNV and CNV in the blood using targeted NGS Gene amplification and mutations can be acquired during treatment cfdna reflects CTC heterogeneity but CTC analysis may provide important information about therapy and response Should blood monitoring be requisite during treatment?

21 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

22 ONCOMINE TM LUNG CFDNA EARLY ACCESS Horizon Multiplex cfdna reference 5%, 1%, 0.1%, 0% dilutions Consistent detection down to 0.1% VAF

23 ACKNOWLEDGEMENTS Professor Jacqui Shaw Dr Karen Page Dr Daniel Fernandez-Garcia Lindsay Primrose Joan Riley Vilas Mistry Professor Charles Coombes Professor Justin Stebbing Professor Simak Ali Allison Hills Olivia Ogle Brenda Rosales Vedia Shahin Kate Goddard

24 Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.