Liquid Biopsy. Jesus Garcia-Foncillas MD PhD. Director

Liquid Biopsy Jesus Garcia-Foncillas MD PhD Director

Main issues about liquid biopsies New paradigm: Precision Medicine Heterogeneity & Dynamics Surrogate mirror for the tumor CTCs in colon cancer ctdna: Technical approaches Heterogeneity & Dynamics Disease course Therapy monitoring

Precision Oncology: Genomics-driven cancer medicine

Enumeration Immunofluorescence Enumeration Immunofluorescence Fluorescence in situ hybridization Next Generation Sequencing Mutation Detection Gene Expression

%Probability of Survival Predictive Value: OS of MCRC Patients with <3 or >3 CTC at Baseline (N=413) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 9.4 Months CTC / 7.5mL Median OS in at Baseline N (%) Months (95% C.I.) <3 CTC 305 (74%) 18.5 (15.5 to 21.2) >3 CTC 108 (26%) 9.4 ( 7.5 to 11.6) Logrank p < 0.0001 Cox Hazard Ratio = 2.5 chi-square = 31.48 (p-value < 0.0001) 18.5 Months 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time from Baseline Blood Draw (Months)

A Reduction in CTC Below 3 After the Initiation of Therapy Predicts Longer OS whereas an Increase in CTC Count to 3 or above Predicts a Shorter OS 100% 90% Median OS in Group Description N (%) Months (95% C.I.) 1 <3 CTC at All Draws 303 (70%) 18.6 (15.9 to 22.5) 2 >3 CTC at BL & <3 CTC at Last Draw 74 (17%) 11.7 ( 9.4 to 18.7) 3 <3 CTC at Early Draw & >3 CTC at Last Draw 29 ( 7%) 7.1 ( 6.3 to 10.8) 4 >3 CTC at All Draws 24 ( 6%) 3.9 ( 2.5 to 5.4) %Probability of Survival 80% 70% 60% 50% 40% 30% 20% Curve Logrank Comparison p-value* 1 vs. 2 0.0007 1 vs. 3 <0.0001 1 vs. 4 <0.0001 2 vs. 3 0.0078 2 vs. 4 <0.0001 3 vs. 4 0.0001 2 10% 1 0% 4 3 *p-values not adjusted for multiple hypothesis tests *p-values not adjusted for multiple hypothesis tests 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time from Baseline Blood Draw (Months)

Wild-type signal BEAMing Pre- Amplification Emulsion PCR Hybridization Flow Cytometry Flow cytometry analysis Wild-type DNA Mutant & Wild-type DNA Mutant DNA Mutant signal

Digital PCR

Cell-free tumor DNA in stage IV (n = 177)

Cell-free tumor DNA in stage IV Localized vs Metastatic Disease (n = 223) Bettegowda et al, Sci Tran Med Feb 2014

ctdna: Monitoring of tumor burden 162 plasma samples from 18 pts (CRC pts) Cell-free tumor DNA No detectable mutations in plasma (n = 4) p=0.0006 Detectable mutations in plasma (n = 14) CEA Detectable CEA (n = 8) p=0.03 No detectable CEA (normal range boundary) (n = 10) Diehl et at. Nat Med. 2008

ctdna: monitoring response Baseline Imaging 53 mcrc (ctdna vs CEA and tumor burden) 8 weeks later Courtesy of: Jeanne Tie, Peter Gibbs: Ann Oncol 2015