Role of liquid biopsies and circulating tumor DNA. Pierre Laurent-Puig European Georges Pompidou Hospital Paris Descartes University
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1 Role of liquid biopsies and circulating tumor DNA Pierre Laurent-Puig European Georges Pompidou Hospital Paris Descartes University
2 DISCLOSURE AMGEN ASTRAZENECA BIOCARTIS BOERINGHER INGELHEIM INTEGRAGEN MERCK SERONO RAINDANCE TECHNOLOGIES NOT yet BIORAD ROCHE SANOFI
3 Detection of circulating tumor DNA By searching for the genetic or epigenetic alterations which characterize the DNA coming from tumor cells Tumor specific mutations Tumor specific structural changes (i.e. rearrangement) Copy Number Variations MicroRNA Methylated DNA Zonta et al. Advances in Clinical Chemistry, Volume 70, 2015, Pages
4 Why detection of ctdna is challenging? Circulating tumor DNA is mixed with circulating normal DNA The detection of circulating tumor DNA and its validation as a biomarker of cancer have long been limited due to inefficient methods of detection Zonta et al. Advances in Clinical Chemistry, Volume 70, 2015, Pages
5 Allele frequency measured by NGS Allele frequency measured by NGS Allele frequency measured by NGS Proportion of advanced cancer patients with less than 2% of ctdna in plasma mutations from 273 plasma samples 69 mutations from 100 plasma samples mutations from 293 plasma samples % lung cancer % pancreatic cancer % colon cancer Adapted from Pecuchet N, Laurent-Puig in Clin Chem 2016
6 See Williams, R. et al. Nature Methods Massively digital PCR in emulsion Droplet-based digital PCR can overcome PCR-based procedure limitations.
7 Digital Droplet PCR Sensitivity demonstrated for 1 mutant sequence in ~200,000 wild type sequences Quantitative procedure and relatively simple to set up 100 µm Pekin D. et al., Quantitative and sensitive detection of rare mutations using droplet-based microfluidics, Lab on Chip, 2011; 11: Taly V. et al., Detecting biomarkers with microdroplet technology,trends in Molecular Medicine, 2012;18:
8 Somatic mutation detection for ctdna monitoring? The follow-up is linked to our ability to detect specific mutation(s) within the tumor The example of colon cancer 45.1% 48% 54.8% Patient follow up will benefit from unique assays allowing to screen as much patients as possible! 1 see cbioportal 2 Laurent-puig, Taly et al., unpublished data 30 assays
9
10 Massively parallel sequencing Different methods have been used to improve sensitivity of NGS CAPP seq, Safe Seq BPER method Circle sequencing All these methods tend to improve sensitivity of detection by differentiating true mutation to background noise Less than 0.1%
11 First question : is liquid biopsy ready for colorectal cancer screening? THE ANSWER IS NO BUT..
12 Circulating Tumor DNA according to Tumor stage Adapted from Garrigou S et al. Clin Chem 2016;62
13 SEPT9 methylation as a marker of circulating tumor DNA 7941 asymptomatic individuals >50 years old 53 cases of colorectal cancers Sensitivity of the SEPT9 test for CRC : 48.2% CI95% 32.4%-63.6% Stage I 35%, Stage II 63%, Stage III 46%, Stage IV 77.4% Specificity of the SEPT9 plasma detection 91.5% CI95% 89.7%-93.1% Sensitivity for advanced adenoma was low 11.2% Based on this study FDA approved the test in April 2016 with this labelling «The epi procolon test is indicated to screen adults of either sex, 50 years or older, defined as average risk for crc, who have been offered and have a history of not completing crc screening. The Epi procolon test is not intended to replace colorectal cancer screening tests that are recommended by appropriate guidelines» Church TR et al. Gut 2014;63:
14 Second question : Is the liquid biopsy ready to search for the RAS gene mutations?
15 KRAS detection : plasma versus tumor Sensitivity Specificty and Accurracy Authors Journal Detection Methods N Sensitivity Specificity Accurracy Yen Clin Cancer Res 2009 Membrane arrays KRAS codon % 95.3% 90% Thierry Nature Med 2014 Intplex system KRAS 12 & 13 7 frequent mutations 95 92% 98% 96% Bettegowda Sci Transl Med 2014 SafeSeqS KRAS 12 & % 99.9% 95%
16 Second question : Is the liquid biopsy ready to search for the RAS gene mutations? THE ANSWER IS PROBABLY YES waiting for prospective series in all RAS mutation
17 Second question : Is the liquid biopsy ready to search for the RAS gene mutations? THE ANSWER IS PROBABLY YES waiting for prospective series in all RAS mutation So we do it
18 RASANC study Prospective biospecimen collection study in previously untreated advanced mcrc (NCT ) AGEO French collaborative study Plasma collected in Streck tubes Plasma NGS performed using established panel Bachet et al. Submitted; ASCO 2017 Poster discussion
19 BPER METHOD Primary analysis Presence of a mutation in 1 of the 22 tested genes Absence of a mutation in 1 of the 22 tested genes Presence of ctdna RAS mutated RAS non mutated Bachet et al. Submitted; ASCO 2017 Poster discussion
20 Presence of a mutation in 1 of the 22 tested genes BPER METHOD Primary analysis Absence of a mutation in 1 of the 22 tested genes RAS mutation in tumor Absence No. Presence No. Total No. RAS mutation in plasma sample Absence No. 167 (41%) 58 (14%) 225 (55%) Presence No. 3 (1%) 184 (45%) 187 (45%) Total No. 170 (41%) 242 (59%) 412 (100%) Presence of ctdna RAS mutated N=187 (45.4%) RAS non mutated N=225 (56.4%) Bachet et al. Submitted; ASCO 2017 Poster discussion
21 Secondary analysis Presence of a mutation in 1 of the 22 tested genes BPER METHOD Absence of a mutation in 1 of the 22 tested genes RAS mutation in tumor Absence No. Presence No. RAS mutation in plasma sample Absence No. 128 (39%) 14 (4%) Presence No. 3 (1%) Total No. 131 (40%) 184 (56%) 198 (60%) Total No. 142(43%) 187 (45%) 329 (100%) Presence of ctdna Digital PCR for detecting methylation universal biomarkers WIF1 & NPY No detection of methylation RAS mutated N=187 (45.4%) RAS non mutated N=142(43%) Inconclusive results n=83 (20.1%) Bachet et al. Submitted; ASCO 2017 Poster discussion
22 Secondary analysis Presence of a mutation in 1 of the 22 tested genes BPER METHOD Absence of a mutation in 1 of the 22 tested genes RAS mutation in tumor Absence No. Presence No. RAS mutation in plasma sample Absence No. 128 (39%) 14 (4%) Presence No. 3 (1%) Total No. 131 (40%) 184 (56%) 198 (60%) Total No. 142(43%) 187 (45%) 329 (100%) Presence of ctdna Digital PCR for detecting methylation universal biomarkers WIF1 & NPY No detection of methylation RAS mutated N=187 (45.4%) RAS non mutated N=225 (56.4%) Inconclusive results n=83 (20.1%) Bachet et al. Submitted; ASCO 2017 Poster discussion
23 Bachet et al: RASANC study Tumor DNA shed is related to a range of clinical and biologic factors Thus, this is a tool that is most effective when used in the right patients Bachet et al. Submitted; ASCO 2017 Poster discussion
24 Bachet et al: RASANC study Tumor DNA shed is related to a range of clinical and biologic factors Thus, this is a tool that is most effective when used in the right patients Bachet et al. Submitted; ASCO 2017 Poster discussion
25 Third question: Is the liquid biopsy a prognostic marker for colorectal cancer in early and advanced stage?
26 Relapse free survival in stage II patients n=250 patients Published by AAAS Tie et al., Sci Transl Med 2016;8:346ra92
27 The [ctdna] is associated with tumor burden 53 patients receiving standard chemotherapy in first-line Tie J, Ann Oncol 2015
28 mcrc patients (n=82) First-line (n=68) or second-line (n=14) chemotherapy regimen Figure 1 Mutation analysis in tumor Identification of targetable mutation* in the tumor n=43 (52.4%) No targetable mutation* identified in the tumor n=39 (47.6%) Alternative strategy based on the hypermethylation detection of WIF1 or NPY Testing plasmatic DNA at baseline for a targetable mutation n= 36/43 positive at baseline (83.7%) Testing plasmatic DNA at baseline for WIF1 and NPY hypermethylation n=27/39 positive at baseline (69.2%) Garlan et al. Clin Can Res 2017 Circulating tumor DNA detected at baseline n=63 (76.8%)
29 Garlan et al. Clin Can Res 2017 [ctdna] at baseline
30 [ctdna] variation during the follow-up is a prognostic indicator of PFS and 0S Decreased ctdna 0.1ng/mL N=40 Decreased ctdna but >0.1ng/mL N=26 Increased ctdna N=7 0 C 1or2 0 C 1or2 0 C 1or2 55% 36% 9% Garlan et al. Clin Can Res 2017
31 [ctdna] variation during the follow-up is a prognostic indicator of PFS and 0S Garlan et al. Clin Can Res 2017
32 [ctdna] variation during the follow-up is a prognostic indicator of PFS and 0S Decreased ctdna 0.1ng/mL N=40 Decreased ctdna but >0.1ng/mL N=26 Increased ctdna N=7 0 C 1or2 0 C 1or2 0 C 1or2 55% 36% 9% Garlan et al. Clin Can Res 2017
33 [ctdna] variation during the follow-up is a prognostic indicator of PFS A B Garlan et al. Clin Can Res 2017
34 [ctdna] variation during the follow-up is a prognostic indicator of PFS and 0S A B Garlan et al. Clin Cancer Res in revision
35 Conclusions Improvement of methods for detecting ctdna allows them to be used in a clinical setting The presence of ctdna in early stage cancer after surgery is an indicator of early recurrence Quantification of ctdna is a probably a surrogate marker of tumor burden and therefore a prognostic marker in advanced stage Evolution of ctdna concentration under treatment is likely an early indicator of chemotherapy efficacy Finally, ctdna allows the identification of resistance mutation and could be a decision-making tool
36 Thanks to... Fundings- Supports Institut National pour la recherche sur le Cancer (INCa) Association pour la Recherche contre le Cancer (ARC) Région Alsace Université de Strasbourg Centre National de la Recherche Scientifique (CNRS) Institut National de la santé et la recherche médicale (INSERM) Université Paris-Descartes Hopital Européen Georges Pompidou (hegp) Agence Nationale de la recherche (ANR) UMRS-1147/ UdS V. Taly JB Bachet L. Benhaim Dr H. Blons Dr V. Boige A. Didelot Dr E. Fabre F. Garlan S. Garrigou T. Hor D. Le Corre Dr P. Nizard C. Normand Dr N. Pecuchet RainDance Technologies Dr D. Link Dr B. Hutchison J. Olson S. Kotsopoulos Q. Zhong I. Atochin Dr D. Pekin Dr K. Perez Toralla Dr Y. Skhiri Dr A. Zaanan Dr E. Zonta Oncologists, pathologists and biostaticians Prof. O. Bouché R. Niarra Dr. B. Landi P. Aucouturier Prof. JF. Emile G. Chatellier Dr F. Bibaut Dr Y. Rozenholc X. Li A. Corner C. Milbury M. Samuels
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