Lukas Bubendorf Pathologie. Liquid biopsies
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1 Lukas Bubendorf Pathologie Liquid biopsies
2 Liquid biopsies 1. Circulating cell-free tumor-dna (ctdna) 2. Circulating tumor cells (CTC) Source: Sysmex
3 CTCs ctdna ctrna exosomes Quantification Protein RNA DNA analysis Drug testing in vitro Drug testing in vivo (Xenograft) Quantification Somatic mutations Copy number variations (array CGH, NGS ) Modified from Hegemann et al, BJU Int 2016
4 Applications of Liquid biopsies Minimally invasive biomarker analysis Prognosis (ctdna/ctcs) Minimal residual disease after surgery to select for adjuvant treatment Monitoring during/after treatment early detection of recurrence / resistance Detection of resistance mutations
5 Liquid biopsies 1. Circulating cell-free tumor-dna (ctdna) 2. Circulating tumor cells (CTC) Source: Sysmex
6 diagnosis 1 st line therapy minimal residual disease relapse Targeted therapy resistance ct-dna level Clinical relevance of ctdna monitoring relapse and resistance t
7 Special aspects of ctdna Low proportion of whole DNA (<1% - >90%) Highly sensitive technologies needed Sensitivity depends on tumor stage/load Plasma better suited than serum Short half-life in the blood ( hrs) ctdna represents all tumor sites in the body
8 Plasma is superior to serum Tumor Plasma Serum AF (%) KRAS:G12C EGFR:indel KRAS:G12A Patient 09 Patient 11 Patient 21 0 N = 3 In line with: El Messaoudi et al, Clini Chim Acta 2013; Vallée A et al; Lung Cancer 2013
9 Tubes for blood collection (ctdna) Tube Blood Collection Tubes («BCT» Streck) PAXgene (Qiagen) K2-EDTA Time to plasma preparation Manufacturer data 14 days 7 days in-house data Up to 3 days (optimal) Up to 5 days (maximum) 7 days 1 hour Observations increased hemolysis compared to Streck Price per tube 8 CHF 12 CHF 0.30 CHF IVD yes no yes All blood samples for cfdna should be shipped and stored on room temperature until plasma preparation Plasma should be long term stored at -80 C to prevent degradation of cfdna
10 Commercial kits for extracting cfdna Thermo Fisher Qiagen Promega MagMax cfdna Kit CNA Kit QIAsymphony Maxwell ccfdna Plasma Kit Manual, beads Manual, column Automated, beads Automated, beads cfdna 72% 56% Recovery ca. 25% 40% Hands-on time Price per sample 1-2h 1-2h 20min 20min 9 CHF 25 CHF ca. 30 CHF 16 CHF Difficulties in downstream NGS observed «gold standard» Currently only customized kits available Hands-on time increases if plasma volume > 1ml
11 Technologies for ctdna detection Principle PCR-based, e.g. qpcr Type of alteration Known point mutations Average Sensitivity (AF) Advantages Limitations >1% Ease of use Limited number of genomic loci Digital PCR Known point mutations, rearrangements 1% % depending on probe High sensitivity limited genomic loci (1 sample: max 10 loci) Targeted deep sequencing (Liquid biopsy NGS panels) Selected SNV, CNV rearrangements >1% rel. inexpensive Difficult for low frequency variants Adapted from Qin et al, Chin J Cancer 2016
12 Digital droplet PCR (ddpcr) Droplet generation High sensitivity Precise quantification
13 Common techniques and platforms for ctdna analysis Commercial solutions NGS IonTorrent Oncomine Panels (Thermo Fischer) Lung- and breast cancer cfdna Panel. Each investigates >150 hotspots, sensitivity 1% ddpcr PrimePCR Mutation Assay (BioRad) Probes available for EGFR T790M, L858R, BRAF, PIK3CA, Multiplex KRAS G12X assay dpcr QuantStudio Assays (Thermo Fisher) High inter-sample variability, high hands-on workload, rather expensive qpcr Therascreen EGFR Plasma RGQ PCR Kit (Qiagen) EGFR T790M and L858R and EGFR deletion detection in one reaction ARMS PCR with Scorpion probes (sensitivity ca. 1%) qpcr Cobas (Roche) 42 mutations in exons 18-21, including L858R, exon 19 deletions, L861Q and T790M. ARMS PCR with TaqMan probes (sensitivity in plasma 100 copies/ml; FFPE 5% AF) Laboratory developed tests (LDTs) NGS panels (LDT) dpcr (LDT)
14 Diagnostic workflow in pathology liquid biopsy workflow DNA isolation DNA sequencing Quantification (AF) FFPE EDTA plasma iontorrent TM PGM / Proton QX200 TM Droplet Digital TM PCR System therascreen EGFR Rotor-Gene Qiagen >2% 0.1% 0.8% % allelic frequency detection limit
15 Testing for the sensitivity of NGS and ddpcr for mutation detection Standards : EGFR T790M (0.1%, 1%, 5%, WT) Standard Expected allelic freq. NGS ddpcr EGFR Therascreen wt negative 0.1% negative 1% negative 5% positive Feb 2016: 1 st CE IVD approved assay
16 Clinical applications of ctdna analysis Prognosis Presence / amount of ctdna Monitoring during treatment Early detection of residual disease or progression Predictive marker testing Instead of biopsy in frail, untreated patients At the time of resistance ( resistance mutation)
17 Post-operative detection of residual disease Example: pancreatic adenocarcinoma (ctdna; dpcr) Pre-OP Post-OP (n=22) (n=29) (n=10) (n=10) Translatable to other cancer types (z.b. bladder, lung)? Hypothesis: post-op ctdna positive = high risk adjuvant treatment Sausen M et al, Nat Comm 2015
18 Clinical applications of ctdna analysis Prognosis Presence / amount of ctdna Monitoring during treatment Early detection of residual disease or progression Predictive marker testing Instead of biopsy in frail, untreated patients At the time of resistance ( resistance mutation)
19 New Engl J Med 2015 n=138 n=62 Osimertinib Partial of full remission in approx. 60% of the patients Approved for T790M mutated NSCLC
20 qpcr (cobas) vs. digital PCR (BEAMing) EGFR mutation detection in plasma ctdna Exon 19 deletion Sensitivity Specificity L858R Sensitivity Specificity T790M Sensitivity Specificity Cobas EGFR Mutation Test 82% (23/28) 97% (30/31) 87% (20/23) 97% (35/36) 73% (30/41) 67% (16/24) BEAMing dpcr 82% (23/28) 97% (30/31) 87% (20/23) 97% (35/36) 81% (33/41) 58% (14/24) FDA approval of cobas system Thress KS, Lung Cancer 2016
21 Mutation alleles/ml of Plasma Plasma ddpcr EGFRex19 del EGFR L858R EGFR T790M KRAS G12X pos neg pos neg pos neg pos neg Tissue Genotype Prospective study (n=180 pts; 120 newly diagnosed; 60 resistance) Median TAT: 3 days Sensitivity: 74-82% for EGFR & 64% for KRAS PPV: 100% for EGFR Ex19/21 but 79% for T790M Sacher AG, JAMA Oncology 2016
22 Plasma NGS (Hot Spot Panel) NGS of ctdna from untreated melanoma & lung cancer patients (Ion Ampliseq Cancer Hot Spot Panel) Routine NHS diagnostic service Melanoma Kaisaki PJ et al, Plos One2016
23 NGS/ddPCR for detection of T790M in ctdna (The Basel experience) Percentage of patients (%) Allelic Frequency (%) Mutations (n=35) EGFR T790M testing (n=23) T790M pos T790M neg EGFR T790M Driver Mutations Allelic frequencies of resistance and oncogenic driver mutations in blood plasma
24 First patient T790M in ctdna (Basel) Months Therapy Tissue Biopsy
25 Liquid Biopsy (zellfreie DNA)
26 Liquid biopsies 1. Circulating cell-free tumor DNA (ct-dna) 2. Circulating Tumor Cells (CTC) Rare (1:10⁶ nucleated cells) Advanced tumors Prognosis Prediction: AR-V7 Source: Sysmex
27 Technologies for CTC detection Alix-Panabières C et al, Nat Rev Cancer, 2014
28 CTCs as prognostic marker (CellSearch system) CTC / 7.5ml blood FDA approved Kiki C et al, Mol Oncol 2015
29 Antonarakis et al, New Engl J Med 2014
30 Detection of AR-V7 in Plasma-derived exosomal RNA by ddpcr AR-V7 negative AR-V7 n=22 wt AR n=14 AR-V7 positive Del Re M et al Eur Urol 2016, Epub
31 Summary Liquid biopsies for detection of (resistance) mutations has entered clinical practice (T790M) No general gold standard for ctdna testing further validation as a continuing process. ddpcr/ngs are particularly promising. Re-biopsy remains an option. CTC analysis is a developing field (AR-V7 testing in advanced PrCA)
32 Liquid biopsies in SAKK clinical trials SAKK 08/14 (open; 84 patients) Castration-resistant prostate cancer: enzalutamide vs. enzalutamide & metformin prostate mutation panel (ctdna) & AR-V7 (CTCs) SAKK 63/12 (opening in 2017; 300 patients) Advanced PrCA (biobanking study) prostate mutation panel (ctdna) & AR-V7 (CTCs)
33 Christian Ruiz Luca Quagliata Luigi Terracciano Spasenija Savic Michel Bihl Novartis Astra Zeneca Ivana Bratic Hench Martin Schöbel Thank you
34 Typical plasma response patterns Sacher AG, JAMA Oncology 2016
35 Ignatiadis M et al, Clin Cancer Res 2015
36
37 Thress KS, Lung Cancer 2016
38 Physical capture of tumor cell clusters (microfluidic device) Sarioglu AF et al, Nat Methods 2015;12:685
39 Number of mutations by tumor type tumor-type specific gene panels Vogelstein B, Science 2013
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