NGS in tissue and liquid biopsy

Transcription

1 NGS in tissue and liquid biopsy Ana Vivancos, PhD Referencias

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3 So, why NGS in the clinics? 2000 Sanger Sequencing (1977-) 2016 NGS (2006-) ABIPrism (Applied Biosystems) Up to 2304 per day (96 sequences per hour) HiSeq X (Illumina) Up to 2 billion sequences per day (6 billion in 3 days) MiSeq (Illumina) Up to 50 million sequences per day

4 TCGA has been possible thanks to NGS 32 tumor types

5 Lessons Learnt from the TCGA Data The long tail of mutations in Cancer PanCancer project, TCGA, Nature Genetics 2013 Van Allen et al., Nature Medicine 20, (2014)

6 Lessons Learnt from the TCGA Data kataegis MSI Alexandrov et al., Signatures of mutational processes in human cancer. Nature 500,

7 Lessons Learnt from the TCGA Data McGranahan et al., Clonal status of actionable driver events and the timing of mutational processes in cancer evolution. Sci Trans Res,2015

8 What can be detected? You name it! Yes, but not all at the same time! Dienstmann et al. J Clin Oncol 2013

9 Modern times: NGS in practice or in research? CLINICAL PRACTICE Amplicon-seq Capture approaches Including Exome-seq Whole genome sequencing Specific Up to 50 regions Mb (200k are probes) multiplex-pcr are sequencing-ready amplified and sequenced. in 1 working day. Allows good intron-exon Quick coverage. and cheap. MUTATIONS & INDELS Allows panels containing hundreds of cancer genes. Up to WES. MUTATIONS, INDELS, (CNA), (FUSIONS), RESEARCH The whole thing. MUTATIONS, INDELS, CNA, REARRANGEMENTS (FUSIONS) TAT, cost, difficulty to interpret results

10 Small gene panels vs Exomes Assessing the clinical utility of cancer genomic and proteomic data across tumor types. Y Yuan et al. Nature Biotechnology 32, (2014)

11 Increasing scope, germline sample is more and more necessary Molecular analysis Patient population T N Tissue Panels WES WGS

12 The sample factor 99% FFPE 1% fresh/frozen tissue 1- TUMOR PURITY 2- DNA QUALITY: FRAGMENTATION AND CHEMICAL DAMAGE 3- AMOUNTS plasma 1- DILUTED ctdna 2- ORGANIZING SERUM EXTRACTION, STORAGE AND ddbb

13 Tumor purity limits sensitivity NGS sensitivity: 5% minimum mutated alleles Mutated allele TUMOR CELLS Mutations may be present in all tumor cells: CLONAL OR May be present in a subset of cells: SUBCLONAL. In some instances, resistance mechanisms ex. EGFR T790M N (2n): stroma, lymphcytes, normal surrounding tissue Some tumor types are stroma enriched, ex. pancreas

14 FFPE DNA & RNA fragmented and chemically damaged 150bp Leading to test failure! GE Fresh/ frozen FFPE Allele frequencies for variants in Amplicon-seq: 80bp cfdna False positives Nt-level damage Average MAF C>T False positives True positives 6% 32% 66% (41/62) 42% (96/228) Average coverage data from 17 independent 150 bplong amplicons (GC=55.7%) vs 30 independent 80-bp (GC=62.5%) per sample.

15 Training the MDs on how to decifer a report Nonsynonymous point mutations: Change in a nucleotide that has an effect at the protein level missense (change of aminoacid) EGFR:NM_005228:exon21:c.2573T>G:p.L858R nonsense (STOP codon introduction) TP53:NM_000546:exon6:c.592G>T:p.E198X Indels: Insertion or deletion of a number of nucleotides that has an effect at the protein level in frame (insertion of aa in the protein) TP53:NM_000546:exon7:c.741_746delCTCCGG:p.N247_R249delinsK frameshifts (normally leading to premature STOP codons and truncated proteins) FGFR3:NM_000142:exon4:c.444_445del:p.T148fs

16 NGS IMPORTANT TIPS CAN T DO EVERYTHING WITH A SINGLE APPLICATION DEPENDING ON WHAT WE WANT TO STUDY, A CAREFUL PLANNING IS REQUIRED, TAKING INTO ACCOUNT: SPECIMEN, SAMPLE PREPARATION, COVERAGE REQUIREMENTS BE AWARE OF LIMITATIONS AND BIASES OF OUR EXPERIMENT

17 Liquid biopsy

18 Now we also need plasma

19 Circulating-free DNA (cfdna) is a naturally occuring DNA that is present in the bloodstream. We can isolate it from the plasmatic fraction of blood. Liquid biopsy and ctdna Total cfdna may be elevated for various reasons: Inflammation Wound healing Malignant lesions Menstruation Sport Circulating-free tumor DNA (ctdna) is a part of cfdna, cell-free DNA released from a solid tumor and, therefore, carries mutations or other genomic alterations. ctdna CTCs cfdna

20 High sensitivity is required for reliable ctdna profiling

21 ctdna prevalence: to shed or not to shed Bettegowda et al, Sci Tran Med Feb 2014

22 The Concordance issue Plasma MU Tumor T ctdna shedding Intratumor heterogeneity Targeted therapies

23 Liquid biopsy as biomarker of response BELLE-2, ctdna better predictor than tissue

24 Digital techniques allow Conventional PCR Digital PCR high sensitivity and Wild-type DNA Mutant DNA Partitioning of DNA into many individual reactions quantitative results Relative signal readout Digital signal readout Vogelstein et al. PNAS 1999; mod. Diehl et al Curr Opin Oncol. 2007

25 Main limitation in the routine application to clinics is that only hotspots in oncogenes can be studied Digital PCR & hotspot mutations for the routine Oncogenes AKT1 Others: BRAF, KRAS, PIK3CA Mutation Hotspot ddpcr Tumor supressors TP53 1 test = 1 mutation Others: PTEN, NF1, VHL indels

26 NGS in plasma yes, but please bear in my mind all the previous Multiplex-PCR Noninvasive diagnosis of actio nable mutations by deep sequ encing of circulating free DNA in lung cancer from neversmokers: a proof-of-concept study from BioCAST/IFCT In-solution capture Bias-corrected targeted nextgeneration sequencing for rapid, multiplexed detection of actionable alterations in cell-free DNA from advanced lung cancer patients. Couraud et al., Clin Cancer Res Paweletz et al., Clin Cancer Res. 2015

27 Monitoring of tumor burden/ minimal residual disease 250 SUBJECTS STAGE II COLON CANCER +/- ADJUVANT CHEMOTHERAPY (CLINICIAN S DISCRETION) REQUIRES FOLLOW-UP OF PATIENT-SPECIFIC CLONAL MUTATION

28 The test may not deliver on desired application, the sample may be challenging Liquid biopsy Tumor shedding and sensitivity Many, many, open questions

29 Thanks!