Qué es la Biopsia Líquida? Circulantes y Ácidos Nucleicos Circulantes. Federico Rojo
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1 Qué es la Biopsia Líquida? Células Tumorales Circulantes y Ácidos Nucleicos Circulantes Federico Rojo
2 Liquid Biopsy in Cancer
3 Liquid Biopsy in Cancer Publication Date: April 5, 2016 Blood Tests replace Surgical Biopsiesto create anew multibillion dollar market opportunity...
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5 Liquid Biopsy in Cancer Analysis of therapeutic targets and drug resistance conferring gene mutations on peripheral blood samples: Estimation of risk for metastatic relapse or progression Understanding metastatic development Prediction of targeted therapy response Monitoring(minimal residual) disease Tracking secondary( acquired ) resistance Assessing intratumor heterogeneity Tumor Targets CTCs cfdna cfrna Platelets Exosomes Origins Selectedviable tumor cells leaving actively primary and/or metastasis Necroticand apoptotic tumor cells Necroticand apoptotictumor cells Active incorporationof exosomes Active secretionof encapsulated particlesbytumor cells Definition Tumor cells Fragmented genomes released from dying tumor cells of primary and/or metastasis FragmentedRNA released from dying tumor cells Circulating platelets Circulating encapsulated particles Analytes DNA, RNA (mrna, mirna), protein DNA RNA RNA (mrna, mirna) RNA (mrna, mirna), protein
6 FirstDescriptionsof CirculatingTumor Cells Ashworth, T. R (1869). "A case of cancer in which cells similar to those in the tumourswere seen in the blood after death". Australian Medical Journal 14: Steven Paget, S. (1889). Distribution of secondary growths in cancerof thebreast. Seedans soiltheoryof cancer. The Lancet 1:99-101
7 CirculatingTumor Cells: fromprimarytumor todistantorgans ChafferCL et al.science2011
8 CirculatingTumor Cells: Mesenchimalphenotypein epithelialcells
9 CirculatingTumor CellClusters
10 CirculatingTumor CellClusters
11 FirstDescriptionsof CirculatingFree DNA
12 FirstDescriptionsof CirculatingTumor DNA
13 Prenatal diagnosis bycirculatingfetal DNA
14 Clinicaluse of CirculatingTumor DNA
15 Exercisephysiology, inflammationand other conditionsare monitorizedbycirculatingdna
16 Liquid Biopsy and Cell-Free Tumor DNA Cellfree tumor DNA releasedfroma solid tumor cfdna CTCs Origin: Necroticorapoptotictumor cells; active secretion Small DNA fragments( bp) Halflife~2h Clearance by kidney Healthy individual: 3,000 5,000 Genomic Equivalents/ml Cancer: >10,000 GenomicEquivalents/ml Concentration: 0.01% to60% of total DNA Total DNA elevatedforvariousreasons: inflammation, wound healing, malignant lesions, menstruation, sport
17 Survey of ctdnain Advanced Human Cancer Cases withdetectable cdna, StageIV Cases withdetectable cdna, localized vs metastatic Bettegowdaet al, SciTran Med Feb 2014
18 Cerebrospinal fluid derived ctdnabetter represents genomic alterations than plasma in brain metastasis De Mattos-Arruda, L et al. NatComm2015
19 Biological limitations for Precision Oncology Molecular heterogeneity in cancer Tumor plasticity and dynamic adaptativemolecular phenotype Tumor representation in tissue samples Feasibility of disseminated disease for re-biopsy Low prevalence of targetable mutations in certain tumor types
20 Biological limitations for molecular testing: Tumourspatial and temporal molecular heterogeneity
21 Biological limitations for molecular testing: Tumourspatial and temporal molecular heterogeneity
22 Circumvent biological limitations of molecular testing by circulating biomarkers: Liquid biopsy Circulating tumor DNA and CTCs might capture tumor spatialand temporal molecular heterogeneity Primary tumor Metastasis CTCs ctdna
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25 Diagnostic applications of blood-based molecular testing Early Detection Diagnosis Prognosis Assessment of Molecular Heterogeneity Monitoring Response Response Prediction Monitoring Resistance Monitoring of Minimal Residual Disease T790M-mutant ctdna
26 Diagnostic applications of blood-based molecular testing Early Detection Diagnosis Prognosis Assessment of Molecular Heterogeneity Monitoring Response Response Prediction Monitoring Resistance Monitoring of Minimal Residual Disease Tumor Load Digital PCR Surgery Therapy Threshold of diagnosis
27 Diagnostic applications of blood-based molecular testing Early Detection Diagnosis Prognosis Assessment of Molecular Heterogeneity Monitoring Response Response Prediction Monitoring Resistance Monitoring of Minimal Residual Disease Tumor Load Digital PCR Surgery Therapy Threshold of diagnosis
28 KRAS/NRAS Panel for Response in mcrc: Opus and Crystal Study ASCO 2014 For metastasized colon cancer patients, clinical validation of the RAS panel OPUS (WT treated with Cetuximaband FOLFOX, N=179) -BEAMingrevealed RAS mutations in additional 26%of patients, previously classified as KRAS Exon2 WT -OS and PFS of the patient cohort with additional mutations in RAS was lower than of the patient cohort of mutated codon2 patients treated with CT only, however not statistically significant as patient cohort was too small. Crystal (WT treated with Cetuximaband Folfiri, N=666) -BEAMingrevealed RAS mutations in an additional 14.7%(63/470) of patients, previously classified as KRAS Exon2 WT KRAS Exon3 3,3% KRAS Exon4 5,6% NRAS Exon3 2,9% NRAS Exon3 2,8% NRAS Exon3 0,9% -In the 1st-line treatment of mcrc, patients with RAS wild type tumors derived a marked benefit from the addition of cetuximab to FOLFIRI -Patients with RAS tumor mutations (KRAS exon2 + new RAS) no benefit from the addition of cetuximab to FOLFIRI was apparent Ciardello, F et al. ASCO 2014 Bokemeyer, C et al. ASCO 2014
29 Diagnostic applications of blood-based molecular testing Early Detection Diagnosis Prognosis Assessment of Molecular Heterogeneity Monitoring Response Response Prediction Monitoring Resistance Monitoring of Minimal Residual Disease Tumor Load Digital PCR Surgery Therapy Threshold of diagnosis
30 Monitoring Response: Residual Disease and Tumor Burden N= 162 plasma samples (18 patients) Cell-Free Tumor DNA Levels Before and After Surgery Before surgery Day of Surgery After surgery Day 3 After surgery Day 244 Normal signal Residual Mutant cfdna Diehl et al. Nature Medicine 2008 Mutant signal
31 Monitoring Response: Residual Disease and Tumor Burden Cell free mutant tumor DNA ( Copies / 2 ml plasma) Diehl et al. Nature Medicine 2008
32 Diagnostic applications of blood-based molecular testing Early Detection Diagnosis Prognosis Assessment of Molecular Heterogeneity Monitoring Response Response Prediction Monitoring Resistance Monitoring of Minimal Residual Disease Tumor Load Digital PCR Surgery Therapy Threshold of diagnosis
33 Resistance Monitoring during Treatment: Emergence of KRAS mutations and acquired resistance to anti-egfr therapy in colorectal cancer TISSUE PLASMA Bettegowdaet al, SciTran Med Feb 2014 Misale et al. Nature 2012 Diaz et al. Nature 2012
34 Cancer Is A Disease Of The Genome Carcinogens Virus and infection Radiation Mistakes in DNA copying If we precisely define the cancer genome, we will understand and cure cancer Founder mutations -first genomic mutation These are often lesions that lead to genomic/chromosomal instability (p53, RB, etc.) and are often not fully transforming Driver mutations -these are mutations that are required for expression of fully transformed phenotype Driver mutations are the mutations we would like to target and inhibit their function Passenger mutations -these mutations are collateral damage resulting from genomic instability and are not required for maintaining the transformed phenotype
35 Biological limitations for molecular testing (1): Tumour(spatial) heterogeneity
36 Biological limitations for molecular testing (1): Tumour(spatial)heterogeneity
37 Biological limitations for molecular testing (4): Feasibility (and opportunity) for re-biopsy in disseminated disease Percentage of pat tients Hidalgo, A et al. AbdomImaging 2005 Gomez-Roca, CA et al. Ann Oncol Chouaid, C et al. Lung Cancer 2014 Khasraw, M et al. Current Oncology Reports 2011 Overman, MJ et al. J ClinOncol2013 Jang, DR et al. PLoSOne 2015 Zhu, YY et al. TumourBiol2015 Re-biopsy No tumor representation Change in therapy (no data for prostate)
38 Biological limitations for molecular testing (5): Low prevalence of oncogenicdrivers in certain tumor types InToGeN, 2016
39 PreanalyticalChallenges for cfdna Testing Blood Plasma cfdna isolation Analysis Factors influencing cfdna recovery: Tube type: size, preservative agents, polarity of tube walls Type of anticoagulant Time delay and storage of blood sample Tube Stability Storage EDTA 1-2h 4ºC Cell-free DNA BCT (Streck) Liquid Biopsy Blood CollectionKit (ThermoFisher) 14d 96h RT RT Bonin, S et al. VirchowsArch2010 Bronkhorst, A et al. Clinica Clhemical Acta 2015 El Messaoudi, S et al. Clinica Chemical Acta 2013
40 PreanalyticalChallenges for cfdna Testing Blood Plasma Factors influencing cfdna recovery: Centrigugation Temperature of storage Tube type Thawing cycles of sample cfdna isolation Analysis Bonin, S et al. VirchowsArch2010 Bronkhorst, A et al. Clinica Clhemical Acta 2015 El Messaoudi, S et al. Clinica Chemical Acta 2013
41 PreanalyticalChallenges for cfdna Testing Blood Plasma Factors influencing cfdna recovery: Method for DNA isolation Presence of detrimental factors Tube type Automatization Elution regime and volume Storage of isolated DNA Quantification cfdna isolation Analysis Bonin, S et al. VirchowsArch2010 Bronkhorst, A et al. Clinica Clhemical Acta 2015 El Messaoudi, S et al. Clinica Chemical Acta 2013
42 Analytical Challenges for Plasma DNA Testing Blood Plasma cfdna isolation Analysis Diaz& Bardelli, J ClinOncol2014
43 TherascreeenEGFR mutation analysis in plasma 29 mutations in EGFR Sensitivity for activating mutations: 65.7% Specificity: 99.8%
44 CobasEGFR mutation analysis in plasma 42 mutations in EGFR Semi-quantitative Index (SQI) Sensitivity for activating mutations: 82% Specificity: 98%
45 BEAMing Beads, Emulsions, Amplification & Magnetics mutation analysis in plasma Emulsion Characteristics Droplet size: 3-10 µm diameter fl volume Droplet density: ~ compartments / µl ~ beads / µl Up to 300 million PCR compartments per reaction Vogelstein et al. PNAS 1999; mod. Diehl et al Curr Opin Oncol Beads in Water-in-Oil Emulsions Magnetic beads (1 µm diameter) Light Microscopy of Emulsions (200x)
46 Example of BEAMingFlow CytometryAnalysis Wild-type Mutant
47 EGFR mutation detection in plasma ctdna: A cross-platform comparison of technologies to support clinical development of TAGRISSO (osimertinib, AZD9291) in NSCLC Phase I trial with AZD9291 in NSCLC (AURA): 38 pre-dose plasma samples and matched tumor biopsies, after first development of resistance and 2 nd look biopsy have been evaluated cfdnawas tested for 3 EGFR mutations (T790M, L858R, Exon19 del) by 3methods and compared with tissue Roche cobas PCR Qiagen Therascreen ARMS Sysmex Inostics BEAMing Exon 19 Deletion Assays Sensitivity 86% 82% 90% Specificity 100% 100% 100% Concordance 89% 87% 93% L858R (Exon 21) Assays Sensitivity 90% 78% 100% Specificity 100% 100% 91% Concordance 97% 95% 93% T790M (Exon 20) Assay Sensitivity 41% 29% 69% Specificity 100% 100% 67% Concordance 57% 48% 68% Thress, KS et al. LungCancer2015
48 EGFR mutation detection in plasma ctdna: A cross-platform comparison of technologies to support clinical development of TAGRISSO (osimertinib, AZD9291) in NSCLC Levels of EGFR T790M in plasma by BEAMing predict response to AZD9291 Thress, KS et al. ESMO 2014
49 Mensajesfinales Elevada complejidad molecular en cáncer: heterogeneidad espacial y temporal, elevado número de alteraciones (y de baja prevalencia) Re-biopsia: acceso limitado, morbilidad, representatividad y momento (recaída vs. progresión) El cfdnarecapitula las alteraciones presentes en el tumor (y de las lesiones diseminadas) Necesidad de estandarización de los procedimientos de estudio del cfdna
50 EGFR mutation detection in ctdnafrom NSCLC patient plasma: A cross-platform comparison of technologies to support the clinical development of AZD9291 Mutationsdetectedin NSCLC patientstreatedwithrociletinibondistantmetastatic(m1b) vs local (M1a/M0) disease Goldman, S et al. AACR 2015 Karlovish, CS et al. ClinCancerRes 2016
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56 Monitoring of EGFR mutations in TKI treatment for NSCLC 25 Proportion of mutated DNA /06/ /09/ /11/ /01/2016 Pret Gefitinib Pretreatment Partial response Progression Del19 T790M
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61 NatRevClinOncol.2014 Mar;11(3): doi: /nrclinonc Epub2014 Jan21. Molecular analysis of circulating tumour cells-biology and biomarkers. KrebsMG 1, MetcalfRL 1, Carter L 1, Brady G 1, BlackhallFH 1, DiveC 1.
62 CirculatingTumor Cells Schilling, D et al. Nat Rev Urol2012
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