Molecular diagnostics for targeted treatments in non small cell lung cancer Winand N.M. Dinjens linical Scientist in Molecular Pathology (SMP) Head Molecular Diagnostics Department of Pathology w.dinjens@erasmusmc.nl ourse Basic and Translational Oncology 2017 Postgraduate School Molecular Medicine Rotterdam, 23-10-2017
Disclosures Translational research fees: Financial support: Member advisory board GI cancer: onsultancy: AstraZeneca Thermo Fisher, Life Amgen BV Roche, Bristol-Myers Squibb
ANER BIOLOGY DOGMA: ANER IS A DISEASE OF THE DNA Tumor cells differ from normal cells by the presence of genomic aberrations Determination of DNA aberrations has clinical value * Malignancy yes/no: lympho-proliferations * Primary/metastasis: multiple tumors * Tumor type: lymphoma, sarcoma * Which treatment: tumors lung, breast, targeted therapy colorectum, melanoma GIST, etc, etc.
Treatment tumors modern: personalized therapy : targeted therapy : patient-tailored therapy : precision therapy : pharmacogenetics : pharmacogenomics : Therapy based on the molecular characteristics of the tumor (and the patient) right drug, right dose, right patient, right time, right diet, right dosage form
Het is dus van belang de DNA bouwsteen (base) volgorde (sequentie) te bepalen van specifieke delen (targeted) van het DNA om afwijkingen te detecteren: DNA sequencing --GTG GG G GG GGT GTG GG-- -- Val Gly Ala Gly Gly Val Gly-- --GTG GG G GT GGT GTG GG-- -- Val Gly Ala Val Gly Val Gly--
EGF EGFR pathway normaal EGFR RAS PI3K PTEN RAF AKT MEK mtor ERK gereguleerde proliferatie en gereguleerde remming celdood
EGFR EGF EGFR pathway geactiveerd door EGFR mutatie RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood
EGFR EGF erlotinib gefitinib Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood
EGF EGFR pathway geactiveerd door KRAS mutatie EGFR RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood
EGFR EGF erlotinib gefitinib Door KRAS mutatie geactiveerde pathway wordt niet geremd door EGFR-TKI RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood
DNA isolatie Paraffine blokje Paraffine coupe H&E gekleurde coupe Immunohistochemisch gekleurde coupe (gekleurd) cytologiepreparaat
DNA isolatie
mutatie wildtype
Tumor DNA Fragment A Normaal DNA Fragment A 4 mutant 12 wildtype
PR Fragment A
Letterlijk één molecuul per agarose bead
Letterlijk één agarose bead per micel Emulsie PR (clonering)
Emulsie PR (clonering)
hip sequencing Fragment A Letterlijk één agarose bead per well Per well wordt DNA sequentie bepaald 60 wells wildtype signaal 20 wells mutatie
mutatie A wildtype A mutatie B wildtype B
Eén tumor Tumor DNA Fragment A Tumor DNA Fragment B Mutant Wildtype Mutant Wildtype
Fragment A Multiplex (2) PR Fragment B
Letterlijk één molecuul per agarose bead
Emulsie PR (clonering)
hip sequencing (elke well één bead): Fragment A: wildtype Fragment B: wildtype Fragment A: mutatie Fragment B: mutatie
Amplicon 1 Amplicon 2 Amplicon 3 Amplicon 4 Sample 1 Sample 2 Sample 3 Sample 4
Amplicon 1 Amplicon 2 Amplicon 3 Amplicon 4 Sample 1 Sample 2 Sample 3 Sample 4
NEXT GENERATION SEQUENING ION TORRENT Personal Genome Machine PGM S5XL
Analyse NGS resultaten Integrative Genomics Viewer (IGV) coverage A = nucleotide variant KRAS p.g12; c.34g>t Referentie sequentie
EGFR EGF erlotinib gefitinib Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood
EGFR EGF erlotinib gefitinib Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI: Resistentie door 2 e EGFR mutatie RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood
EGFR EGF erlotinib gefitinib Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI: Resistentie door 2 e EGFR mutatie: Geremd door 2 e -lijns TKI RAS PI3K PTEN osimertinib RAF AKT MEK mtor ERK Proliferatie Remming celdood
Massive parallel Single molecule 100s-1000s fragments / analysis Output 50 >1000 x 10 6 bases Short amplicons (<200bp) Lab developed panels Enriched with SNP amplicons Dubbink et al., J Mol Diagn. 2016. doi: 10.1016/j.jmoldx.2016.06.002 Low amount of input DNA (<<10 ng) High sensitivity (<5%) Mean coverage 500-1500x >Semi-quantitative Pooling of samples Bio-informatics support Next Generation Sequencing (NGS) Ion Torrent S5XL
ell free (cf) DNA: ell free tumor (ct) DNA: Wan et al., Nature Reviews ancer, 17, 223-238, April 2017 low concentration low concentration in background normal DNA (ctdna down to 0.1% range)
Liquid biopsies: Advantages: * Minimaly invasive, easy to obtain, also longitudinal * Better representation of malignant burden (heterogeneity, multiple localisations) * Disease monitoring, resistance detection Disadvantage: * Need for extreme sensitive assays: <<1% mutant Wan et al., Nature Reviews ancer, 17, 223-238, April 2017
Oncomine cfdna panels: Lung: ALK (25), NRAS (23), PIK3A (3), ROS1 (1), EGFR (39), MET (18), BRAF (7), KRAS (12), MAP2K1 (13), TP53 (34), HER2 (1) Total 176 hotspots Breast: SF3B1 (1), PIK3A (18), FBXW7 (2), ESR1 (7), EGFR (7), KRAS (10), AKT1 (1), TP53 (97), HER2 (2), HER3 (16) Total 161 hotspots olon: NRAS (22), PIK3A (14), FBXW7 (8), BRAF (3), AP (36), EGFR (10),, KRAS (13), AKT1 (1), TNNB1 (6), HER2 (9) MAP2K1 (10), TP53 (97), HER2 (9), SMAD4 (8), GNAS (5) Total 242 hotspots Preselected hotspots. We adopted analyses to evaluate sequencing results of all genomic positions covered by the panels
Workflow hips: 520: 5 miljoen reads output 530: 20 miljoen reads output 540: 80 miljoen reads output ctdna analyses mean coverage: >25,000
Liquid biopsies: Disadvantage: * Need for extreme sensitive assays: <<1% mutant 0.1% mutant in background of 99.9% wildtype Limit of detection: PR mistakes PR duplicates
Limit of detection: Unique Molecular Identifier (UMI) tagging (single molecule molecular tag)
Limit of detection: combination of amount of DNA input and sequencing coverage Detection 0.1% variant: 20ng input ~ 6000 haploïd genomes ~ 6000 templates 25,000x coverage 6000 unique molecules 0.1% = 6 molecules variant practice +/- 50% efficiency 0.1% = 3 molecules variant
Woman, 57 years, in 2008 lung cytology: NSL
Woman, 57 years, in 2008 lung cytology: NSL 60 ytology 2010 lung brush 2,1 2,1 Indicated are percentages variant, (number of unique molecules) ng input DNA : mutations in IS: on the same molecule
Woman, 57 years, in 2008 lung cytology: NSL ytology 2010 lung brush 2,1 2,1 60 51 ytology 2014 lung brush 6,3 6,3 Indicated are percentages variant, (number of unique molecules) ng input DNA : mutations in IS: on the same molecule
Woman, 57 years, in 2008 lung cytology: NSL ytology 2010 lung brush 2,1 2,1 60 ytology 2014 lung brush 6,3 51 Blood plasma August 2016 18 18 49 Indicated are percentages variant, (number of unique molecules) ng input DNA : mutations in IS: on the same molecule : mutations in IS: on the same molecule
Woman, 57 years, in 2008 lung cytology: NSL ytology 2010 lung brush 2,1 2,1 60 ytology 2014 lung brush 6,3 51 Blood plasma August 2016 18 18 49 Blood plasma October 2016 21 52 Indicated are percentages variant, (number of unique molecules) ng input DNA : mutations in IS: on the same molecule : mutations in IS: on the same molecule
Woman, 57 years, in 2008 lung cytology: NSL ytology 2010 lung brush 2,1 2,1 ytology 2014 lung brush 6,3 Blood plasma August 2016 18 18 60 51 49 Blood plasma 52 October 2016 21 Blood plasma 53 November 2016 19 3,22 (27) 3,23 (62) 3,53 (58) 1,58 (26) 0,51 (12) 0,15 (2) T T Indicated are percentages variant, (number of unique molecules) ng input DNA : mutations in IS: on the same molecule : mutations in IS: on the same molecule T: mutations in TRANS: on different molecules
T790M in cis 797S
Limit of detection, specificity, sensitivity ctdna analysis: 1. Amount input DNA 2. Sequencing coverage 3. Information mutations in tumor tissue 4. Number of (simultaneously detected) mutations 5. Molecular barcoding (UMIs) 6. integrated Digital Error Suppression (ides) 7. Genomic position-specific error correction 8. Mutations/variants in cis or in trans
NEAR FUTURE ctdna analyses: Longitudinal monitoring multiple tumor types based on ctdna analysis of (clonal) mutations identified in tumor tissue Detection of translocations/fusions (DNA, RNA based) Detection of genomic imbalances: deletions and amplifications (shallow sequencing, comparable to NIPT): resistance mechanisms DISTANT FUTURE ctdna analysis: Screening on medical indication (complaints, imaging, etc) Population screening of healthy individuals????
Erasmus M ctdna molecular diagnostics: Pathology, molecular diagnostics: Peggy Atmodimedjo Senior technician Ronald van Marion Senior technician Laura Moonen technician Niels Krol Bio-informatician Jan von der Thüsen Pathologist Erik Jan Dubbink linical Scientist in Molecular Pathology Pulmonary Medicine Prof. dr. Joachim Aerts linical hemistry Evert de Jonge Medical Oncology Dr. Maurice Jansen linical hemistry Prof. dr. Ron van Schaik
Thank you for your attention