Molecular diagnostics for targeted treatments in non small cell lung cancer Winand N.M. Dinjens Clinical Scientist in Molecular Pathology (CSMP) Head Molecular Diagnostics Department of Pathology w.dinjens@erasmusmc.nl Course Basic and Translational Oncology 2017 Postgraduate School Molecular Medicine Rotterdam, 23-10-2017
Disclosures Translational research fees: AstraZeneca Financial support: Thermo Fisher, Life Member advisory board GI cancer: Amgen BV Consultancy: Roche, Bristol-Myers Squibb
CANCER BIOLOGY DOGMA: CANCER 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 ” : Therapy based on the molecular characteristics “ patient- tailored therapy“: of the tumor (and the patient) “precision therapy” : “pharmacogenetics“ : “ pharmacogenomics ” : 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 GGC GCC GGC GGT GTG GGC-- -- Val Gly Ala Gly Gly Val Gly-- --GTG GGC GCC GTC GGT GTG GGC-- -- Val Gly Ala Val Gly Val Gly--
EGFR pathway normaal EGF EGFR PI3K RAS PTEN RAF AKT MEK mTOR ERK gereguleerde proliferatie en gereguleerde remming celdood
EGFR pathway geactiveerd door EGFR mutatie EGF EGFR PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferatie Remming celdood
Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI EGF EGFR erlotinib gefitinib PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferatie Remming celdood
EGFR pathway geactiveerd door EGF KRAS mutatie EGFR PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferatie Remming celdood
Door KRAS mutatie geactiveerde pathway EGF wordt niet geremd door EGFR-TKI EGFR erlotinib gefitinib PI3K RAS 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
PCR Fragment A
Letterlijk één molecuul per agarose bead
Letterlijk één agarose bead per micel Emulsie PCR (clonering)
Emulsie PCR (clonering)
Chip sequencing Fragment A Letterlijk één agarose bead per well Per well wordt DNA sequentie bepaald 60 wells wildtype signaal 20 wells mutatie
mutatie A mutatie B wildtype B wildtype A
Eén tumor Tumor DNA Fragment A Tumor DNA Fragment B Mutant Mutant Wildtype Wildtype
Multiplex (2) PCR Fragment A Fragment B
Letterlijk één molecuul per agarose bead
Emulsie PCR (clonering)
Chip 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 SEQUENCING ION TORRENT Personal Genome Machine S5XL PGM
Analyse NGS resultaten – Integrative Genomics Viewer (IGV) KRAS p.G12C; c.34G>T coverage A = nucleotide variant Referentie sequentie
Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI EGF EGFR erlotinib gefitinib PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferatie Remming celdood
Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI: EGF EGFR Resistentie door 2 e EGFR erlotinib mutatie gefitinib PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferatie Remming celdood
Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI: EGF EGFR Resistentie door 2 e erlotinib EGFR mutatie: gefitinib Geremd door 2 e -lijns TKI PI3K osimertinib RAS PTEN RAF AKT MEK mTOR ERK Proliferatie Remming celdood
Next Generation Sequencing (NGS) Ion Torrent S5XL “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
Wan et al., Nature Reviews Cancer, 17, 223-238, April 2017 Cell free (cf) DNA: low concentration Cell free tumor (ct) DNA: 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 Cancer, 17, 223-238, April 2017
Oncomine cfDNA panels: Lung: ALK (25), NRAS (23), PIK3CA (3), ROS1 (1), EGFR (39), MET (18), BRAF (7), KRAS (12), MAP2K1 (13), TP53 (34), HER2 (1) Total 176 hotspots Breast: SF3B1 (1), PIK3CA (18), FBXW7 (2), ESR1 (7), EGFR (7), KRAS (10), AKT1 (1), TP53 (97), HER2 (2), HER3 (16) Total 161 hotspots Colon: NRAS (22), PIK3CA (14), FBXW7 (8), BRAF (3), APC (36), EGFR (10),, KRAS (13), AKT1 (1), CTNNB1 (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 Chips: 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: PCR mistakes PCR 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: NSCLC
Woman, 57 years, in 2008 lung cytology: NSCLC 60 Cytology 2010 lung brush 2,1 C C 2,1 Indicated are percentages variant, (number of unique molecules) ng input DNA C: mutations in CIS: on the same molecule
Woman, 57 years, in 2008 lung cytology: NSCLC Cytology 2010 60 lung brush 2,1 C C 2,1 51 Cytology 2014 6,3 lung brush C C 6,3 Indicated are percentages variant, (number of unique molecules) ng input DNA C: mutations in CIS: on the same molecule
Woman, 57 years, in 2008 lung cytology: NSCLC Cytology 2010 60 2,1 lung brush C C 2,1 Cytology 2014 51 lung brush 6,3 C 6,3 C Blood plasma 49 August 2016 18 18 C C C C Indicated are percentages variant, (number of unique molecules) ng input DNA C: mutations in CIS: on the same molecule C: mutations in CIS: on the same molecule
Woman, 57 years, in 2008 lung cytology: NSCLC Cytology 2010 60 2,1 lung brush C C 2,1 Cytology 2014 51 C lung brush 6,3 C 6,3 Blood plasma 49 C C August 2016 18 18 C C Blood plasma 52 October 2016 21 C C C C Indicated are percentages variant, (number of unique molecules) ng input DNA C: mutations in CIS: on the same molecule C: mutations in CIS: on the same molecule
Woman, 57 years, in 2008 lung cytology: NSCLC Cytology 2010 60 2,1 lung brush C C 2,1 Cytology 2014 51 lung brush 6,3 C C 6,3 Blood plasma 49 August 2016 18 C 18 C C C Blood plasma 52 October 2016 21 C C C C 3,22 (27) 0,51 (12) 0,15 (2) 3,23 (62) 3,53 (58) 1,58 (26) Blood plasma 53 November 2016 19 T C C C T C Indicated are percentages variant, (number of unique molecules) ng input DNA C: mutations in CIS: on the same molecule C: mutations in CIS: on the same molecule T: mutations in TRANS: on different molecules
in cis T790M C797S
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
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