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 2018 Postgraduate School Molecular Medicine Rotterdam, 08-10-2018
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, brain * Which treatment: tumors lung, breast, “targeted therapy” colorectum, melanoma “druggable aberrations” 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
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--
DNA isolation from routine Pathology (FFPE) specimens Mix of tumor and normal cells, highly degraded DNA Paraffin block Immuno stained section Paraffin section (stained) cytology preparation H&E stained section
DNA isolatie
Normal cell DNA Tumor cell DNA DNA ➔ isolation DNA amplification (PCR) Tumor cells : normal cells = 1 : 1 Mutant DNA 2x (25%) Wild type DNA 6x (75%) Single molecule cloning and sequencing
One molecule per agarose bead
one agarose bead per micell Emulsion PCR (cloning)
Emulsion PCR (cloning)
Chip sequencing one agarose bead per well Per well determination of DNA sequence 60 wells wild type signal 20 wells mutation
One molecule per agarose bead
Chip sequencing (each well one bead): Fragment A: wild type Fragment B: wild type Fragment A: mutant Fragment B: mutant
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
Pathology Erasmus MC Cancer Institute Targeted NGS Custom made Diagnostics V4 panel 328 amplicons CDKN2A Complete CDS PTEN TP53 ID3 Hotspots SNPs CTNBB1 ex3 RAF1 ex7 1p 11 SNPs BRAF ex11+15 POLE ex9+13 8p 9 SNPs EGFR ex18-21 POLD1 ex12 19q 9 SNPs ERBB2 ex19-21 Amel_X chr7 9 SNPs FOXL2 ex3 Amel_Y APC 9 SNPs GNA11 ex4+5 APC ex14 ARID1A 8 SNPs GNAS ex8+9 CHEk2 ex4, 5, 12, 13 ATM 9 SNPs GNAQ ex4+5 FGFR1 ex4, 7, 12 (voor ampl. analyse) BRCA1 9 SNPs HRAS ex2-4 FGFR2 ex7+9 (voor ampl. analyse) BRCA2 9 SNPs KIT ex8, 9, 11, 13, 17 FGFR3 ex7+9 (voor ampl. analyse) CDKN2A 9 SNPs KRAS ex2-4 EZH2 ex16 FHIT 9 SNPs NRAS ex2-4 FBXW7 ex9+10 PTEN 9 SNPs PDGFRa ex12, 14, 18 MYD88 ex5 RB1 9 SNPs PIK3CA ex10+21 NOTCH1 ex26+27 SMAD4 9 SNPs MET ex2, 14, 19 RET ex11+16 STK11 9 SNPs IDH1 ex4 RNF43 ex3, 4, 9 TP53 9 SNPs IDH2 ex4 SMAD4 ex3, 9, 12 VHL 9 SNPs ALK ex20+22-25 ROS1 ex38 AKT1 ex3 STK11 ex4, 5, 8 ARAF ex7
Analysis NGS results – Integrative Genomics Viewer (IGV) KRAS p.G12C; c.34G>T coverage A = nucleotide variant Reference sequence
Next Generation Sequencing (NGS) Ion GeneStudio S5 Prime System “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, PMID: 27461031 Low amount of input DNA (<<10 ng) High sensitivity (<5%) Mean coverage 500-1500x >Semi-quantitative Pooling of samples Bio-informatics support: Lab developed bioinf. Pipeline and SeqNext
Wan et al., Nature Reviews Cancer, 17, 223-238, April 2017 Liquid biopsy: blood Plasma cell free tumor DNA (ctDNA): low concentration of tumor DNA in background of 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
ctDNA analysis sensitivity: 0.1% mutant DNA in background of 99.9% wildtype DNA Single molecule molecular barcoding
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
EGFR pathway normal EGF EGFR PI3K RAS PTEN RAF AKT MEK mTOR ERK regulated proliferation and regulated inhibition cell death
EGFR pathway activated by EGFR mutation EGF EGFR PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferation Inhibition cell death
Through EGFR mutation activated pathway Inhibited by EGFR-TKI EGF EGFR erlotinib gefitinib PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferation Inhibition cell death
EGFR pathway activated by EGF KRAS mutation EGFR PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferation Inhibition cell death
Through KRAS mutation activated pathway EGF No inhibition by EGFR-TKI EGFR erlotinib gefitinib PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferation Inhibition cell death
Through EGFR mutation activated pathway Inhibited by EGFR-TKI EGF EGFR erlotinib gefitinib PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferation Inhibition cell death
Through EGFR mutation activated pathway Inhibited by EGFR-TKI: EGF EGFR Resistence through erlotinib 2nd EGFR mutation gefitinib PI3K RAS PTEN RAF AKT MEK mTOR ERK Proliferation Inhibition cell death
Through EGFR mutation activated pathway Inhibited by EGFR-TKI: EGF EGFR Resistence through erlotinib 2nd EGFR mutatie: gefitinib Inhibited by 2nd-line TKI PI3K RAS osimertinib PTEN RAF AKT MEK mTOR ERK Proliferation Inhibition cell death
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 Cytology 2014 51 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
NEAR FUTURE ctDNA analyses: ▪ Longitudinal monitoring multiple tumor types based on ctDNA analysis of (clonal) mutations identified in tumor tissue DISTANT FUTURE ctDNA analysis: ▪ Screening on medical indication (complaints, imaging, etc) ▪ Population screening of healthy individuals????
Erasmus MC ctDNA molecular diagnostics: ▪ Pathology, molecular diagnostics: Peggy Atmodimedjo Ronald van Marion Laura Moonen Niels Krol Jan von der Thüsen Erik Jan Dubbink Senior technician Senior technician technician Bio-informatician Pathologist Clinical Scientist in Molecular Pathology Pulmonary Medicine Clinical Chemistry Prof. dr. Joachim Aerts Evert de Jonge Medical Oncology Clinical Chemistry Dr. Maurice Jansen Prof. dr. Ron van Schaik
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