SLIDE 1 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
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Disclosures
Translational research fees: AstraZeneca Financial support: Thermo Fisher, Life Member advisory board GI cancer: Amgen BV Consultancy: Roche, Bristol-Myers Squibb
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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.
SLIDE 4 Treatment tumors modern: “personalized therapy” : “targeted therapy” : “patient-tailored therapy“: “precision therapy” : “pharmacogenetics“ : “pharmacogenomics” :
right drug, right dose, right patient, right time, right diet, right dosage form
Therapy based on the molecular characteristics
- f the tumor (and the patient)
SLIDE 5 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--
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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
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DNA isolatie
SLIDE 8 Mutant DNA 2x (25%) Wild type DNA 6x (75%)
Tumor cell DNA Normal cell DNA
➔
DNA isolation DNA amplification (PCR)
Single molecule cloning and sequencing
Tumor cells : normal cells = 1 : 1
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One molecule per agarose bead
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agarose bead per micell Emulsion PCR (cloning)
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Emulsion PCR (cloning)
SLIDE 12 Chip sequencing
Per well determination of DNA sequence 60 wells wild type signal 20 wells mutation
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One molecule per agarose bead
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Chip sequencing (each well one bead): Fragment A: wild type Fragment B: wild type Fragment A: mutant Fragment B: mutant
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Sample 1 Sample 2 Sample 3 Sample 4 Amplicon 1 Amplicon 2 Amplicon 3 Amplicon 4
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Sample 1 Sample 2 Sample 3 Sample 4 Amplicon 1 Amplicon 2 Amplicon 3 Amplicon 4
SLIDE 18 CDKN2A PTEN TP53 ID3 1p 11 SNPs 8p 9 SNPs 19q 9 SNPs chr7 9 SNPs APC 9 SNPs ARID1A 8 SNPs ATM 9 SNPs BRCA1 9 SNPs BRCA2 9 SNPs CDKN2A 9 SNPs FHIT 9 SNPs PTEN 9 SNPs RB1 9 SNPs SMAD4 9 SNPs STK11 9 SNPs TP53 9 SNPs VHL 9 SNPs CTNBB1 ex3 BRAF ex11+15 EGFR ex18-21 ERBB2 ex19-21 FOXL2 ex3 GNA11 ex4+5 GNAS ex8+9 GNAQ ex4+5 HRAS ex2-4 KIT ex8, 9, 11, 13, 17 KRAS ex2-4 NRAS ex2-4 PDGFRa ex12, 14, 18 PIK3CA ex10+21 MET ex2, 14, 19 IDH1 ex4 IDH2 ex4 ALK ex20+22-25 AKT1 ex3 ARAF ex7 RAF1 ex7 POLE ex9+13 POLD1 ex12 Amel_X Amel_Y APC ex14 CHEk2 ex4, 5, 12, 13 FGFR1 ex4, 7, 12 (voor ampl. analyse) FGFR2 ex7+9 (voor ampl. analyse) FGFR3 ex7+9 (voor ampl. analyse) EZH2 ex16 FBXW7 ex9+10 MYD88 ex5 NOTCH1 ex26+27 RET ex11+16 RNF43 ex3, 4, 9 SMAD4 ex3, 9, 12 ROS1 ex38 STK11 ex4, 5, 8
Pathology Erasmus MC Cancer Institute Targeted NGS Custom made Diagnostics V4 panel 328 amplicons
Complete CDS Hotspots SNPs
SLIDE 19 KRAS p.G12C; c.34G>T
coverage
A = nucleotide variant
Reference sequence
Analysis NGS results – Integrative Genomics Viewer (IGV)
SLIDE 20 “Massive parallel” “Single molecule” 100s-1000s fragments / analysis Output 50 – >1000 x 106 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:
Next Generation Sequencing (NGS) Ion GeneStudio S5 Prime System
Lab developed bioinf. Pipeline and SeqNext
SLIDE 21 Wan et al., Nature Reviews Cancer, 17, 223-238, April 2017
Plasma cell free tumor DNA (ctDNA): low concentration of tumor DNA in background of normal DNA (ctDNA down to 0.1% range) Liquid biopsy: blood
SLIDE 22 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
SLIDE 23 ctDNA analysis sensitivity: 0.1% mutant DNA in background of 99.9% wildtype DNA
Single molecule molecular barcoding
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Limit of detection: Unique Molecular Identifier (UMI) tagging (single molecule molecular tag)
SLIDE 25 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
SLIDE 26 ERK RAS RAF PI3K mTOR MEK AKT PTEN
EGF
regulated proliferation and regulated inhibition cell death
EGFR
EGFR pathway normal
SLIDE 27 ERK RAS RAF PI3K mTOR MEK AKT PTEN
Proliferation Inhibition cell death
EGFR EGF
EGFR pathway activated by EGFR mutation
SLIDE 28 ERK RAS RAF PI3K mTOR MEK AKT PTEN
Proliferation Inhibition cell death
EGFR EGF erlotinib gefitinib
Through EGFR mutation activated pathway Inhibited by EGFR-TKI
SLIDE 29 ERK RAS RAF PI3K mTOR MEK AKT PTEN
Proliferation Inhibition cell death
EGF EGFR
EGFR pathway activated by KRAS mutation
SLIDE 30 ERK RAS RAF PI3K mTOR MEK AKT PTEN
Proliferation Inhibition cell death
EGF EGFR erlotinib gefitinib
Through KRAS mutation activated pathway No inhibition by EGFR-TKI
SLIDE 31 ERK RAS RAF PI3K mTOR MEK AKT PTEN
Proliferation Inhibition cell death
EGFR EGF erlotinib gefitinib
Through EGFR mutation activated pathway Inhibited by EGFR-TKI
SLIDE 32 ERK RAS RAF PI3K mTOR MEK AKT PTEN
Proliferation Inhibition cell death
EGFR EGF erlotinib gefitinib
Through EGFR mutation activated pathway Inhibited by EGFR-TKI: Resistence through 2nd EGFR mutation
SLIDE 33 ERK RAS RAF PI3K mTOR MEK AKT PTEN
Proliferation Inhibition cell death
EGFR EGF erlotinib gefitinib
Through EGFR mutation activated pathway Inhibited by EGFR-TKI: Resistence through 2nd EGFR mutatie: Inhibited by 2nd-line TKI
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Woman, 57 years, in 2008 lung cytology: NSCLC
SLIDE 35 Woman, 57 years, in 2008 lung cytology: NSCLC
2,1
Indicated are percentages variant, (number of unique molecules) ng input DNA 60
Cytology 2010 lung brush
2,1 C C C: mutations in CIS: on the same molecule
SLIDE 36 Woman, 57 years, in 2008 lung cytology: NSCLC
2,1 6,3
Indicated are percentages variant, (number of unique molecules) ng input DNA 60 51
Cytology 2010 lung brush
2,1
Cytology 2014 lung brush
6,3 C C C C C: mutations in CIS: on the same molecule
SLIDE 37 Woman, 57 years, in 2008 lung cytology: NSCLC
2,1 6,3 18
Indicated are percentages variant, (number of unique molecules) ng input DNA 60 51 49
Cytology 2010 lung brush
2,1
Cytology 2014 lung brush
6,3
Blood plasma August 2016 18
C C C C C C C C C: mutations in CIS: on the same molecule C: mutations in CIS: on the same molecule
SLIDE 38 Woman, 57 years, in 2008 lung cytology: NSCLC
2,1 6,3 18
Indicated are percentages variant, (number of unique molecules) ng input DNA 60 51 49
Cytology 2010 lung brush
2,1
Cytology 2014 lung brush
6,3
Blood plasma August 2016 18
52
Blood plasma October 2016 21
C C C C C C C C C C C C C: mutations in CIS: on the same molecule C: mutations in CIS: on the same molecule
SLIDE 39 Woman, 57 years, in 2008 lung cytology: NSCLC
2,1 6,3 18
Indicated are percentages variant, (number of unique molecules) ng input DNA 60 51 49
Cytology 2010 lung brush
2,1
Cytology 2014 lung brush
6,3
Blood plasma August 2016 18
52
Blood plasma October 2016 21
53
Blood plasma November 2016 19
3,22 (27) 3,53 (58) 3,23 (62) 1,58 (26) 0,15 (2) 0,51 (12)
C C C C C C C C C C C C C C C C T T C: mutations in CIS: on the same molecule C: mutations in CIS: on the same molecule T: mutations in TRANS: on different molecules
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T790M C797S
in cis
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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????
SLIDE 42 ▪ Pathology, molecular diagnostics:
Pulmonary Medicine Clinical Chemistry
Evert de Jonge Medical Oncology Clinical Chemistry
- Dr. Maurice Jansen
- Prof. dr. Ron van Schaik
Erik Jan Dubbink
Clinical Scientist in Molecular Pathology
Ronald van Marion
Senior technician
Peggy Atmodimedjo
Senior technician
Erasmus MC ctDNA molecular diagnostics:
Jan von der Thüsen
Pathologist
Niels Krol
Bio-informatician
Laura Moonen
technician
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Thank you for your attention
