Molecular diagnostics for targeted treatments in non small cell lung cancer

Transcription

1 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 ourse Basic and Translational Oncology 2017 Postgraduate School Molecular Medicine Rotterdam,

2 Disclosures Translational research fees: Financial support: Member advisory board GI cancer: onsultancy: AstraZeneca Thermo Fisher, Life Amgen BV Roche, Bristol-Myers Squibb

3 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.

4 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

5 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--

6 EGF EGFR pathway normaal EGFR RAS PI3K PTEN RAF AKT MEK mtor ERK gereguleerde proliferatie en gereguleerde remming celdood

7 EGFR EGF EGFR pathway geactiveerd door EGFR mutatie RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood

8 EGFR EGF erlotinib gefitinib Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood

9 EGF EGFR pathway geactiveerd door KRAS mutatie EGFR RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood

10 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

11 DNA isolatie Paraffine blokje Paraffine coupe H&E gekleurde coupe Immunohistochemisch gekleurde coupe (gekleurd) cytologiepreparaat

12 DNA isolatie

13 mutatie wildtype

14 Tumor DNA Fragment A Normaal DNA Fragment A 4 mutant 12 wildtype

15 PR Fragment A

16 Letterlijk één molecuul per agarose bead

17 Letterlijk één agarose bead per micel Emulsie PR (clonering)

18 Emulsie PR (clonering)

19 hip sequencing Fragment A Letterlijk één agarose bead per well Per well wordt DNA sequentie bepaald 60 wells wildtype signaal 20 wells mutatie

20 mutatie A wildtype A mutatie B wildtype B

21 Eén tumor Tumor DNA Fragment A Tumor DNA Fragment B Mutant Wildtype Mutant Wildtype

22 Fragment A Multiplex (2) PR Fragment B

23 Letterlijk één molecuul per agarose bead

24 Emulsie PR (clonering)

25 hip sequencing (elke well één bead): Fragment A: wildtype Fragment B: wildtype Fragment A: mutatie Fragment B: mutatie

26 Amplicon 1 Amplicon 2 Amplicon 3 Amplicon 4 Sample 1 Sample 2 Sample 3 Sample 4

27

28 Amplicon 1 Amplicon 2 Amplicon 3 Amplicon 4 Sample 1 Sample 2 Sample 3 Sample 4

29 NEXT GENERATION SEQUENING ION TORRENT Personal Genome Machine PGM S5XL

30 Analyse NGS resultaten Integrative Genomics Viewer (IGV) coverage A = nucleotide variant KRAS p.g12; c.34g>t Referentie sequentie

31 EGFR EGF erlotinib gefitinib Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI RAS PI3K PTEN RAF AKT MEK mtor ERK Proliferatie Remming celdood

32 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

33 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

34 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 doi: /j.jmoldx Low amount of input DNA (<<10 ng) High sensitivity (<5%) Mean coverage x >Semi-quantitative Pooling of samples Bio-informatics support Next Generation Sequencing (NGS) Ion Torrent S5XL

35 ell free (cf) DNA: ell free tumor (ct) DNA: Wan et al., Nature Reviews ancer, 17, , April 2017 low concentration low concentration in background normal DNA (ctdna down to 0.1% range)

36 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, , April 2017

37 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

38 Workflow hips: 520: 5 miljoen reads output 530: 20 miljoen reads output 540: 80 miljoen reads output ctdna analyses mean coverage: >25,000

39 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

40

41 Limit of detection: Unique Molecular Identifier (UMI) tagging (single molecule molecular tag)

42 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

43 Woman, 57 years, in 2008 lung cytology: NSL

44 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

45 Woman, 57 years, in 2008 lung cytology: NSL ytology 2010 lung brush 2,1 2, 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

46 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 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

47 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 Blood plasma October 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

48 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 Blood plasma 52 October Blood plasma 53 November ,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

49 T790M in cis 797S

50 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

51 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????

52 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

53 Thank you for your attention