Molecular diagnostics for targeted treatments in non small cell lung - - PowerPoint PPT Presentation

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” : “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)

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--

ERK RAS RAF PI3K mTOR MEK AKT PTEN

EGF

gereguleerde proliferatie en gereguleerde remming celdood

EGFR

EGFR pathway normaal

ERK RAS RAF PI3K mTOR MEK AKT PTEN

Proliferatie  Remming celdood 

EGFR EGF

EGFR pathway geactiveerd door EGFR mutatie

ERK RAS RAF PI3K mTOR MEK AKT PTEN

Proliferatie  Remming celdood

EGFR EGF erlotinib gefitinib

Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI

ERK RAS RAF PI3K mTOR MEK AKT PTEN

Proliferatie  Remming celdood 

EGF EGFR

EGFR pathway geactiveerd door KRAS mutatie

ERK RAS RAF PI3K mTOR MEK AKT PTEN

Proliferatie  Remming celdood 

EGF EGFR erlotinib gefitinib

Door KRAS mutatie geactiveerde pathway wordt niet geremd door EGFR-TKI

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 wildtype A mutatie B wildtype B

Tumor DNA Fragment A Tumor DNA Fragment B

Mutant Mutant Wildtype Wildtype

Eén tumor

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

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

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

NEXT GENERATION SEQUENCING

ION TORRENT

Personal Genome Machine PGM S5XL

KRAS p.G12C; c.34G>T

coverage

A = nucleotide variant

Referentie sequentie

Analyse NGS resultaten – Integrative Genomics Viewer (IGV)

ERK RAS RAF PI3K mTOR MEK AKT PTEN

Proliferatie  Remming celdood

EGFR EGF erlotinib gefitinib

Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI

ERK RAS RAF PI3K mTOR MEK AKT PTEN

Proliferatie  Remming celdood 

EGFR EGF erlotinib gefitinib

Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI: Resistentie door 2e EGFR mutatie

ERK RAS RAF PI3K mTOR MEK AKT PTEN

Proliferatie  Remming celdood

EGFR EGF erlotinib gefitinib

Door EGFR mutatie geactiveerde pathway geremd door EGFR-TKI: Resistentie door 2e EGFR mutatie: Geremd door 2e-lijns TKI

• simertinib

 

“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. 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

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

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

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

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

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

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

T790M C797S

in cis

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????

• Pathology, molecular diagnostics:

Pulmonary Medicine Clinical Chemistry

• Prof. dr. Joachim Aerts 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

Thank you for your attention