Point-EXACCT transformation from microtiterplate to

10de bijeenkomst Werkgroep “Moleculaire Diagnostiek in de Pathologie”
15 Januari 2016, Utrecht
Disclosures
Behandelkeuze voor targeted-therapie aan de hand
van mutatie-analyse op cel-vrij plasma DNA
Consultant/Advisory Board:
Ed Schuuring
Klinisch Moleculair Bioloog in de Pathologie
Hoogleraar in de Moleculaire Oncologische Pathologie
Hoofd Laboratorium Moleculaire Pathologie
Afdeling Pathologie van UMCG
AstraZeneca, Roche, Pfizer, Novartis, Amgen, BioCartis,
QCMD, ESP, IQNPATH
Speaker’s fee:
Abbott, Novartis, Roche
Stock/Royalties:
None
[email protected]
Behandelkeuze voor targeted-therapie aan de hand van mutatieanalyse op cel-vrij plasma DNA
Can peripheral blood be used to determine the
presence of the tumor ?
• Clinical relevance of cell free DNA testing
- Cell-free DNA from plasma reflects volume/mutation-profile of tumor
- Clinical applications
•
•
•
•
Methods to detect circulating tumor DNA
Collection of plasma (standarisation)
Plasma processing and DNA extraction (standarisation)
Quality control and EQA
1
Circulating tumor DNA in blood plasma
Can peripheral blood be used to determine the
presence of the tumor ?
Plasma:
* DNA (cell-free DNA, cfDNA)
* RNA (exosomes)
* Proteomic/immunological markers
* Pharmacokinetics ([drug])
Leukocytes
Platelets
Schwarzenbach Nat Rev Cancer 2011
Can peripheral blood be used to determine the
presence of the tumor ?
Cell-free plasma:
* DNA and circulating tumor DNA (ctDNA)
* RNA and circulating tumor RNA (ctRNA)
* Proteomic/immunological tumor markers
* Pharmacokinetics ([drug])
Leukocytes and circulating tumor cells (CTC)
Platelets and tumor RNA
Behandelkeuze voor targeted-therapie aan de hand van mutatieanalyse op cel-vrij plasma DNA
• Clinical relevance of cell free DNA testing
- Cell-free DNA from plasma reflects volume/mutation-profile of tumor
- Clinical applications
•
•
•
•
Methods to detect circulating tumor DNA
Collection of plasma (standarisation)
Plasma processing and DNA extraction (standarisation)
Quality control and EQA
2
Plasma-cfDNA increased in colon cancer and
associated with tumor size
Plasma cell-free DNA as a predictor for the presence of NSCLC
Ulivi Cell Oncol 2013
Gorges Biomarkers 2012
Plasma cell-free DNA as a predictor for the presence of NSCLC
Plasma cell-free DNA as a predictor for the presence of NSCLC
In all these studies different assays to determine DNA concentration:
• qPCR
• Picogreen
• Nanodrop
• Qubit (approx 10x lower concentration compared to nanodrop)
• In general: <2 ng/ml plasma (untreated pts !!!)
Ulivi Cell Oncol 2013
Ulivi Cell Oncol 2013
3
DNA concentrations by picogreen (ng/ml plasma) classified per
tumor type at baseline
Liquid biopsies: the detection of cell-free circulating tumor DNA in plasma (ctDNA)
Total amount of cell-free
plasma DNA increases:
•
•
•
•
•
•
Luca:
9-19 ng
~12 ng
Tumor progression
Inflammation
Exercise
Treatment
Pregnancy
Hemolysis
GIST:
4-25 ng
~10 ng
Perkins Plos One 2012
PA UMCG 2015/16 Qubit
Crowly E. et al Nat. Rev. Clin. Oncol. 10, 472-484 (2013)
Tumor mutation testing on liquid biopsies
Tumor mutation testing on liquid biopsies
sample selection: important issues
sample selection: important issues
Plasma collection and storage
- Blood collection in EDTA-tube: processing < 4hrs (no consensus)
- Streck-tube: <24hrs BUT expensive and no routine tube in hospital
Blood collection, plasma processing and storage:
Plasma processing/collecting:
- Centrifugation: 1x slow (~800g) and 1x high (13000g) (no consensus)
-
Storage:
- -20oC, -80oc
- Storage step 1, and 2nd centrigation after thawing
very time-consuming
high hands-on time (many technicians)
Automatisation not suitable
Large storage capacity (-80oC)
- No standards/consensus for processing
- Existing plasma-biobanks NOT suitable (hemolysis)
- No references/EQA available
4
The clinical utility of ctDNA
CT-scanning to identify lung disease
Diagnosis and prediction
Gouden standaard:
> verkrijgen tumorweefsel via biopt om diagnose en behandelkeuze
te kunnen stellen in routine moleculaire pathologie
De detectie van EGFR-mutaties in circulerend tumor DNA in plasma
komt heel goed overeen met DNA uit het pretreatment tumor biopt
> Biopteren niet altijd mogelijk (erg belastend, risicovol neveneffecten, diffuse nodules)
Meta-analysis of 20 studies
comparing tumor with baseline
ctDNA for EGFR mutations
> In 15-25% van pretreatment biopten: geen representatief weefsel
(te weinig weefsel of te weinig neoplastische cellen)
Specifiteit: 99.8%
Pooled specificity: 92.2%
Pooled sensitivity: 69.1%
D
Vals-positiviteit: 1/546
Sensitiviteit: 34% niet gedetecteerd in plasma
A small biopsy; B core-needle biopsy; C surgical specimen; D cytology
E biopsy with very few tumor cells
E
Luo, Sc Reports 2014
Douillard JTO 2014
5
Molecular diagnostics of lung cancer for treatment
planning using gene-targeted therapy in NL
dutch guidelines
In 2007: starting with EGFR-mutation screening
In 2013: Dutch guideline in NSCLC Only EGFR-mutation analysis
Validation of EGFR mutation testing (G719S, exon19del,
L858R, T790M) in ctDNA plasma (ongoing)
comparing ddPCR, cobas-EGFR, NGS and Idylla
• ddPCR (BioRad vs in house primers probes)
 Relative cheap (~50-100 ?)
 TAT: resp 8-12 hrs
 Hands-on time: 2-3 hrs
In 2013: ALK-translocation (Registration crizotinib July 2012)
• Validation on tumor tissue (MD-sneltest)
In July 2015: revisited Dutch guideline for NSCLC:
• Biobanking plasma/platelets of all lung cancer patients at t=0,
2, 6, 12 weeks (starting July 2015)
(1) EGFR, ALK; (2) HER2, BRAF, RET and ROS1
> testing ctDNA from plasma if no appropriate biopsy is available
• Plasma vs pretreatment tumor (preliminary):
- EGFR-L858R: 3/5 positive
- EGFR-ex19del: 2/3 positive
Bosman, Tibbesma, Elst, Groen, Schuuring
Validation of EGFR mutation testing (G719S, exon19del,
L858R, T790M) in ctDNA plasma (ongoing)
comparing ddPCR, cobas-EGFR, NGS and Idylla
Validation of EGFR mutation testing (G719S, exon19del,
L858R, T790M) in ctDNA plasma (ongoing)
comparing ddPCR, cobas-EGFR, NGS and Idylla
Commercial systems:
NGS iontorrent of tumor:
•



Cobas EGFR plasma (dec 2015)
Relative expensive (~200-300 ?)
TAT: 4 hrs
Hands-on time: 1-2 hrs
•



Idylla EGFR plasma (2016)
Relative expensive (~200-300 ?)
TAT: 90 min
Hands-on time: 5-10 min
L858R
No mutation
G719S
Ex19del + T790M
G719S + S768I
Ex19del + T790M
L858R
Ex19del
Bosman, Tibbesma, Elst, Groen, Schuuring
6
Cobas EGFR mutation blood test (in development)
sensitivity/specificity
The clinical utility of ctDNA
Monitoring treatment response
Meldgaard BMC Cancer 2014
Specificity: 94%
Sensitivity: 71% (39% not detected in plasma)
Agreement: 91%
False-positive rate: 6/168
Procedure of plasma cfDNA mutation testing
Mutation testing using plasma cell free DNA
applications in lung cancer (treatment response monitoring)
tumor
Tumor
volume
plasma
Resistant mutations
Druggable
mutation
Druggable
mutation
DNA
Tumor
volume
DNA
sequencing
Define tumor
specific mutation
Aim:
•Success of treatment response upon decreased plasma mutation levels of druggable mutation
cfDNA
quantification
ctDNA
ddPCR
•Early detection of therapy resistance (prior to clinical manification) by increased plasma mutation levels of druggable mutation
•Early detection of therapy-resistent plasma mutation levels (prior to clinical manification) enabling new treatment opportunities
Adapted from Diaz, J Clin Oncol 2014
Adapted from Diehl Nat Med 2008
Mutant DNA concentration
7
Disappearance of EGFR mutations during TKI
treatment using cobas-ctDNA test in NSCLC
KRAS-mutations are associated with acquired resistance upon
anti-EGFR-treatment using sensitive digital beaming assay
CRC patients with KRAS-wt in
pretreatment biopsy
ctDNA testing in plasma before start
treatment and after resistence
Bettegowda, Sc Transl Med 2014
Marchetti JTO 2015
13-04-2015
30-03-2015
25-03-2015
16-03-2015
15-12-2014
12-12-2014
13-10-2014
09-2013
22-09-2014
25-05-2013
10-09-2014
KWF-Alpe d’Huez: Pieter Boonstra, Marco Tibbesma, Arja ter Elst, Ed Schuuring, An Reyners
(GIST-consortium UMCG, Radboud, LUMC, ErasmasMC and NKI/AvL)
01-09-2014
Example of workflow of GIST-patient during KIT-mutation targeted therapy
25-08-2014
GALLOP-study: Assessment of mutations in tumors and circulating tumor
DNA and measurement of TKI plasma exposure to optimize treatment
(KIT)
Tumor volume
Resistant mutations
Operatie
Druggable
mutation
Sunitinib
37.5mg
Imatinib
400mg
Druggable
mutation
Tumor
volume
Adapted from Diaz, J Clin Oncol 2014
Recidief
Partiële
respons
Progressie
Boonstra Tibbesma Elst Schuuring Reyners Gallop
8
Location of mutations/deletions in exon 11 of
KIT gene in GIST in two hotspot regions
Design of multiplex ddPCR drop-off assay to detect mutations in
hotspot 1 or 2 of exon 11 KIT gene
Boonstra Tibbesma Elst Schuuring Reyners Gallop
KIT exon 11 drop-off ddPCR assay to detect hotspot 1 or 2
mutations in pretreatment biopsies of GIST
Boonstra Tibbesma Elst Schuuring Reyners Gallop
KIT exon 11 drop-off ddPCR assay to detect hotspot 1 or 2
mutations in pretreatment biopsies of GIST
Patient
NGS+/ddPCR+: 16
NGS+/ddPCR-: 2 (outside hotspot)
NGS+/ddPCR-: 1 (false-negative)
NGS-/ddPCR-: 9
Agreement: 27/28 (96%)
Boonstra Tibbesma Elst Schuuring Reyners Gallop
Mutatie
Type mutatie
Baseline
Twee weken
Zes weken
2
KIT 11
c.1667_1669del; p.Q556_W557>R
-
-
-
3
KIT 11
c.1669T>C; p.(W557R)
-
-
-
4
KIT 11
c.1727_1729del; p.(L576del)
8%
-
-
5
KIT 11
c.1671_1676delGAAGGT; p.W557_V559>C
0%
-
-
6
KIT 11
c.1655_1660delTGTATG; p.(M552_E554delinsK)
-
-
7
KIT 11
c.1726_1728del; p.L576del
0%
-
-
9
KIT 11
c.1676T>A; p.V559D
0%
0%
0%
10
KIT 11
c.1669_1674del; p.(W557_K558del)
-
-
-
13
KIT 11
c.1671_1672delinsTG
0%
0%
0%
14
KIT 11
c.1669_1674del; p.(W557_K558del)
0%
-
16
KIT 11
insertie 36bp
-
-
-
17
KIT 11
c.1679_1680delinsAG; p.V560E
0%
0%
0%
18
KIT 11
c.1673_1717del
0%
-
-
19
KIT 11
c.1662_1674delinsGGAAGAA;
2,5%
0,0%
0,7%
22
KIT 11
c.1669_1674del; p.(W557_K558del)
-
-
-
26
KIT 11
c.1649_1663del; p.K550_V555>I
-
-
-
28
KIT 11
c.1649_1663del.; p.(K550_V555delinsI)
-
-
-
29
KIT 11
c.1669_1674del; p.(Trp557_Lys558del)
16,5%
61,0%
3,0%
30
KIT 11
c.1676T>A; p.V559D
0,9%
5,5%
0%
31
KIT 11
c.1676T>A; p.(V559D)
0,1%
-
-
32
KIT 11
c.1669_1674del; p.(W557_K558del)
-
7,2%
-
Only 7/12 baseline plasma samples positive
-
-
Boonstra Tibbesma Elst Schuuring Reyners Gallop
9
mutation detection using ddPCR during imatinib-treatment of GIST patient
mutation detection using ddPCR during imatinib-treatment of GIST patient
Tumor
FA: 35%
Baseline
FA: 0.7%
Pretreatment plasma
ctDNA: FA = 16%
Pretreatment tumor:
FA = 88%
Tumor :
20x14cm
2 week
FA: 5.5%
6 week
FA: 0%
4 wk after TKI-treatment
Plasma ctDNA: FA = 3%
2 wk after TKI-treatment
Plasma ctDNA: FA = 61%
KWF-Alpe d’Huez: Boonstra, Tibbesma, ter Elst, Schuuring, Reyners
(GIST-consortium UMCG, Radboud, LUMC, ErasmasMC and NKI/AvL)
KWF-Alpe d’Huez: Boonstra, Tibbesma, ter Elst, Schuuring, Reyners
(GIST-consortium UMCG, Radboud, LUMC, ErasmasMC and NKI/AvL)
GALLOP-study: Assessment of mutations in tumors and circulating tumor
DNA and measurement of TKI plasma exposure to optimize treatment
Tumor :
7.5x12cm
Clinical utility of ctDNA testing
(KIT)
Tumor volume
Druggable
mutation
Resistant mutations
Druggable
mutation
Tumor
volume
Adapted from Diaz, J Clin Oncol 2014
Detection of resistant mutations (switching treatment)
•
•
•
•
•
•
•
Detection of early disease
Assessment of molecular heterozygeneity of overall disease
Identification of molecular markers for targeted therapy
Evaluation of early treatment response
Monitoring treatment response
Diagnosis of minimal residual disease
Detection of resistant mutations
NGS of pretreatment GIST: activating KIT exon 11 del (Q556_V560delinsH) 86%
NGS of TKI resistant GIST: resistant KIT mutation (Y823D) 92%
NGS of plasma at progression: both mutations (resp 18% and 17%)
10
Technologies for detection and characterization of ctDNA
Assays to detect low copy number mutations in plasma cfDNA
Haber and Velculescu, Cancer Disc 2014
Power In Partitioning
Digital droplet PCR technologie
ddPCR
Nanodroplet PCR reactions are
independent, single
amplification events
One measurement
Many thousands
of discrete measurements
ddPCR-assay: primer-set with HEX-probe for mutation and FAM-probe for wt
11
Rare Mutation Detection: Example 2
(KRAS G12V)
mutant only
mut
1% mutant
0.1% mutant
0.001% mutant
0% mutant
High analytical sensitivity of mutation detection using ddPCR
20 ng input A549
(KRAS-G12S)
20ng input H1650
(EGFR-E746_A750del)
mut + wt
wt
0.01% mutant
wt
Biorad
DNA concentrations by picogreen (ng/ml plasma) classified per
tumor type at baseline
0.1%
20 ng input A549
(KRAS-G12S)
2 ng input A549
(KRAS-G12S)
Luca:
9-19 ng
~12 ng
GIST:
4-25 ng
~10 ng
Perkins Plos One 2012
0.01%
20 ng input H1650
(EGFR-E746_A750del)
2 ng input H1650
(EGFR-E746_A750del)
 Using 2 ng (from ~1 ml plasma) the sensitivity decreases 10x
 For sensitivity of 0.1/0.01% at least 10ml plasma is needed per test !!!
PA UMCG 2015/16 Qubit
12
Other methods to detect low copy mutations
in plasma ctDNA at UMCG
• Digital droplet PCR (BioRad)
• Cobas EGFR mutation plasma test (launched nov 2015) (reflab)
• Biocartis (in development; reflab) for EGFR, BRAF, KRAS mutation
• NGS (IonTorrent-in house-panel) and with Maier (Leipzig), Bubnoff
(Freiburg), Dinjens (ErasmusMC) and Ligtenberg (RadboudMC)
Quality and assessment of proficiency
• Proper control and reference samples (artificial)
• Spiking plasma with plasmid or degraded cellular DNA
Assessment:
• Processing plasma and ctDNA extraction (no plasma for
so many labs ???)
• Detection of mutations in plasma (IQNPath-pilot-program)
(in development)
• European consensus meeting in summer 2016
Disadvantages of ctDNA testing
• Yield of total cell-free DNA might be low
• To detect low copy-mutations 2-4 ml plasma is needed
(equivalent of 10 ml EDTA tube)
• Because of low copies quantitative assay is needed
• Processing blood/plasma is laboreous
• No standarisation of collection/processing blood plasma
Cell-free, circulating tumor DNA testing in plasma
•
•
•
•
Non-invasive
Routine blood tubes (EDTA buis)
Cheap
Multiple testing
UMCG is reference-lab for EQA-IQNPath, cobas and Biocartis
13
Illumina Launches GRAIL, Focused on Blood-Based Cancer Screening
MONDAY, JANUARY 11, 2016
Dank voor uw aandacht
[email protected]
Illumina has joined investors that include Bill Gates and Jeff Bezos in committing more
than $100 million toward launching a new company with the ambitious goal of
screening for the most frequent types of early-stage cancers through a simple test that
measures circulating tumor DNA (ctDNA) in the blood.
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