Genpanels in de hematologie en oncologie1 genpanels in de hematologie en oncologie peter...
Transcript of Genpanels in de hematologie en oncologie1 genpanels in de hematologie en oncologie peter...
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genpanels in de hematologie en
oncologie
peter vandenberghe
centrum voor menselijke erfelijkheid, UZ
Leuven
544-496 v.C.
The Art of War
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Nowell and Hungerford, J Natl Canc Inst, 1960;
Science 132, 1960, 1497.
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NGS applications• DNA
• Whole genome sequencing (WGS)
• Whole exome sequencing (WES)
• Targeted resequencing
• Captured genes
• Amplicons
• Copy number profiling
• RNA sequencing
• Point mutations and fusion transcripts
• Gene expression profiling
• …
The mutational landscape in acute myeloid leukemia
The Cancer Genome Atlas Research Network. N Engl J Med 2013;368:2059-2074.
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The Cancer Genome Atlas Network Nature 487, 330-337 (2012) doi:10.1038/nature11252
The mutational landscape in human CRC
The traditional PCR workflow ziekte patiënt 1 patiënt 2 patiënt 3
gen A mutatie a test 1 test 1 test 1
mutatie b test 2 test 2 test 2
gen B mutatie c test 3 test 3 test 3
gen C mutatie d test 4 test 4 test 4
mutatie e test 5 test 5 test 5
mutatie f test 6 test 6 test 6
gen D mutatie g test 7 test 7 test 7
……. ……. ……. ……. …….
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NGS applications• DNA
• Whole genome sequencing (WGS)
• Whole exome sequencing (WES)
• Targeted resequencing
• Captured genes
• Amplicons
• Copy number profiling
• RNA sequencing
• Point mutations and fusion transcripts
• Gene expression profiling
• …
Targeted resequencing
workflow
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Keuze van doelwitten • “Must have”
o Diagnostisch criterium
o Diagnostische “standard of care”
• Therapeutische keuzes, bv terugbetaling van specifieke doelgerichte medicatie
• Prognostische waarde en geassocieerde therapeutische opties
• “Nice to have”
o Ondersteunende diagnostische waarde
o Ondersteuning bij therapeutische beslissingen
o Inclusie in baskettrials met experimentele therapeutica
• “Very long shots”
Solid tumor panels EGFR 18-21 MASTR
GIST MASTR
SOMATIC 1 MASTR v2
HaloPlex Cancer
Research Panel (~50)
GeneRead DNAseq Targeted Panels V2 (8-160 genen)
Ion AmpliSeq™ Cancer
Hotspot Panel v2 (50)
Ion AmpliSeq™
Comprehensive
Cancer Panel (~409)
Ion AmpliSeq™ Colon
and Lung Cancer
Research Panel v2 (22)
Tumor Hotspot Mastr Plus
SOMATIC 2 MASTR +
TruSight™
Cancer (~100)
TruSight
Tumor 26
TruSeq® Amplicon
Cancer Panel (48)
TruSight
Tumor 15
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KIT en/of PDGFRA(vanaf juli 2015)
EGFR(jan-dec 2015)
KRAS/NRAS/BRAF(jan-dec 2015)
Maligne melanoma n=10
7 (WT)
2 (1 mutatie)
1 (2 mutaties)
n=160
36 (WT)
4 (NOK)
73 (1 mutatie BRAF) (46%)
1 (1 mutatie KRAS)
46 (1 mutatie NRAS) (29%)
GIST n=28
5 (WT)
20 (1 mutatie) (70%)
3 (2 mutaties)
longadenocarcinoma n=216
165 (WT)
4 (NOK)
36 (1 mutatie) (16%)
11 (2 mutaties) (5%)
pre-screening KRAS (WTB-PCR)
mCRC n=423
151 (WT)
33 (NOK) (8%)
37 (1mutatie BRAF) (9%)
181 (1 mutatie KRAS) (43%)
16 (1 mutatie NRAS) (4%)
5 (2 mutaties)
Papillair schildkliercarcinoma n=27
11 (WT)
1 (NOK)
12 (1 mutatie BRAF) (44%)
1 (1 mutatie KRAS)
2 (1 mutatie NRAS)
ISO 15189http://economie.fgov.be/nl/ondernemingen/leven_onderneming/kwaliteitsbeleid/Accreditatie/
Stalen NGS 2015 (n= 864) – CME/PO
TruSight Tumor panel 26 (Illumina)
EGFR 18-21 MASTR: 4 exons
SOMATIC 1 MASTR v2: 9 exons
26 genen
82 exonen
174 amplicons
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Hematology panels
TruSight Myeloid
Sequencing Panel (54 genen, 568 amplicons)
Clear Seq AML (20 genen)
GeneRead DNAseq Targeted Panels V2
(myeloid neoplasms 50 genen, 2536 amplicons)
Ion AmpliSeq™ AML
Research Panel (19 genen, 264 amplicons)
CLL Mastr Plus (9 genen, 251 amplicons)
Uitdagingen• Technisch
o FFPE-materiaal voor solide tumoren
o Beperkte hoeveelheid materiaal – voor diverse diagnostische analyses
• Kosten en RIZIV-terugbetaling
• Multipele mutaties en moleculaire diversiteit in “pathologische entiteiten”
o tussen patiënten, “long tail of cancer”
o binnen patiënten (intralesioneel, interlesioneel)
o tumorselectie en –evolutie, spontaan en onder therapie
• Herkenning van nieuwe premaligne entiteiten “clonal hematopoiesis of
indeterminate potential” (CHIP)
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clonal hematopoiesis of indeterminate potential (CHIP)
Genovese G et al. N Engl J Med 2014;371:2477-2487.
Dosis sola facit venenum (Paracelsus)
1971 : carpet bombing
2001: targeted therapy
2010 : personalised therapy
20?? : precision medicine
Precision diagnosisPersonalized drug development
Precision treatment
Conclusie
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targeted resequencing in oncology• Multiplicom MASTRs (PCR-based) on Illumina MiSeq platform:
• EGFR (exon 18-21)
• SOMATIC1v2 (KRAS exon 2-3-4/ NRAS exon 2-3-4/ BRAF exon 15)
• GIST MASTR (KIT exons 9, 11, 13, 14, 15, 16, 17/ PDFGRA exons 8, 10, 12, 14, 18).
• Detection limit: 5% mutant alleles (10% tumor cells)
• Workflow in the molecular lab; 2 runs/w
o Reception of sample from pathology lab
o Day 1&2: DNA extraction → Qubit → qPCR (Illumina)
o Day 3: Multiplicom MASTRs (2-steps PCR protocol) → PCR3
o Day 4: pooling of samples → pooling of different MASTR libraries→ run on Miseq
o Day 5: data analysis and report
o TAT: =<10d
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