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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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