A new microarray design used as a universal cancer diagnostic tool for detection of fusion genes

Rolf I. SkotheimpHealth, June 26, 2009

Department of Cancer PreventionInstitute for Cancer Research

The Norwegian Radium Hospital, Oslo University Hospital

• Caused by e.g. chromosomal translocations, deletions, and inversions.

• Particularly common in haematological cancers, sarcomas, and prostate cancer.

• Identification of certain fusion genes are currently performed for differential diagnosis or therapeutic decision-making.

• Several technological limitations.

Fusion genes in cancer

Fusion genes in prostate cancer

SLC45A3

TMPRSS2

HERV-K_22q11.23

C15orf21

HNRPA2B1

ERG

ETV1

ETV4

References: Chinnaiyan and coworkers: Science 2005, Cancer Res. 2006, Nature 2007, Cancer Res. 2008 & Hermans et al.,

Cancer Res., 2008. Rickman et al., Cancer Res. 2009

ETV5

CANT1

KLK2

FOXP1

EST14

HERVK17

ELK4

Fusion gene microarray

Fusion gene microarray

Pilot design, fusion gene microarray

• Databases/literature– 275 known fusion genes at time of pilot array design

• Sequences and exon annotation from Biomart.org• Generation of chimeric sequences (~60 000)

– Automised by script programmed in Python

• Oligo design– 34-40mers– Chimeric oligos with matching Tm from up- and downstream

fusion partners– Intragenic oligos

• Microarray platform:

Production of the oligo microarray

• Custom manufacturing• Variable lengths isothermic probes• Maximum of 2.1 million oligos per slide• Pilot microarray with 4 x 70 000 oligos

digital micromirrors

Samples in pilot

Proof of principle– selected samples with one known fusion gene each

• Two leukaemia cell lines– RCH-ACV with TCF3:PBX1– REH with ETV6:RUNX1

• Four prostate cancers, all with TMPRSS2:ERG

Visualisation of results

exons TMPRSS2

exonsERG

exons ERG

chimeric oligos

before

after

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1

2

3

4

5

6

7

8

9

10

11

1 2 3 4 5 6 7 8 9 10 11

intragenic oligos(longitudinal profiles)A prostate cancer sample with

TMPRSS2:ERG fusion gene

Visualisation of results

exons TCF31 2 3 4 5 6 7 8 9 10 11 12 13 14

exonsPBX1

1

2

3

4

5

6

7

8

15 16 17 18

chimeric oligos

before

afterbefore

after

exons TCF3 exons PBX1

relativeexpression

A leukaemic cell line with TCF3:PBX1 fusion gene

PBX1, exon 3TCF3, exon 15

TCF3 PBX1

TCF3:PBX1 chimeric sequence. Validation by cDNA sequencing.

Automated scoringFor each possible exon-exon junction between up- and downstream

genes (A and B genes):

Fusion score = C * P(B-tr) * P(B-ex)

C = normalised expression value for chimeric junction oligo.P(B-tr) and P(B-ex) = Probabilities that the B-gene has a breakpoint at the same site.

T-test based on all probes in transcript [P(B-tr)] and based on probes on the immediately proximal exons [P(B-ex)].

http://www.molecular-cancer.com/content/8/1/5Pilot:

AcknowledgementsOslo University HospitalRikshospitalet

Norwegian Radium HospitalInstitute for Cancer ResearchDepartment of Cancer Prevention

Marthe EkenGard O. S. ThomassenLina CekaiteTrude H. ÅgesenGuro E. LindStine Aske DanielsenMette EknæsSharmini AlagaratnamAnita SveenRagnhild A. Lothe

Department of Cancer GeneticsFrancesca MicciSverre Heim

Inst. Medical Microbiology / CMBNGard O. S. ThomassenTorbjørn Rognes

Portuguese Oncology InstituteFranclim R. RibeiroManuel R. Teixeira

www.rr-research.no/cancerprevention