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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