Transcript Extending FISH Probes for Paediatric Tumours

Extending FISH Analysis of
Paediatric Tumours
Rachel Newby
Trainee Cytogeneticist
Northern Genetics Service
Paediatric Tumours
Alveolar Rhabdomyosarcomas
 Ewing’s Tumour
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Diagnosis - critical for correct treatment
- specific translocations
- classical cytogenetics problematic
 FISH and RT-PCR are pivotal in getting
CORRECT diagnosis
A-RMS
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t(2;13)(q35;q14) & t(1;13)(p36;q14)
Vysis FKHR (FOXO1) - 13q14, PAX3 - 2q35, PAX7 - 1p36
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Zytovision RMSI t(2;13) & RMSII t(1;13)
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Problems with current A-RMS FISH
probes
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FKHR not always split! - other rearrangements
involving PAX genes but not FKHR exist
- AFX1-PAX3 fusion (AFX1 Xq13)
- NCOA1-PAX3 - 2p23 in t(2;2)(q35;p23)
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Risk of FALSE negative results
Solution?
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‘breakapart’ probes - PAX3 (2q35) and PAX7
(1p36)
Identify rearrangements NOT involving FKHR.
BACs - Ensembl -> FISH probes
Verified by FISH and PCR
Test cases
PAX3 +ve control case
PAX7 +ve control case
Cell line RH30
M05/22
RT-PCR - FKHR/PAX3 +ve
RT-PCR – FKHR/PAX7 +ve
der(13)
der(13)
der(1)
der(2)
Normal 2
Normal 1
Interesting Case 1
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Case received from Nottingham
Pathology – A-RMS
Complex karyotype
No visible t(2;13) or t(1;13) translocations
Vysis FKHR ‘breakapart’ probe NOT split
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Rearrangement which does not involve FKHR?
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PAX3 and PAX7 results
PAX3 ba - NOT split
PAX7 ba - NOT split
Signal pattern 3F and 4F
Signal pattern 3F and FF
Interesting Case 2
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Case NG – 5 year old boy - ?A-RMS
FISH – FKHR NOT split 100% cells
RT-PCR – NO PAX3-FKHR or PAX7-FKHR
fusion transcript
Rearrangement which does not involve FKHR?
PAX3 and PAX7 results
PAX3 ba - NOT split
PAX7 ba - NOT split
Signal pattern – multiple fusion
signals
Signal pattern multiple fusions
A-RMS results
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No novel PAX3 and PAX7 rearrangements
discovered yet...
Ewing’s Tumour
Ewing’s– t(11;22)(q24;q12)
- accounts for 85% of cases
- EWS (22q12) and FLI1 (11q24)
 10-15 % cases t(21;22)(q22;q12) – EWS and ERG (21q22)
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Problems with current EWS FISH
EWS probe splits - which other chromosome is
involved?
 EWS is not always split!
 Risk of false negative result!
- detrimental effect on treatment & patient
prognosis.
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Solution?
Fusion probes for common EWS partners
- EWS-FLI1
- EWS-ERG
 ERG ‘breakapart’ probe for rearrangements of
21q22.
 Useful when no metaphases or EWS NOT split
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Interesting case 1
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ZA – 16 year old girl
46,XX,add(16)(q13)
EWS probe NOT split
RT-PCR +ve EWS-FLI1
Birmingham FISH EWS-FLI1 POSITIVE
Confirmed with new
EWS-FLI1 probe
A
B
C
D
E
Type I (327 bp)
EWS-FLi1
cDNA check
Interesting case 1 cont.
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Diminished FLI1 signal
Fusion on der(22)
FLI1 on
Normal 11
EWS on Normal 22
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EWS-FLI1 +ve but EWS
did not split!
? Portion of FLI1
inserted into EWS
No RT-PCR, No FISH
= false negative result
Interesting Case 2
Cell-Line – CADO-ES
47,XX,dup(1)(q2?5q42),+8,
i(8)(q10),add(18)(p11)
 EWS probe - abnormal
Signal pattern – FFR
 Metaphase – extra red on
G-group chromosome
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?
FF – 2 x Normal 22’s
Vysis EWS – ‘breakapart’ probe
Signal pattern - FFR
Interesting Case 2 cont.
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EWS-ERG +ve by PCR
EWS-ERG ‘in-house’
probe - Signal pattern
FRGG
Portion of EWS has
inserted into ERG
der(21)
Diminished
EWS signal
Fusion on der(21)
ERG on
Normal 21
EWS on
Normal 22
ERG on 21
EWS
on 22
Ewing’s results
EWS-FLI1 and EWS-ERG fusion probes
- good results on positive controls and archived
cases
 ERG ‘breakapart’ probe did not split! WHY?
 Would expect ERG to split in ~ 10% case
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ERG
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ERG 3’ to 5’
EWS 5’ to 3’
ERG inverted for in-frame fusion gene with
EWS
More complex than a translocation
EWS or ERG translocates by insertion-invertion
mechanism
ERG never split?
Summary
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Probes will benefit the service we provide
PAX3 and PAX7 – to be used routinely on new
cases
PAX probes - FKHR is not split.
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Reduce - false negative results
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Summary
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ERG – better understanding of complexity
EWS split -> EWS-FLI1 and EWS-ERG
Increased confidence
Microinsertions
Commercial probes - a false negative result
Without extended FISH or RT-PCR =
PITFALLS
Addendum
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Published Bacterial artificial chromosomes (BAC) clones and P1-derived artificial chromosomes (PAC) clones
were selected for use in this project, and BAC clones mapping to specific genes of interested were also
identified using ‘Ensembl’ (www.ensembl.org). The BACs selected are listed in Table
Probe set
Clone
Location
Label
Paper
RP11-71J24
BAC
Proximal portion of PAX3 locus
SpectrumOrange
Nishio et al (2006)
RP11-384O8
BAC
Distal portion of PAX3 locus
SpectrumGreen
Nishio et al (2006)
RP11-16P6
BAC
Distal portion of PAX3 locus
SpectrumGreen
Ensembl
RP1-93P18
PAC
Proximal portion of PAX7 locus
SpectrumOrange
Nishio et al (2006)
RP1-8B22
PAC
Distal portion of PAX7 locus
SpectrumGreen
Nishio et al (2006)
RP11-476D17
BAC
Proximal to ERG locus
SpectrumOrange
Shing et al (2003)
RP11-95I21a
BAC
Distal to ERG locus
SpectrumGreen
Shing et al (2003)
RP11-24A11
BAC
Proximal portion of ERG locus
SpectrumOrange
Ensembl
RP11-153L15
BAC
Distal portion of ERG locus
SpectrumGreen
Ensembl
RP1-259N9
BAC
Proximal to WT1
SpectrumOrange
Ensembl
RP4-760G15
BAC
Distal portion to WT1 locus
SpectrumGreen
Ensembl
RP1-74J1
BAC
Spans WT1 locus
SpectrumOrange
Ensembl
RP11-612D3
BAC
Spans EWS locus
SpectrumGreen
Ensembl
RP11-744N12
BAC
Spans FLI1 locus
SpectrumOrange
Ensembl
RP11-760G3b
BAC
Proximal to FLI1 locus
SpectrumOrange
Ensembl
RP11-405P15b
BAC
Distal to FLI1 locus
SpectrumOrange
Ensembl
Table shows the BACs selected and the probe sets for identifying Rhabdomyosarcomas and Ewing;s tumours.
(a)This BAC was also labelled SpectrumOrange for use in the EWS-ERG fusion probe set.
(b)These were selected for FLI1 after RP11-744N12 originally hybridised to the wrong chromosome and was FLI1 negative by PCR validation
Addendum
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BAC’s were ordered from BACPAC CHORI (Children’s Hospital Oakland Research Institute) grown up and
DNA extracted using the Qiagen plasmid preparation kit and then fluorescent labelled with either
SpectrumOrange or SpectrumGreen using the Vysis Nick translation Kit.
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Ref:
Danielle C. Shing, Dominic J. Mc.Mullan, Paul Roberts, Kim Smith, Suet-Feung Chin, James Nicholson, Roger
M. Tillman, Pramila Ramani, Catherine Cullinane, and Nicholas Coleman. FUS/ERG Gene fusions in Ewing’s
Tumours, Cancer Research 63, 4568-4576, August 1, 2003.
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Jun Nishio, Pamel A Althof, Jacqueline M Bailey, Ming Zhou, JamesR Neff, Frederic G Barr, David M Parham,
Lisa Teot, Stephen J Qualman and Julia A Bridge. Use of Novel FISH assay on paraffin-embedded tissues as
an adjunct to diagnosis of alveolar rhabdomyosarcoma. Laboratory Investigation (2006) 86, 547-556.
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Georges Maire, Christopher W. Brown, Jane Bayani, Carlos Pereira, Denis H. Gravel, John C. Bell, Maria
Zielenska, Jeremy A. Squire. Complex rearrangement of chromosomes 19, 21, and 22 in Ewing sarcoma
involving a novel reciprocal inversion-insertion mechanism of EWS-ERG fusion gene formation; a case
analysis and literature review. Cancer Genetics and Cytogenetics 181 (2008) 81-92
Acknowledgements
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Thanks to
- Nick Bown
- Fiona Harding
- Steve Hellens
- Malignancy Section at the Northern Genetics Service
- Meg Heath, Kate Martin & Tom McCulloch
Nottingham Cytogenetics lab
- Dom McMullan – Birmingham Cytogenetics Lab