Extending FISH Extending FISH Analysis of Paediatric Analysis of Paediatric

TumoursTumoursRachel NewbyRachel Newby

Trainee CytogeneticistTrainee Cytogeneticist

Northern Genetics ServiceNorthern Genetics Service

Paediatric TumoursPaediatric Tumours

Alveolar RhabdomyosarcomasAlveolar Rhabdomyosarcomas Ewing’s Tumour Ewing’s Tumour

Diagnosis - critical for correct Diagnosis - critical for correct treatmenttreatment

- specific translocations- specific translocations - classical cytogenetics problematic- classical cytogenetics problematic FISH and RT-PCR are pivotal in FISH and RT-PCR are pivotal in

getting CORRECT diagnosisgetting CORRECT diagnosis

A-RMSA-RMS

t(2;13)(q35;q14) & t(1;13)(p36;q14)t(2;13)(q35;q14) & t(1;13)(p36;q14) Vysis FKHR (FOXO1) - 13q14, PAX3 - 2q35, Vysis FKHR (FOXO1) - 13q14, PAX3 - 2q35,

PAX7 - 1p36PAX7 - 1p36

Zytovision RMSI t(2;13) & RMSII t(1;13)Zytovision RMSI t(2;13) & RMSII t(1;13)

Problems with current A-RMS Problems with current A-RMS FISH probesFISH probes

FKHR not always split! - other FKHR not always split! - other rearrangements involving PAX genes rearrangements involving PAX genes but not FKHR existbut not FKHR exist

- AFX1-PAX3 fusion (AFX1 Xq13)- AFX1-PAX3 fusion (AFX1 Xq13)

- NCOA1-PAX3 - 2p23 in t(2;2)(q35;p23) - NCOA1-PAX3 - 2p23 in t(2;2)(q35;p23)

Risk of FALSE negative resultsRisk of FALSE negative results

Solution?Solution?

‘‘breakapart’ probes - PAX3 (2q35) breakapart’ probes - PAX3 (2q35) and PAX7 (1p36)and PAX7 (1p36)

Identify rearrangements NOT Identify rearrangements NOT involving FKHR.involving FKHR.

BACs - Ensembl -> FISH probesBACs - Ensembl -> FISH probes Verified by FISH and PCR Verified by FISH and PCR

Test casesTest casesPAX3 +ve control case

Cell line RH30

RT-PCR - FKHR/PAX3 +ve

PAX7 +ve control case

M05/22

RT-PCR – FKHR/PAX7 +ve

der(13)

der(2)

Normal 2

der(1)

der(13)

Normal 1

Interesting Case 1 Interesting Case 1

Case received from NottinghamCase received from Nottingham Pathology – A-RMSPathology – A-RMS Complex karyotypeComplex karyotype No visible t(2;13) or t(1;13) No visible t(2;13) or t(1;13)

translocationstranslocations Vysis FKHR ‘breakapart’ probe NOT split Vysis FKHR ‘breakapart’ probe NOT split

Rearrangement which does not involve Rearrangement which does not involve FKHR?FKHR?

PAX3 and PAX7 resultsPAX3 and PAX7 results

PAX7 ba - NOT split

Signal pattern 3F and FF

PAX3 ba - NOT split

Signal pattern 3F and 4F

Interesting Case 2Interesting Case 2

Case NG – 5 year old boy - ?A-RMSCase NG – 5 year old boy - ?A-RMS FISH – FKHR NOT split 100% cellsFISH – FKHR NOT split 100% cells RT-PCR – NO PAX3-FKHR or PAX7-RT-PCR – NO PAX3-FKHR or PAX7-

FKHR fusion transcriptFKHR fusion transcript

Rearrangement which does not Rearrangement which does not involve FKHR?involve FKHR?

PAX3 and PAX7 resultsPAX3 and PAX7 results

PAX7 ba - NOT split

Signal pattern multiple fusions

PAX3 ba - NOT split

Signal pattern – multiple fusion signals

A-RMS resultsA-RMS results

No novel PAX3 and PAX7 No novel PAX3 and PAX7 rearrangements discovered rearrangements discovered yet...yet...

Ewing’s TumourEwing’s Tumour

Ewing’s– t(11;22)(q24;q12)Ewing’s– t(11;22)(q24;q12) - accounts for 85% of cases - accounts for 85% of cases - EWS (22q12) and FLI1 (11q24) - EWS (22q12) and FLI1 (11q24) 10-15 % cases t(21;22)(q22;q12) – EWS and ERG 10-15 % cases t(21;22)(q22;q12) – EWS and ERG

(21q22)(21q22)

Problems with current EWS Problems with current EWS FISHFISH

EWS probe splits - which other EWS probe splits - which other chromosome is involved?chromosome is involved?

EWS is not always split!EWS is not always split! Risk of false negative result! Risk of false negative result!

- detrimental effect on treatment & - detrimental effect on treatment & patient prognosis.patient prognosis.

Solution?Solution?

Fusion probes for common EWS Fusion probes for common EWS partnerspartners

- EWS-FLI1- EWS-FLI1

- EWS-ERG- EWS-ERG ERG ‘breakapart’ probe for ERG ‘breakapart’ probe for

rearrangements of 21q22.rearrangements of 21q22. Useful when no metaphases or EWS Useful when no metaphases or EWS

NOT splitNOT split

Interesting case 1Interesting case 1

ZA – 16 year old girlZA – 16 year old girl 46,XX,add(16)(q13) 46,XX,add(16)(q13) EWS probe NOT EWS probe NOT

splitsplit RT-PCR +ve EWS-RT-PCR +ve EWS-

FLI1FLI1 Birmingham FISH - Birmingham FISH -

EWS-FLI1 POSITIVEEWS-FLI1 POSITIVE Confirmed with new Confirmed with new

EWS-FLI1 probeEWS-FLI1 probe

EWS-FLi1 cDNA check

Type I (327 bp)

A B C D E

Interesting case 1 cont.Interesting case 1 cont.

EWS-FLI1 +ve but EWS-FLI1 +ve but EWS did not split!EWS did not split!

? Portion of FLI1 ? Portion of FLI1 inserted into EWSinserted into EWS

No RT-PCR, No No RT-PCR, No FISH = false FISH = false negative resultnegative resultEWS on Normal

22

Diminished FLI1 signal

FLI1 on

Normal 11

Fusion on der(22)

Interesting Case 2Interesting Case 2

Cell-Line – CADO-ESCell-Line – CADO-ES

47,XX,dup(1)(q2?47,XX,dup(1)(q2?5q42),+8,i(8)5q42),+8,i(8)(q10),add(18)(p11)(q10),add(18)(p11)

EWS probe - EWS probe - abnormal Signal abnormal Signal pattern – FFRpattern – FFR

Metaphase – extra Metaphase – extra red onred on

G-group chromosomeG-group chromosome

Vysis EWS – ‘breakapart’ probe

Signal pattern - FFR

FF – 2 x Normal 22’s

?

Interesting Case 2 cont.Interesting Case 2 cont.

EWS-ERG +ve by EWS-ERG +ve by PCRPCR

EWS-ERG ‘in-EWS-ERG ‘in-house’ probe - house’ probe - Signal pattern Signal pattern FRGGFRGG

Portion of EWS has Portion of EWS has inserted into ERGinserted into ERG

der(21)

ERG on

Normal 21

EWS on

Normal 22

Fusion on der(21)

ERG on 21

EWS on 22

Diminished EWS signal

Ewing’s resultsEwing’s results

EWS-FLI1 and EWS-ERG fusion EWS-FLI1 and EWS-ERG fusion probesprobes

- good results on positive controls and - good results on positive controls and archived casesarchived cases

ERG ‘breakapart’ probe did not split! ERG ‘breakapart’ probe did not split! WHY? WHY?

Would expect ERG to split in ~ 10% Would expect ERG to split in ~ 10% casecase

ERGERG

ERG 3’ to 5’ERG 3’ to 5’ EWS 5’ to 3’EWS 5’ to 3’ ERG inverted for in-frame fusion ERG inverted for in-frame fusion

gene with EWSgene with EWS More complex than a translocationMore complex than a translocation EWS or ERG translocates by EWS or ERG translocates by

insertion-invertion mechanisminsertion-invertion mechanism ERG never split?ERG never split?

SummarySummary

Probes will benefit the service we Probes will benefit the service we provideprovide

PAX3 and PAX7 – to be used PAX3 and PAX7 – to be used routinely on new cases routinely on new cases

PAX probes - FKHR is not split.PAX probes - FKHR is not split.

Reduce - false negative resultsReduce - false negative results

SummarySummary

ERG – better understanding of ERG – better understanding of complexitycomplexity

EWS split -> EWS-FLI1 and EWS-ERG EWS split -> EWS-FLI1 and EWS-ERG Increased confidenceIncreased confidence MicroinsertionsMicroinsertions Commercial probes - a false negative Commercial probes - a false negative

result result

Without extended FISH or RT-PCR = Without extended FISH or RT-PCR = PITFALLSPITFALLS

AddendumAddendum

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 EnsemblTable 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

Published Bacterial artificial chromosomes (BAC) clones and P1-derived artificial Published Bacterial artificial chromosomes (BAC) clones and P1-derived artificial chromosomes (PAC) clones were selected for use in this project, and BAC clones chromosomes (PAC) clones were selected for use in this project, and BAC clones mapping to specific genes of interested were also identified using ‘Ensembl’ (mapping to specific genes of interested were also identified using ‘Ensembl’ (www.ensembl.orgwww.ensembl.org). The BACs selected are listed in Table). The BACs selected are listed in Table

AddendumAddendum

BAC’s were ordered from BACPAC CHORI (Children’s Hospital Oakland Research 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 Institute) grown up and DNA extracted using the Qiagen plasmid preparation kit and then fluorescent labelled with either SpectrumOrange or SpectrumGreen using the then fluorescent labelled with either SpectrumOrange or SpectrumGreen using the Vysis Nick translation Kit.Vysis Nick translation Kit.

Ref:Ref: Danielle C. Shing, Dominic J. Mc.Mullan, Paul Roberts, Kim Smith, Suet-Feung Chin, 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 James Nicholson, Roger M. Tillman, Pramila Ramani, Catherine Cullinane, and Nicholas Coleman. FUS/ERG Gene fusions in Ewing’s Tumours, Coleman. FUS/ERG Gene fusions in Ewing’s Tumours, Cancer Research 63Cancer Research 63, 4568-, 4568-4576, August 1, 2003.4576, August 1, 2003.

Jun Nishio, Pamel A Althof, Jacqueline M Bailey, Ming Zhou, JamesR Neff, Frederic G 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 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 FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma. rhabdomyosarcoma. Laboratory Investigation (2006) 86Laboratory Investigation (2006) 86, 547-556., 547-556.

Georges Maire, Christopher W. Brown, Jane Bayani, Carlos Pereira, Denis H. Gravel, Georges Maire, Christopher W. Brown, Jane Bayani, Carlos Pereira, Denis H. Gravel, John C. Bell, Maria Zielenska, Jeremy A. Squire. Complex rearrangement of John C. Bell, Maria Zielenska, Jeremy A. Squire. Complex rearrangement of chromosomes 19, 21, and 22 in Ewing sarcoma involving a novel reciprocal inversion-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 insertion mechanism of EWS-ERG fusion gene formation; a case analysis and literature review. review. Cancer Genetics and Cytogenetics 181 (2008)Cancer Genetics and Cytogenetics 181 (2008) 81-92 81-92

AcknowledgementsAcknowledgements

Thanks toThanks to

- Nick Bown- Nick Bown

- Fiona Harding- Fiona Harding

- Steve Hellens- Steve Hellens

- Malignancy Section at the Northern - Malignancy Section at the Northern Genetics ServiceGenetics Service

- Meg Heath, Kate Martin & Tom McCulloch - Meg Heath, Kate Martin & Tom McCulloch Nottingham Cytogenetics labNottingham Cytogenetics lab

- Dom McMullan – Birmingham - Dom McMullan – Birmingham Cytogenetics LabCytogenetics Lab