Extending FISH Analysis of Paediatric Tumours

Rachel Newby Trainee Cytogeneticist Northern Genetics Service

Paediatric Tumours

 

Alveolar Rhabdomyosarcomas Ewing’s Tumour



Diagnosis - critical for correct treatment

- specific translocations - classical cytogenetics problematic  FISH and RT-PCR are pivotal in getting CORRECT diagnosis

A-RMS

  t(2;13)(q35;q14) & t(1;13)(p36;q14) Vysis FKHR (FOXO1) - 13q14, PAX3 - 2q35, PAX7 - 1p36  Zytovision RMSI t(2;13) & RMSII t(1;13)

Problems with current A-RMS FISH probes

 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)  Risk of FALSE negative results

Solution?

 ‘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 Cell line RH30 RT-PCR - FKHR/PAX3 +ve der(13) PAX7 +ve control case M05/22 RT-PCR – FKHR/PAX7 +ve der(13) der(1) der(2) Normal 2 Normal 1

Interesting Case 1

 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  Rearrangement which does not involve FKHR?

PAX3 and PAX7 results

PAX3 ba - NOT split Signal pattern 3F and 4F PAX7 ba - NOT split Signal pattern 3F and FF

Interesting Case 2

 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 Signal pattern – multiple fusion signals PAX7 ba - NOT split Signal pattern multiple fusions

A-RMS results

 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)

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.

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

Interesting case 1

      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 Type I (327 bp)

A B C

EWS-FLi1

D E

cDNA check

Interesting case 1 cont.

Diminished FLI1 signal FLI1 on Normal 11 EWS on Normal 22 Fusion on der(22 )    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 ?

FF – 2 x Normal 22’s Vysis EWS – ‘breakapart’ probe Signal pattern - FFR

Interesting Case 2 cont.

   EWS-ERG +ve by PCR EWS-ERG ‘in-house’ probe - Signal pattern FRGG Portion of EWS has inserted into ERG der(21) Fusion on der(21) ERG on Normal 21 ERG on 21 EWS on Normal 22 Diminished EWS signal 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

ERG

 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

 Probes will benefit the service we provide  PAX3 and PAX7 – to be used routinely on new cases  PAX probes - FKHR is not split.

 Reduce - false negative results

Summary

     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

 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 RP11-71J24 RP11-384O8 RP11-16P6 RP1-93P18 RP1-8B22 RP11-476D17 RP11-95I21

a

RP11-24A11 RP11-153L15 Clone BAC BAC BAC PAC PAC BAC BAC BAC BAC Location Proximal portion of PAX3 locus Distal portion of PAX3 locus Distal portion of PAX3 locus Proximal portion of PAX7 locus Distal portion of PAX7 locus Proximal to ERG locus Distal to ERG locus Proximal portion of ERG locus Distal portion of ERG locus Label SpectrumOrange SpectrumGreen SpectrumGreen SpectrumOrange SpectrumGreen SpectrumOrange SpectrumGreen SpectrumOrange SpectrumGreen Paper Nishio et al (2006) Nishio et al (2006) Ensembl Nishio et al (2006) Nishio et al (2006) Shing et al (2003) Shing et al (2003) Ensembl Ensembl RP1-259N9 RP4-760G15 RP1-74J1 RP11-612D3 BAC BAC BAC BAC Proximal to WT1 Distal portion to WT1 locus Spans WT1 locus Spans EWS locus SpectrumOrange SpectrumGreen SpectrumOrange SpectrumGreen Ensembl Ensembl Ensembl Ensembl RP11-744N12 RP11-760G3 b BAC BAC Spans FLI1 locus Proximal to FLI1 locus SpectrumOrange SpectrumOrange Ensembl Ensembl RP11-405P15 b 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

 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.

  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.

 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.

 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

 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