Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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 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 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 PAX7 ba - NOT split Signal pattern 3F and 4F 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 PAX7 ba - NOT split Signal pattern – multiple fusion signals 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 A B C D E Type I (327 bp) EWS-FLi1 cDNA check Interesting case 1 cont. Diminished FLI1 signal Fusion on der(22) FLI1 on Normal 11 EWS on Normal 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) 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 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 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 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