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 Getting the diagnosis right between alveolar Rhabdomyosarcoma and Ewing’s sarcoma is critically important for selecting the right treatment regime to give the patient the best chance of survival. It’s well established that finding specific translocations in the tumour cells can be a powerful aid to the Pathologist in making the diagnosis. Classical cytogenetics, however, is an unreliable approach, so it’s necessary to supplement it with other techniques such as FISH and RT-PCR.

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) The translocations involved in aveolar Rhabdos are either 1;13 or 2;13 These fuse Forkhead at 13q14 to either PAX3 at 2q35 or PAX7 at 1p36. there is a commercial FISH probe available - the Vysis FKHR ‘breakapart’ probe, which registers splitting of 13q14 by either of these translocations There are also single fusion probe sets available from Zytovision which can specifically identify the individual translocations.

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 Problem – small minority of alveolar rhabdos don’t show FKHR involvement, i.e. FKHR is not split! Other rearrangements have been published in alveolar rhabdos which do not involve FKHR but do involve PAX genes and other members of the Forkhead family. So by relying on FKHR FISH, we are potentially overlooking rare – but diagnostically crucial – alternative translocations.

Solution? ‘breakapart’ probes - PAX3 (2q35) and PAX7 (1p36) Identify rearrangements NOT involving FKHR. BACs - Ensembl -> FISH probes Verified by FISH and PCR I set out to design PAX breakapart probes using BAC clones for PAX3 and PAX7. BAC clones were selected from Ensembl. And labelled for FISH. The identities of the BACs and technical details are in an addendum slide at the end of this presentation Probes were tested on normal chromosomes and the BAC DNA was verified by PCR.

Test cases PAX3 +ve control case Cell line RH30 RT-PCR - FKHR/PAX3 +ve M05/22 RT-PCR – FKHR/PAX7 +ve der(13) der(2) Normal 2 der(1) der(13) Normal 1 The PAX3 probe was tested on rhabdo cell line RH30 which is known to have the FKHR-PAX3 gene fusion. PAX7 was tested on a case known to have FKHR-PAX7 fusion. In each case, as hoped, the fusion signal was present on chromosomes 2 and 1 and separate red and green signals were seen on the translocation products So appears that we have created a functioning pair of breakapart FISH probes

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? So we then went on to test them on archived cases: First case of interest was kindly supplied by the Nottingham lab. – the Pathology on the case suggested that an alveolar rhabdo was the most favoured diagnosis However, on chromosome analysis it had a complex karyotype but no visible 1;13 or 2;13 rearrangements and the FKHR probe was not split. Could this be a case where there is a rearrangement associated with a Rhabdo but no rearrangement of Forkhead??

PAX3 and PAX7 results PAX3 ba - NOT split Signal pattern 3F and 4F Signal pattern 3F and FF Tested the case with the PAX probes – PAX3 ‘breakapart’ did not split and the signal pattern showed cells to contain either four fusions or three fusions. PAX7 also did not split – and the signal pattern showed cells to have either 3 fusion signals or 2 fusion signals. This was the end of the road with this case, i.e. if really an alveolar RMS, then the cytogenetics is unresolved and remains intriguing.

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? So lets look at another case – NG Failed on G-banding Forkhead FISH showed that the signal was intact in 100% of cells i.e. the signal was not spit And RT-PCR result showed no evidence of a PAX3-FKHR or PAX7-FKHR fusion transcript. So another contender for a possible novel translocation or novel PAX gene fusion

PAX3 and PAX7 results PAX3 ba - NOT split Signal pattern – multiple fusion signals PAX7 ba - NOT split Signal pattern multiple fusions With PAX3 the signal was not split but there were multiple fusion signals present PAX7 – also did not split and again multiple fusion signals were present – suggesting multiple copies of chromosome 1’s and 2’s.

A-RMS results No novel PAX3 and PAX7 rearrangements discovered yet... Disappointingly, as yet we haven’t discovered one of these rare alternative translocations in an alveolar Rhabdo. But we feel happier now because having these probes may help avoid a false-negative pitfall in the diagnosis of a future case.

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) Turning to Ewing’s Tumour – These are defined by specific translocations, most commonly 11;22 translocation and this involves the EWS gene at 22q12 and the FLI1 gene at 11q24 A further 10-15% of cases are caused by fusion of EWS and ERG on 21q22 Currently there is a Vysis EWS breakapart probe to look for rearrangements in 22q12, which is split by the 22q12 translocation.

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. Of course, a breakapart probe doesn’t tell you the translocation partner But most importantly there is a risk of giving a false negative result as some EWS gene fusions do not affect the FISH probe This could contribute to a mis-diagnosis, with the patient perhaps receiving inappropriate and ineffective treatment.

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 So we decided to refine our FISH capabilities for Ewing’s Sarcoma by developing fusion probes for the common translocations, and also a breakapart probe for ERG. The probes were tested on positive control cases and then used on some of our archived interesting cases.

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 EWS-FLi1 cDNA check Type I (327 bp) A B C D E Case number one was a 16 year old girl with a likely Ewing’s tumour G- banding showed her tumour to have an add(16)(q) – but chromosomes 11 and 22 looked normal. The Vysis EWS FISH probe was not split suggesting there was no rearrangement in EWS! This might have been reported as not consistent with Ewing’s as it does not contain a EWS rearrangement – However, in Newcastle we carry out RT-PCR and surprisingly the patient was shown to be positive for an EWS-FLI1 fusion transcript! We knew that Dom McMullan and colleagues in Birmingham had EWS-FLI1 FISH probes and they kindly agreed to FISH some of our cell pellet – this confirmed that the tumour was indeed EWS-FLI1 positive So we then tested out newly designed EWS-FLI1 probes on this case

Interesting case 1 cont. EWS-FLI1 +ve but EWS did not split! EWS on Normal 22 Diminished FLI1 signal FLI1 on Normal 11 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 On interphase FISH – Green represents the normal EWS on chromosome 22 Red is normal FLI1 on chromosome 11 Here’s the Fusion is EWS-FLI1– since the 22s were normal by chromosome analysis, this is a cryptic insertion of FLI1 into EWS. In fact, in the majority of cells one of the red signals looks smaller than the other – presumably this is the origin of the relocated FLI1 sequences Alarmingly in this case, EWS did not split so without RT-PCR and confirmation FISH this case would have been reported as not containing an EWS rearrangement – 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 Vysis EWS – ‘breakapart’ probe Signal pattern - FFR FF – 2 x Normal 22’s ? The second case is a Ewing’s cell line – CADO-ES on chromosome analysis, chromosomes 11, 21 & 22 are all visibly normal. EWS breakapart probe gave an abnormal signal pattern. Suggesting two normal chromosome 22’s with EWS intact so what is the extra red signal? From looking at metaphases it appeared to be on a G-group chromosome.

Interesting Case 2 cont. EWS-ERG +ve by PCR der(21) ERG on Normal 21 EWS on Normal 22 Fusion on der(21) ERG on 21 EWS on 22 EWS-ERG +ve by PCR EWS-ERG ‘in-house’ probe - Signal pattern FRGG Portion of EWS has inserted into ERG By RT-PCR this case was positive for EWS-ERG. So you might have expected some visible abnormality of 21 or 22 in the karyotype – not so. We then used the newly developed in-house probes to look at the case. This confirmed an EWS-ERG fusion seen here in both metaphase and interphase Green signal is EWS on chromosome 22, Red is ERG on chromosome 21 Fusion EWS-ERG on the derivative chromosome 21. This time in the majority of cells one of the green signals looks slightly diminished – a portion of the EWS gene has been inserted into ERG. So the previous tumour shows cryptic insertion of FLI1 in to EWS. This cell line shows cryptic insertion of EWS into ERG. Diminished EWS signal

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 The EWS-FLI1 and EWS-ERG fusion probes seem to work well However, the ERG breakapart did not split in any of the cases we tried it on In THE 10% of tumours which have EWS-ERG fusion rather than EWS-FLI1, surely the ERG signal should be split? Why isn’t this so?

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? ERG is not as simple as it first appears! It’s is arranged in the opposite orientation to EWS: 3’ to 5’ rather than 5’ to 3’ For ERG to form an in-frame fusion gene with EWS it must be inverted, so the rearrangements tend to be more complex than simple reciprocal translocations and must involve inversion-insertion mechanisms. This suggests that our ERG breakapart probe will only very rarely be split even by genuine ERG rearrangements?

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 In summary – although the rhabdo side of project didn’t turn up any exciting new translocations, we now have PAX3 and PAX7 probes which will be used routinely in the lab on new cases, with the potential benefits which I’ve already mentioned

Without extended FISH or RT-PCR = PITFALLS 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 For Ewing's Sarcoma, our attempt to develop an ERG ba probe was – On the face of it - unsuccessful and this reflects the complex nature of ERG rearrangements. On the other hand, EWS-FLI1And EWS-ERG fusion probes were developed quite straightforwardly and will increase our diagnostic confidence from now on. We also turned up further evidence that both EWS-FLI1 and EWS-ERG fusions can result from microinsertions which are both cytogenetically cryptic and not visible by Vysis EWS breakapart FISH. Pitfalls like this need to be kept in mind when analysing these type of tumours.

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 Distal portion of PAX3 locus SpectrumGreen RP11-16P6 Ensembl RP1-93P18 PAC Proximal portion of PAX7 locus RP1-8B22 Distal portion of PAX7 locus RP11-476D17 Proximal to ERG locus Shing et al (2003) RP11-95I21a Distal to ERG locus RP11-24A11 Proximal portion of ERG locus RP11-153L15 Distal portion of ERG locus RP1-259N9 Proximal to WT1 RP4-760G15 Distal portion to WT1 locus RP1-74J1 Spans WT1 locus RP11-612D3 Spans EWS locus RP11-744N12 Spans FLI1 locus RP11-760G3b Proximal to FLI1 locus RP11-405P15b Distal to FLI1 locus Table shows the BACs selected and the probe sets for identifying Rhabdomyosarcomas and Ewing;s tumours. This BAC was also labelled SpectrumOrange for use in the EWS-ERG fusion probe set. 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