Joanne Mason, Registered Clinical Scientist West Midlands Regional Genetics Laboratory,

1. Acquired isodisomy of chromosome 21 in an acute myeloid leukaemia (AML) patient as an incidental finding during routine chimaerism analysis, and the introduction of a new RUNX1 screening service. Joanne Mason, Registered Clinical Scientist West Midlands Regional Genetics Laboratory, Birmingham Women’s NHS Foundation Trust,

2. Introduction • AML is a genetic disease • Characterised by enhanced proliferation & differentiation block • ~50% cases have cytogenetically visible aberrations • The remaining cases have genetic aberrations which are only detectable at the molecular level • These genetic lesions help to characterise the subtype of leukaemia, and can be used to guide therapeutic decisions and inform prognosis • Molecularly-targeted therapy (e.g. Glivec in CML) Joanne Mason, WMRGL Birmingham

3. Patient A • Diagnosed with AML in May 2006 • Karyotype analysis: trisomy 13 (47,XY,+13 [10]) • Treated with chemotherapy on the MRC AML15 trial protocol • Relapsed November 2007 (47,XY,+13) • Salvage chemotherapy, followed by stem cell transplant (SCT) in March 2008 Joanne Mason, WMRGL Birmingham

4. CAGA CAGA 3-15CAGA CAGA CAGA Chimaerism monitoring post-SCT • Sex-matched SCT patients are monitored for levels of donor and host DNA post-transplant using polymorphic microsatellite markers. • A pre-requisite for chimaerism analysis is to find at least one informative marker that distinguishes donor from host. Joanne Mason, WMRGL Birmingham

5. Multiplex microsatellite marker PCR and subsequent fragment analysis Joanne Mason, WMRGL Birmingham

6. Chimaerism analysis DONOR HOST PRE-TRANSPLANT POST-TRANSPLANT 74% donor 26% host Joanne Mason, WMRGL Birmingham

7. Microsatellite results • Pre-transplant DNA 13 13 13 Joanne Mason, WMRGL Birmingham

8. D21S11 D21S1270 D21S1411 Penta D Chromosome 21 markersAverage ratio 4:1 Remission DNA Relapse DNA Joanne Mason, WMRGL Birmingham

9. Ch 21 markers : copy number change?Cytogenetics 2 normal copies Ch 21 • Possible explanations for the discrepancy: • 1) Sub-microscopic deletion within chromosome 21 (unlikely as multiple deletions would be required) • 2) A cryptic sub-clone with gain or loss of 21 in some cells, not detected by initial cytogenetic analysis (impossible with a microsatellite ratio of 4:1) • 3)Acquired isodisomy (aka acquired uniparental disomy, or copy number neutral loss of heterozygosity) Joanne Mason, WMRGL Birmingham

10. Acquired isodisomy (AID) • Common mechanism of oncogenesis • Prognostic significance in AML? Joanne Mason, WMRGL Birmingham

11. Mitotic Recombination 21 21 AID 21 Joanne Mason, WMRGL Birmingham

12. Acquired isodisomy (AID) • AID is a mechanism by which homozygosity for a mutation can be achieved without detrimental loss or gain of contiguous chromosome material • It is cytogenetically invisible (both chromosomes look the same) and therefore very difficult to detect unless you specifically look for it. • DNA microarrays – sub-microscopic & cryptic changes Joanne Mason, WMRGL Birmingham

13. AID21: What genes might be affected? • RUNX1 21q22.3 • Transcription factor • Most frequent target for chromosomal translocation in leukaemia • Point mutations • in sporadic AML • In familial platelet disorder/AML (FPD/AML) Joanne Mason, WMRGL Birmingham

14. RUNX1 point mutations in sporadic AML • 1.2% of all AML • Highly associated with • AML FAB M0 • trisomy 21 • trisomy 13 (80-100%) [Patient AS 47,XY,+13] • RUNX1 mutation associated with a poor prognosis in MDS (prognosis in AML not yet known) • Discovery of mutations has implications for • Risk adapted therapy • Molecularly targeted therapy Joanne Mason, WMRGL Birmingham

15. Familial Platelet Disorder with Predisposition to Acute Myeloid Leukaemia (FPD/AML) • Rare autosomal dominant disorder • Characterised by inherited thrombocytopenia, platelet function defect and a lifelong risk of myelodysplastic syndrome (MDS) and AML • Caused by heterozygous germline mutations in RUNX1 • Worldwide, only fifteen pedigrees have been reported to date. • In November 2008, request for ?FPD/AML in a West Midlands AML patient. Joanne Mason, WMRGL Birmingham

16. RUNX1 Point Mutations • RUNX1 mutation screening service • AID21 patient • AML cases with a strong association with RUNX1 mutations (FAB M0, +13) • FPD/AML patient • Sequencing of the entire coding region Joanne Mason, WMRGL Birmingham

17. RUNX1 mutation screening service • cDNA template • PCR under same conditions (‘touchdown PCR’) • M13 tag to facilitate high-throughput sequencing a c b d Primer sequences courtesy of Dicker et al, Leukemia 2007 Joanne Mason, WMRGL Birmingham

18. RUNX1 sequencing results.....so far • Patient A: p.Asp171Gly (D171G, homozygous) • DNA binding domain • Previously reported in two AML patients • 26% of mutations in RUNX1 are homozygous (wild-type RUNX1 is lost) Wild-type Patient AS Joanne Mason, WMRGL Birmingham

19. SNP-based DNA microarrays to investigate cytogenetically cryptic areas of somatically acquired homozygosity (AID) • Postulated that such regions contain homozygousmutations in genes known to be mutational targets in leukaemia. • In 7 of 13 cases with acquired isodisomy, homozygous mutations were identified at four distinct loci (WT1, FLT3, CEBPA,and RUNX1) • The mutation precedes mitotic recombination,which acts as a "second hit" responsible for removal of theremaining wild-type allele. Joanne Mason, WMRGL Birmingham

20. wt B wt C RUNX1 sequencing results.....so far • ?FPD/AML patient and three AML patients with trisomy 13 (i.e. highly likely to have RUNX1 mutations) • Patient B AML 47,XX +13 • p.Val137_Gly138insThr • Patient C AML 50,XY +8,+9,+13,+21 • p.Met25Lys • p.Arg135Lys • All de novo, but two other mutations involving arginine 135 have been reported before Joanne Mason, WMRGL Birmingham

21. Further work • Complete the sequence analysis of all four fragments comprising the coding region of RUNX1 • Effect of mutations? • Inheritance pattern in familial cases • Confirm RUNX1 mutations are acquired and not constitutional by sequencing stored remission DNA Joanne Mason, WMRGL Birmingham

22. Summary Unexpected microsatellite pattern in pre-transplant DNA taken at relapse Molecular data + cytogenetic data = acquired isodisomy 21 Candidate gene = RUNX1 RUNX1 mutation D171G Sequencing service for other sporadic AML patients, and for suspected FPD/AML referrals. Joanne Mason, WMRGL Birmingham

23. Birmingham, WMRGL: Val Davison Mike Griffiths Fiona Macdonald Susanna Akiki Paula White Natalie Morrell Charlene Crosby Birmingham Clinicians: Dr Prem Mahendra Prof Charlie Craddock Acknowledgements Thank you for your attention Joanne Mason, WMRGL Birmingham