Presentation on theme: "Acquired isodisomy of chromosome 21 in an acute myeloid leukaemia (AML) patient as an incidental finding during routine chimaerism analysis, and the introduction."— Presentation transcript:
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,
Joanne Mason, WMRGL Birmingham 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 Patient A Diagnosed with AML in May 2006 Karyotype analysis: trisomy 13 (47,XY,+13 ) 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 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. CAGA 3-15 CAGACAGA
Joanne Mason, WMRGL Birmingham Multiplex microsatellite marker PCR and subsequent fragment analysis
Joanne Mason, WMRGL Birmingham Microsatellite results Pre-transplant DNA 13
Joanne Mason, WMRGL Birmingham Chromosome 21 markers Average ratio 4:1 D21S11 Penta D D21S1411 D21S1270 Remission DNA Relapse DNA
Joanne Mason, WMRGL Birmingham 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) Ch 21 markers : copy number change? Cytogenetics 2 normal copies Ch 21 –3) Acquired isodisomy (aka acquired uniparental disomy, or copy number neutral loss of heterozygosity)
Joanne Mason, WMRGL Birmingham Acquired isodisomy (AID) Common mechanism of oncogenesis Prognostic significance in AML?
Joanne Mason, WMRGL Birmingham Mitotic Recombination 21 AID 21
Joanne Mason, WMRGL Birmingham 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 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 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 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 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 RUNX1 mutation screening service cDNA template PCR under same conditions (‘touchdown PCR’) M13 tag to facilitate high-throughput sequencing a b c d Primer sequences courtesy of Dicker et al, Leukemia 2007
Joanne Mason, WMRGL Birmingham 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 SNP-based DNA microarrays to investigate cytogenetically cryptic areas of somatically acquired homozygosity (AID) Postulated that such regions contain homozygous mutations 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 the remaining wild-type allele.
Joanne Mason, WMRGL Birmingham 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 wt B wt C 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 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 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 Acknowledgements Birmingham, WMRGL: –Val Davison –Mike Griffiths –Fiona Macdonald –Susanna Akiki –Paula White –Natalie Morrell –Charlene Crosby Birmingham Clinicians: –Dr Prem Mahendra –Prof Charlie Craddock Thank you for your attention