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.

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 Womens NHS Foundation Trust,

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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)

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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

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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.

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Multiplex microsatellite marker PCR and
subsequent fragment analysis
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Chimaerism analysis
DONOR
HOST PRE-TRANSPLANT
POST-TRANSPLANT
74 donor 26 host
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Microsatellite results

• Pre-transplant DNA

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Chromosome 21 markersAverage ratio 41
Remission DNA
Relapse DNA
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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 41)

• 3) Acquired isodisomy (aka acquired uniparental
  disomy, or copy number neutral loss of
  heterozygosity)

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Acquired isodisomy (AID)

• Common mechanism of oncogenesis
• Prognostic significance in AML?

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Mitotic Recombination
21
21
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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

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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)

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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

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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.

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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

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RUNX1 mutation screening service
a
c
b
d

• cDNA template
• PCR under same conditions (touchdown PCR)
• M13 tag to facilitate high-throughput sequencing

Primer sequences courtesy of Dicker et al,
Leukemia 2007
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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
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• 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.

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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

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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

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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.

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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