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A Molecular Oncology Case Study

A Molecular Oncology Case Study. Making a difference to patient treatment. Chris Bowles. Birmingham Lab. SCOBEC Case Study Day 2008. Chronic Myeloid Leukaemia.

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A Molecular Oncology Case Study

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  1. A Molecular Oncology Case Study Making a difference to patient treatment Chris Bowles. Birmingham Lab. SCOBEC Case Study Day 2008.

  2. Chronic Myeloid Leukaemia • Leukaemia is used to describe a number of acquired diseases leading to the misregulation of proliferation of blood cells and their progenitors. • Myeloid or Lymphoid depending on which type of cell line they affect, and further divided into acute and chronic depending upon the speed of progression of the disease. • Chronic Myeloid Leukaemia is a stem cell cancer representing about 15-20% of adult leukaemia. Average age at presentation 45-55.

  3. Chronic Myeloid Leukaemia • If left untreated eventually leads to an acute disease. • During the chronic phase the progenitor cells retain their capacity to differentiate normally resulting in an accumulation of mature cells, whereas blast crisis is characterized by loss of differentiation capacity, resulting in an accumulation of blast cells. • Eventually the immature cells overrun the bone marrow and spill out into the blood stream, leading to eventual organ infiltration.

  4. What causes CML? • 95% of cases have a common t(9;22)(q34;q11) chromosome translocation, resulting in an abnormally short chromosome 22, the Philadelphia Chromosome. • Results in the fusion of two genes: • BCR on chromosome 22 • ABL on chromosome 9 • Usually exon 13 or 14 of BCR fuses with exon 2 of ABL • Fusion protein codes for a constitutively active tyrosine kinase, part of a pathway leading to uncontrolled cell growth, genomic instability and protection of DNA damaged cells from apoptosis.

  5. Normal Treatment • Originally Hydroxyurea and Interferon a to reduce symptoms • Bone Marrow Transplant • Imatinib (Glivec) • Recommended 400mg daily starting dose • Haematological response in 95% patients (vs 55%) • Cytogenetic response in 76% patients (vs 15%) • Major Molecular response in 40% patients after 12 months (vs 2%) • Five year survival rate of 89%

  6. Imatinib • Designer drug based on knowledge of pathogenicity. • Drug competitively binds at ATP binding site, preventing tyrosine kinase from working. • Occasional no response/loss of response to Imatinib.

  7. AKD mutation • Mutations of the ABL kinase domain can prevent Imatinib from working • Directly changing an imatinib binding site • Indirect preventing imatinib access to domain • Direct sequencing can detect variants, and the mutation detected will have varying effects on drug efficiency. • New drugs (Dasatinib, Nilotinib) emerging that have a different method of binding so provide an alternative – but more expensive!

  8. Diagnosis • Preferably Marrow sample • Karyotype • Metaphase cells • FISH • Molecular Diagnostics

  9. CML • Involves many different techniques • RT-PCR • RQ-PCR • Fragment Analysis • Sequencing • Co-operation with Cytogenetics! • Conventional Karyotyping • FISH • And a little bit of immunology.....

  10. RT and RQ PCR • RT – PCR on cDNA. • RNA extracted and converted to cDNA. Can determine whether or not fusion gene is present. • RQ – Real Time Quantitative • Measure quantity of gene in exponential phase of PCR • Probe containing fluorescence and quencher molecule • As PCR progress fluorescence released from quencher to give signal • Fluorescence increases exponentially and is indicative of transcript level

  11. Introduction to patient • 44 year old male • Originally referred for diagnostic testing • Splenomegaly • Increased White Blood Cell Count • Increased Platelet Count • Single step RT-PCR analysis of marrow showed a b3a2 BCR-ABL gene fusion. • RQ-PCR analysis was positive with a BCR/ABL to ABL percentage ratio of 165%.

  12. Other Analysis • Karyotyping was carried out on 20 cells confirming a t(9;22)(q34;q11). • The patient also had a sub-clone of cells (13/20) with an additional Ph chromosome. • Additional chromosomal abnormalities can sometimes indicate the disease is moving out of the chronic phase towards blast crisis. • However Immunological analysis of the bone marrow showed no excess of blast cells in the patient’s sample, which would suggest that the CML was still in the chronic phase.

  13. Limited initial response to Imatinib • After 24 months had a BMT – immediate reduction in BCR-ABL • However MRD monitoring indicated steady increase in fusion gene • Re-emergence of sub-clone population • Seen at much lower level by cytogenetic analysis

  14. AKD analysis • Sequencing ABL gene from cDNA • Can use multiple samples – disease is clonal and dynamic • Three samples analysed • No mutation detected • E255V Mutation detected at less then 100% • E255V Mutation detected at 100% • Common mutation that has been shown to have no response to Imatinib treatment, and partial response to Dasatinib and Nilotinib.

  15. Points of interest • Monitor residual levels of disease • Early warning system • Can help make decisions on patient treatment • Overall better quality treatment for patients • Better quality of life for patients

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