CHIMERISM. Principles and practise.

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Normal haemopoiesis
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ABNORMALITIES IN THE HEMOPOIETIC SYSTEM

• CAN LEAD TO
• HEMOGLOBINOPATHIES
• HEMOPHILIA
• DEFECTS IN HEMOSTASIS/THROMBOSIS
• HEMATOLOGICAL MALIGNANCY

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MUTATIONS AND DNA

• VARIOUS TYPES OF MUTATIONS CAN OCCUR LEADING TO
  DISEASE PHENOTYPE
• POINT MUTATIONS
• INSERTIONS OR DELETIONS
• TRANSLOCATIONS
• COMPLEX CHROMOSOMAL REARRANGEMENTS

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EXAMPLE OF COMMON MUTATIONS IN HUMAN DISEASE
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Sickle cell disease
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Sickle cell disease, morphology and molecular
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• VARIABILITY IN GENETIC DISEASES
• ONE DISEASE, ONE GENE, ONE MUTATION
• ONE DISEASE, ONE GENE, MANY MUTATIONS
• ONE DISEASE, MORE THAN ONE GENE, MANY
• MUTATIONS

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HAEMOPHILIA X LINKED RECESSIVE
DISORDER HAEMOPHILIA A MUTATIONS IN FACTOR
VIII GENE HAEMOPHILIA B MUTATIONS IN FACTOR IX
GENE SIMPLE AND COMPLICATED MUTATIONS THE FLIP
TIP MUTATION
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F8B
A
CEN
TEL
B
TEL
E1 E22 E23
E26
CEN
F8A
C
E1
E22
E23 E26
TEL
CEN
INVERSION 22
FIGURE 4 THE IVS 22 MUTATION IN HAEMOPHILIA A.
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Genetic factors and deep vein thrombosis

• FACTOR V LEIDEN MUTATION
• PROTHROMBIN MUTATION
• ? OTHER FACTORS IN THE PROTEIN C PATHWAY
• FVL LEADS TO SIGNIFICANT INCREASE IN RISK OF DVT,
  PARTICULARLY IN ASSOCIATION WITH OTHER
  ENVIRONMENTAL FACTORS EG OCP

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CANCER DEVELOPMENT ITS IN THE GENES
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HOW DOES A CELL BECOME TUMORIGENIC?

• THREE PROCESSES ARE INVOLVED
• IMMORTALISATION
• TRANSFORMATION
• METASTASIS

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IMMORTALISATION

• PROCESS BY WHICH THE CELLS ARE INDUCED TO GROW
  INDEFINITELY

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TRANSFORMATION

• CELLS ARE NOT CONSTRAINED IN TERMS OF GROWTH
  CHARACTERISTICS AND TEND TO BECOME FACTOR
  INDEPENDENT

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METASTASIS

• CANCER CELLS GAIN THE ABILITY TO INVADE NORMAL
  TISSUE AND ESTABLISH OTHER FOCI OF MALIGNANCY

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WHAT CAUSES CELL TRANSFORMATION?

• ENVIRONMENTAL
• CARCINOGENS(INITIATORS AND PROMOTERS)
• GENETIC
• SOMATIC MUTATIONS
• MENDELIAN INHERITANCE

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ONCOGENES

• NORMAL CELLULAR COUNTERPARTS(PROTO-ONCOGENES)
• MUTATION/ACTIVATION LEADS TO TUMOR FORMATION
• HUNDREDS OF ONCOGENES IDENTIFIED
• GAIN OF FUNCTION

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Tumour suppressor genes

• Originally known as recessive oncogenes
• Need to have both copies of the gene affected to
  promote a malignant phenotype
• Knudsons 2 hit hypothesis
• First mutation makes cells susceptiple to
  development of cancer
• 2nd hit leads to a malignant phenotype

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TRANSLOCATIONS AND CANCER

• SEEMS PARTICULARLY RELEVANT IN HEMATOLOGICAL
  MALIGNANCIES
• CHRONIC MYELOID LEUKEMIA
• ACUTE PROMYELOCYTIC LEUKEMIA
• BURKITTS LYMPHOMA
• NON HODGKINS LYMPHOMA

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Leukaemia, the current hypothesis

• Defect in maturation of white blood cells
• May involve a block in differentiation and/or a
  block in apoptosis
• Acquired genetic defect
• Initiating events unclear
• Transformation events involve acquired genetic
  changes
• Chromosomal translocation implicated in many
  forms of leukaemia

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Chronic Myeloid Leukaemia

• Malignancy of the haemopoietic system
• Transformation of the pluripotent stem cell
• 922 translocation giving rise to the
  Philadelphia (Ph) chromosome
• Creation of a leukaemia specific mRNA (BCR-ABL)
• Resistance to apoptosis, abnormal signalling and
  adhesion
• Molecular diagnostics
• Molecular and cellular therapeutics

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Cytogenetic Abnormality of CMLThe Ph Chromosome
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22
x
Y
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The Ph Chromosome t(922) Translocation
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9 q

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Ph ( or 22q-)
bcr
bcr-abl
abl
FUSION PROTEINWITH TYROSINEKINASE ACTIVITY
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Prevalence of the Ph Chromosome in
Haematological Malignancies

• Leukaemia of Ph Patients
• CML 95
• ALL (Adult) 1530
• ALL (Paediatric) 5
• AML 2

Faderl S et al. Oncology (Huntingt).
199913169-184.
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bcr-abl Gene and Fusion Protein Tyrosine Kinases
Chromosome 9
Chromosome 22
2-11
c-bcr
c-abl
1
2-11
p210Bcr-Abl
2-11
p185Bcr-Abl
Exons Introns CML Breakpoints ALL Breakpoints
Adapted from Melo JV. Blood. 1996882375-2384.
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NON HODGKINS LYMPHOMA

• B CELL FOLLICULAR LYMPHOMA
• t(1418)(q21q14)
• BCL 2 AND IMMUNOGLOBULIN GENES INVOLVED
• DYSREGULATION OF BCL 2
• FAILURE OF APOPTOSIS

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Detecting Cancer where to begin?
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Detecting cancer, the need for a marker of disease
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Detecting Cancer different markers for
different diseases?
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Cancer Molecular Diagnostics discriminating
cancers at the gene level
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How Cancer Molecular Diagnostics?

• Chromosome analysis
• Gene analysis
• Gene expression analysis
• Protein analysis
• Gene chip analysis

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Leukaemia diagnostics

• Morphology
• Cytogenetics
• Fluorescent In Situ Hybridisation (FISH)
• Immunophenotyping
• PCR of chromosomal translocations

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New developments in Cancer Molecular
Diagnostics The Gene Chip
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The Gene Chip, a Molecular snap shot of the cell
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MOLECULAR MEDICINE

• A new approach to medicine
• New Diagnostics
• New Therapeutics
• A number of agents now in clinical trials
• Molecular medicine will help identify new targets
  and permit rational drug development