Normal haemopoiesis

ABNORMALITIES IN THE HEMOPOIETIC SYSTEM

• CAN LEAD TO

• HEMOGLOBINOPATHIES

• HEMOPHILIA

• DEFECTS IN HEMOSTASIS/THROMBOSIS

• HEMATOLOGICAL MALIGNANCY

MUTATIONS AND DNA

• VARIOUS TYPES OF MUTATIONS CAN OCCUR LEADING TO DISEASE PHENOTYPE

• POINT MUTATIONS• INSERTIONS OR DELETIONS• TRANSLOCATIONS• COMPLEX CHROMOSOMAL

REARRANGEMENTS

EXAMPLE OF COMMON MUTATIONS IN HUMAN DISEASE

Sickle cell disease

Sickle cell disease, morphology and molecular

VARIABILITY IN GENETIC DISEASES

•ONE DISEASE, ONE GENE, ONE MUTATION

•ONE DISEASE, ONE GENE, MANY MUTATIONS

•ONE DISEASE, MORE THAN ONE GENE, MANY •MUTATIONS

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

F8A

E1 E22 E23 E26

F8A

E1 E22 E23 E26

TEL CEN

CEN

CENTEL

TEL

INVERSION 22

E1E22 E23 E26

A

B

C

FIGURE 4 THE IVS 22 MUTATION IN HAEMOPHILIA A.

F8B

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

CANCER DEVELOPMENT: ITS IN THE GENES

HOW DOES A CELL BECOME TUMORIGENIC?

• THREE PROCESSES ARE INVOLVED

• IMMORTALISATION

• TRANSFORMATION

• METASTASIS

IMMORTALISATION

• PROCESS BY WHICH THE CELLS ARE INDUCED TO GROW INDEFINITELY

TRANSFORMATION

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

METASTASIS

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

WHAT CAUSES CELL TRANSFORMATION?

• ENVIRONMENTAL• CARCINOGENS(INITIATORS AND

PROMOTERS)

• GENETIC• SOMATIC MUTATIONS• MENDELIAN INHERITANCE

ONCOGENES• NORMAL CELLULAR

COUNTERPARTS(PROTO-ONCOGENES)

• MUTATION/ACTIVATION LEADS TO TUMOR FORMATION

• HUNDREDS OF ONCOGENES IDENTIFIED

• GAIN OF FUNCTION

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

TRANSLOCATIONS AND CANCER

• SEEMS PARTICULARLY RELEVANT IN HEMATOLOGICAL MALIGNANCIES

• CHRONIC MYELOID LEUKEMIA

• ACUTE PROMYELOCYTIC LEUKEMIA

• BURKITTS LYMPHOMA

• NON HODGKINS LYMPHOMA

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

Chronic Myeloid Leukaemia• Malignancy of the haemopoietic system• Transformation of the pluripotent stem cell• 9;22 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

Cytogenetic Abnormality of CML:The Ph Chromosome

1 2 3 4 5

6 7 8 10 119 12

13 14 15 16 17 18

19 20 21 22 x Y

The Ph Chromosome: t(9;22) Translocation

22

bcr

abl

Ph ( or 22q-)

bcr-abl

FUSION PROTEINWITH TYROSINEKINASE ACTIVITY

9 9 q+

Prevalence of the Ph Chromosome in Haematological Malignancies

Leukaemia % of Ph+ Patients

CML 95

ALL (Adult) 15–30

ALL (Paediatric) 5

AML 2

Faderl S et al. Oncology (Huntingt). 1999;13:169-184.

bcr-abl Gene and Fusion Protein Tyrosine Kinases

Adapted from Melo JV. Blood. 1996;88:2375-2384.

p210Bcr-Abl

p185Bcr-Abl2-11

2-11

Chromosome 9

c-bcr

Chromosome 22

c-abl

Exons

Introns

CML Breakpoints

ALL Breakpoints

1

2-11

NON HODGKINS LYMPHOMA

• B CELL FOLLICULAR LYMPHOMA

• t(14;18)(q21;q14)

• BCL 2 AND IMMUNOGLOBULIN GENES INVOLVED

• DYSREGULATION OF BCL 2

• FAILURE OF APOPTOSIS

Detecting Cancer – where to begin?

Detecting cancer, the need for a marker of disease

Detecting Cancer – different markers for different diseases?

Cancer Molecular Diagnostics – discriminating cancers at the gene level

How Cancer Molecular Diagnostics?

• Chromosome analysis

• Gene analysis

• Gene expression analysis

• Protein analysis

• Gene chip analysis

Leukaemia diagnostics

• Morphology

• Cytogenetics

• Fluorescent In Situ Hybridisation (FISH)

• Immunophenotyping

• PCR of chromosomal translocations

New developments in Cancer Molecular DiagnosticsThe Gene Chip

The Gene Chip, a Molecular snap shot of the cell

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