Genetics and leukaemia Flashcards
What are the 2 main kinds of leukaemia?
Lymphoid and myeloid
What is lymphoid leukaemia and what types are there?
Proliferating lymphoid progenitors- block- unable to make lymphocytes
CL and MM
What is myeloid leukaemia and what types are there?
CML-proliferative leukaemia- problem with signalling and accumulation of immature ‘blasts’
AML- block in maturation, no functioning cell types, not enough red blood cells
MDC- less than 20% of blasts are affected
What’s the more common type of leukaemia?
Myeloid
more common and more cell types involved
What are the types of genetic changes associated with leukaemia? What techniques can visualise these changes?
Loss of chromosomal material Gain of chromosomal material Altered gene expression DNA mutations Karyotyping FISH PCR
Which techniques are used specifically to identify chromosome abnormalities?
Cytogenetics
Karyotyping via G band
FISH
DNA lab sequencing
What are the steps involved in preparing a karyotype?
Sample of marrow- add to a culture medium- incubate 2-3 days- add colcemid- add hypotonic solution- add a fixative and do repeated rounds of it- spread cells onto a slide via dropping- G banding via computers
What produces bands seen on a karyotype? How does it produce the light and dark bands
Trypsin - is a protease
Trypsin removes certain proteins from certain parts of the chromosomes-
AT rich, late replication, gene poor or heterochromatic= dark bands= G bands
GC rich, early replication, gene rich, euchromatic = light bands = R bands
What are the types of chromosome abnormalities?
Trisomy monosomy inversion insertion translocation
What’s the main difference between fusion genes and deregulation?
Fusion genes creates a hybrid gene which gives rise to a chimeric protein
e.g t(9;22) hybrid gene has been shown to increase tyrosine kinase activity- which results in neoplastic growth
Deregulation results in a gene being placed under the influence of a new regulatory sequence- expression pattern changes- inappropriately expressed
e.g Burkitts lymphoma
Where is the translocation in Burkitt’s lymphoma and what is the result?
t(8;14)
juxtaposes the myc gene to an immunoglobulin gene
=results in the constitutive overexpression of myc under the aberrant influence of the immunoglobulin regulatory sequence
How can we confidently detect gene fusions?
FISH
there is a need for the rapid confirmation of gene fusion- treatment and prognostic implications
used to use telomeric probes
What is XY centromere FISH?
Probes for the centromeres on the X and Y chromosomes
used to monitor the progress of a stem cell transplant if the transplant came from the opposite sex
What are breakapart FISH probes?
Probes used to see if genes have broken apart
normal cells= signals are fused together
abnormal cells= signals have broken apart- no fusion of signals
very important in leukaemia
What is dual colour dual fusion?
FISH used to detect if 2 genes have fused together
used to detect gene fusions from known translocations
What known translocation can dual colour dual fusion FISH detect?
T(9;22)- BCR/ABL1 fusion
seen in CML
How has the significance of DNA mutation detection changed?
Historically- smaller role in diagnosis and prognosis
last few years- mutations have been discovered in cytogenetically normal leukaemia
in 2005- janus kinase 2 mutation was described in 98% of PV (blood cancer) and in 50-60% of those with ET (rare bone marrow condition) and PMF (uncommon type of chronic leukaemia)
What is RT-qPCR and why is it used?
PCR- with the added quantification of DNA molecules
how has the patient responded to therapy
3 repeated steps:
denaturation, annealing and elongation
How is RT-qPCR used in BCR/ABL1 fusion?
Used to measure the amount of BCR/ABL1 transcripts
used in the monitoring of the disease- whats the % of BCR/ABL1 vs the % of ABL1
Characteristics of BCR/ABL1 fusion
Translocation between 9 and 22
ABL1 is an oncogene and encodes a nuclear tyrosine kinase protein
ABL1 is juxtaposed onto the breakpoint cluster region of the BCR gene
The fusion protein has increased tyrosine kinase activity
New chromosome seen= Philadelphia chromosome, very small and pale
CML= chronic myeloid leukaemia
What are the steps in digital droplet PCR?
ddPCR is a highly sensitive mutation detection technique
probes for WT and mutant DNA
labelled with different fluorescent markers
PCR
primers
DNA of a cell is incorporated in an oil droplet
fluorescence from each droplet measured
green= WT only
blue= mutant
orange= double positive mutant
What is ddPCR used for?
Diagnosis
if the % of abnormality has increased in a patient, they might relapse- need a new therapy
can make earlier predictions
What is next generation sequencing?
Identify mutations in the DNA
use a panel of 10-100s of genes- important genes to interrogate for the patients treatment
much better and in depth than the sanger method
Who has next generation sequencing affected myeloid leuakaemia?
Now have a panel of specific mutations- that werent seen via karyotyping
the mutations have important diagnostic and prognostic effects
can look at all the mutations at once- more cost effective than previously looking at mutations individually via PCR assays
Example of where genetic testing has helped in diagnosis
AML (acute myeloid leukaemia)
55% of patients have a chromosomal abnormality- most commonly t(8;21)
80% are non-random and have specific correlations
each gene fusion is associated with a specific treatment- know the cancer genome- can personalise their treatment
Example of where genetic testing has helped in prognosis?
AML patients-
good prognosis for some genetic changes
poor prognosis- deletions on chromosomes 5,7- need a tougher treatment
patients who have an adverse genetic lesion- 11% survival after 5 years
Examples of genetic testing effecting monitoring of the disease
How we monitor depends on the abnormality
- persistence of genetically abnormal leukemic cells- patients stopped responding to treatment
- % of male to female cells- in bone marrow transplants from the opposite sex
- check for karyotype evolution- gaining more abnormalities- cancer is progressing
What is the profile for APL- acute promyelocytic leukaemia?
Gene fusion between PML and RARA
accumulation of immature cell types
they have DIC (disseminated intravascular coagulation)
PML-RARA fusion= transcriptional activation of an aberrant set of genes in a retinoic acid-dependent manner
What is the treatment of APL?
Very fast treatment needed due to the DIC which can be fatal
Use all-trans retinoic acid (ATRA)
Stops DIC- improves outlook
with this treatment- tend to survive very well
What gene fusions are seen when there is translocation involving chromosome 9 and 22?
Most commonly- CML
other fusion- acute lymphoblastic leukaemia
What testing techniques are used when testing BCR/ABL1 fusion?
FISH
Full cytogenetics
Request EDTA sample for molecular testing
RT-qPCR
