Genetics and leukaemia

Question 1

What are the 2 main kinds of leukaemia?

Answer 1

Lymphoid and myeloid

Question 2

What is lymphoid leukaemia and what types are there?

Answer 2

Proliferating lymphoid progenitors- block- unable to make lymphocytes
CL and MM

Question 3

What is myeloid leukaemia and what types are there?

Answer 3

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

Question 4

What’s the more common type of leukaemia?

Answer 4

Myeloid

more common and more cell types involved

Question 5

What are the types of genetic changes associated with leukaemia? What techniques can visualise these changes?

Answer 5

Loss of chromosomal material
Gain of chromosomal material 
Altered gene expression 
DNA mutations 
Karyotyping
FISH
PCR

Question 6

Which techniques are used specifically to identify chromosome abnormalities?

Answer 6

Cytogenetics
Karyotyping via G band
FISH
DNA lab sequencing

Question 7

What are the steps involved in preparing a karyotype?

Answer 7

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

Question 8

What produces bands seen on a karyotype? How does it produce the light and dark bands

Answer 8

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

Question 9

What are the types of chromosome abnormalities?

Answer 9

Trisomy 
monosomy 
inversion 
insertion
translocation

Question 10

What’s the main difference between fusion genes and deregulation?

Answer 10

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

Question 11

Where is the translocation in Burkitt’s lymphoma and what is the result?

Answer 11

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

Question 12

How can we confidently detect gene fusions?

Answer 12

FISH
there is a need for the rapid confirmation of gene fusion- treatment and prognostic implications
used to use telomeric probes

Question 13

What is XY centromere FISH?

Answer 13

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

Question 14

What are breakapart FISH probes?

Answer 14

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

Question 15

What is dual colour dual fusion?

Answer 15

FISH used to detect if 2 genes have fused together

used to detect gene fusions from known translocations

Question 16

What known translocation can dual colour dual fusion FISH detect?

Answer 16

T(9;22)- BCR/ABL1 fusion

seen in CML

Question 17

How has the significance of DNA mutation detection changed?

Answer 17

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)

Question 18

What is RT-qPCR and why is it used?

Answer 18

PCR- with the added quantification of DNA molecules
how has the patient responded to therapy
3 repeated steps:
denaturation, annealing and elongation

Question 19

How is RT-qPCR used in BCR/ABL1 fusion?

Answer 19

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

Question 20

Characteristics of BCR/ABL1 fusion

Answer 20

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

Question 21

What are the steps in digital droplet PCR?

Answer 21

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

Question 22

What is ddPCR used for?

Answer 22

Diagnosis
if the % of abnormality has increased in a patient, they might relapse- need a new therapy
can make earlier predictions

Question 23

What is next generation sequencing?

Answer 23

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

Question 24

Who has next generation sequencing affected myeloid leuakaemia?

Answer 24

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

Question 25

Example of where genetic testing has helped in diagnosis

Answer 25

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

Question 26

Example of where genetic testing has helped in prognosis?

Answer 26

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

Question 27

Examples of genetic testing effecting monitoring of the disease

Answer 27

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

Question 28

What is the profile for APL- acute promyelocytic leukaemia?

Answer 28

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

Question 29

What is the treatment of APL?

Answer 29

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

Question 30

What gene fusions are seen when there is translocation involving chromosome 9 and 22?

Answer 30

Most commonly- CML

other fusion- acute lymphoblastic leukaemia

Question 31

What testing techniques are used when testing BCR/ABL1 fusion?

Answer 31

FISH
Full cytogenetics
Request EDTA sample for molecular testing
RT-qPCR