chronic leukemia Flashcards
list possible symptoms of chronic leukemia
- general fatigue
- night sweats
- weight loss
- abdominal heaviness (from splenomegaly)
- low hemoglobin or rbc count
does prevalence of CML increase or decrease with age?
increase
are men or women more susceptible to CML?
men
what is the philadelphia chromosome?
-abnormal/shortened copy of chromosome 22 that is linked to CML
does each CML cell have the philadelphia chromosome or just the origional cell?
what does this suggest?
- each CML cell
- suggests a progenitor cell w a shortned chromosome 22 gave rise to all CML cells and the adnormality was passed on
define karyotyping
what is it used for?
when is it good?
- used to examine chromosomes under a microscope
- chromosomes are isolated during metaphase then stained and analyzed
- good for samples with heparin or anticoagulants (inhibit PCR) or want more want to know than one mutation type
how is the philadelphia chromosome formed?
what is the result?
- through reciporical translocation (swapping) of genetic material btwn chromosomes 9 and 22
- one short 22 and one long 9
what is the proper nomenclature for the philadelphia chromosome?
t(9;22)
is t(9;22) 100% specific to CML?
- no
- also seen in some cases of acute leukemia
explain what a somatic mutational event means in relation to the philadelphia chromosome
genetic mutation does not exist in all cells of the CML patient - only in the leukemia cells
how does a somatic mutation differ from a germline/constitutional mutation?
somatic mutations only present in cancer cells, where germline/constitutional mutations would be present in all cells
explain preferential clonal expansion in relation to CML
the genetic changes associated with CML give rise to a fusion protein that provides descendents a competitive advantave over normal cells
how does preferential colonal expansion lead to CML?
allows their progeny to take over the bone marrow and potentially overflow out of the marrow into the peripheral blood, resulting in CML
what is the BCR? where is it found?
- found on chromosome 22 next to the breakpoint
- coiled-coil region typically involved in homotypic protein-protein interactions
what is the ABL gene? where is it found?
- found on chromosome 9 just below the breakpoint
- highly regulated non-receptor tyrosine kinase
- participates in singal transduction pathways and affects gene transcription
- when activated it inhibits apoptosis and promotes cell proliferation
what happens to exons from the BCR and ABL genes during translocation?
-they become juxtaposed on the philadelphia chromosome forming a new gene called the BCR-ABL gene
is the proccess of translocation completely conservative? why or why not?
- not completely conservative but the same every time
- C-term of wild type BCR and N-term of ABL are lost
what is contained by the N-term after translocation?
coiled-coil region of the BCR protein
what is contained by the C-term after translocation?
the catalytic tyrosine kinase domain from the ABL protein
what are the functional consequences of the lost/kept domains for the BCR-ABL protein? (2)
(1) coiled-coil domain retained from BCR allows homodimerization of BCR-ABL (stimulates its own activity) and is thus more catalytically active than wildtype ABL
(2) kinase domain of wild type ABL is retained where inhibitory domain is lost, thus, BCR-ABL protein is constantly in its active state (unregulated)
what are the benefits of karyotyping for CML diagnosis? (3)
- gold standard
- can identify variations of the translocation
- can quantify how many cells are carrying the translocation
what are the limitations of karytoping for CML diagnosis? (5)
- requires viable dividing cells
- labour intensive/expensive
- requires expertise in karyotyping analysis
- low-ish sensitivity
- requires invasive sampling procedure
explain how CML is a neoplasm (5)
(1) HSCs containing the philadelphia chromosome outcompete/exceed normal HSCs for growth & survival
(2) growth of CML cells is independent of normal HSCs
(3) CML cells are autonomous (unregulated)
(4) CML is caused by a heritable mutation where the somatic mutation from a single cell was then inherited by its progeny
(5) CML manifests monoclonality (all CML cells in a leukemia patient have the philadelphia chromosome)
what are three diagnostic testing methods for CML?
(1) FISH
(2) PCR
(3) nested PCR
explain how FISH is used for CML diagnosis
- involves denaturing individual chromosomes yeilding single strands
- large DNA probes created with fluorescent tags attached
- ss chromosomes and DNA probes hybridized together
- colour change indicates where genes have joined
- e.g. BCR (red) and ABL (green) yeild yellow if join
- good for samples with heparin or anticoagulants (inhibit PCR)
what are the benefits of FISH? (4)
- easy to interpret
- quantitative (can determine how often signal is observed)
- more sensitive than karyotyping
- can use peripheral blood (less invasive than bone aspirate)
what are the limitations of FISH? (3)
- expensive
- time consuming
- need to find enough cancer cells to analyze (since they carry the translocation not normal cells)
give a general explanation for how PCR is used for CML diagnosis
- involves the production of forward a BCR primer and a reverse ABL primer
- these primers cannot generate a product is the proteins are separate but they can if proteins have fused
is it possible to create one PCR primer that spans the entire breakpoint region (where mutations occur)?
if so, how do we do this or is not, how do we overcome this?
- not possible
- overcome by using natural splicing mechanisms to splice the breakpoint regions out of the mRNA transcript
- can do this bc breakpoint regions = introns
- thus we only need a few PCR primer sets to amplify spliced regions of the fusion gene
at what exon will processed mRNA transcripts start for ABL? for BCR?
ABL: exon 2
BCR: exon 1, 13, 14 or 19
when using PCR for CML diagnosis, is RNA or DNA being extracted from patient samples?
RNA
how to we comapre samples once they have been amplified using PCR?
electrophoresis agarose gel
what should each sample show on agarose gel?
where do proteins of interest appear in relation to this?
- a PCR internal control fragment at at the same molecular weight across each protein
- protein of interest is shown below this fragment
what are the benefits of using PCR to diagnose CML? (5)
(1) easy to read the output
(2) sensitive (detects trasnlocations in 1/10,000-100,000 cells)
(3) economical and rapid
(4) can use bone marrow or peripheral blood
(5) doesn’t requrire dividing cells
what are the limitations of using PCR to diagnose CML? (2)
(1) requires isolation of RNA (which is an unstable substance)
(2) may miss some rare breakpoints (selective for well-known ones)
explain how Nested PCR is used to diagnose CML
- conducted to increase sensitivity of regular PCR procedure
- do o.g. PCR
- design second set of primers closer together
- ensures that second round of PCR is conducted on the products of o.g. PCR
- amplifies translocation further
- sensitivity increases to a detection rate of ~1/10,000,000
- less specific than other 3 mechanisms though
list the following diagnostic techniques in order of increasing sensitivity:
FISH, Nested PCR, PCR, karyotyping
karyotyping, FISH, PCR, Nested PCR
what is the main goal of CML treatment?
to acheive major molecular remission (significantly lower BCR-ABL levels in the blood)
what is the gold standard of treatment for CML?
- imatinib (drug)
- small molecular compound that acts as a selective tyrosine kinase inhibitor
how does imatinib function?
- functions to antagonize the catalytic function of BCR-ABL
- primarily targets the ABL catalytic site limiting the ability of CML cells to grow/proliferate
- overall reduces the proportion of bone marrow cells containing the philadelphia chromosome
how toxic is imatinib? does it produce many adverse effects?
- relatively non-toxic
- produces few adverse effects
can imatinib reduce the rate of progression to blast crisis by several years and prevent other patients from entering blast crisis at all?
yes
does histology of blood smears remain useful the longer a patient remains on imatinib?
why or why not?
- no
- as the number of CML cells decreases, they become restricted to bone marrow (rather than escape into blood)
what are two tecniques used for monitoring treatment progress? why?
- PCR and karyotyping
- only two techniques that can detect the presence of a mutations rather than the amount of mutated cells
are FISH, karyotyping and PCR qualitative or quantitative tests?
what information do they provide?
how can they do this?
- qualitative tests
- detect the presence or absence of CML
- can do so bc CML has one specific biomarker that is sufficient for diagnosis
do qualitative tests (FISH, PCR, karyotyping) provde information about the amount of a biomarker?
no
give an example of a quantitative assay.
what is this used for?
what does this provide information about?
- quantitative qRT-PCR
- used for monitoring protein levels that fall below those detectable through qualitative testing
- provide informatin about the relative amount of BCR-ABL fusion gene to see how patients are responding to therapy
can CML recurrence happen after treatment?
why or why not?
- yes
- philadelphia chromosome-positive cells can repopulate the blood upon withdrawl of inhibin
is imatinib a full cure?
why or why not?
- no
- imatinib can reduce the number of philadelphia chromosome-positive cells but cannot eliminate all affected cells
why can’t imatinib eliminate all affected CML cells?
- philadelphia chromosome-positive HSCs are relativelt resistant to imatinib treatment and are capable of self-renewal
- thus, even if bulk eliminated, HSCs can eventually repopulate
- additonally, some CMl patients develop imatinib resistance through prolonged use
what are the mechanisms by which patients develop imatinib resistance?
- clones of CML cells develop resistance by missense mutations in the BCR-ABL catalytic site - preventing imatinib from binding
- OR through increased expression of BCR-ABL gene
why do resistant sub-clones of CML cells become more prevalent in the CML populaiton?
what does this lead to?
- because they possess a selective advantage in the context of imatinib treatment
- results in imatinib resistance
which testing method is most effective for measuring and analyzing recurrence?
why?
- PCR
- much more specific than karyotyping so can see recurrence sooner
- also allows you to understand why the patient had a relapse (what specific mutation caused relapse)
- could help suggest further treatment
