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chronic leukemia Flashcards

1
Q

list possible symptoms of chronic leukemia

A
  • general fatigue
  • night sweats
  • weight loss
  • abdominal heaviness (from splenomegaly)
  • low hemoglobin or rbc count
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2
Q

does prevalence of CML increase or decrease with age?

A

increase

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3
Q

are men or women more susceptible to CML?

A

men

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4
Q

what is the philadelphia chromosome?

A

-abnormal/shortened copy of chromosome 22 that is linked to CML

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5
Q

does each CML cell have the philadelphia chromosome or just the origional cell?
what does this suggest?

A
  • each CML cell

- suggests a progenitor cell w a shortned chromosome 22 gave rise to all CML cells and the adnormality was passed on

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6
Q

define karyotyping
what is it used for?
when is it good?

A
  • 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
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7
Q

how is the philadelphia chromosome formed?

what is the result?

A
  • through reciporical translocation (swapping) of genetic material btwn chromosomes 9 and 22
  • one short 22 and one long 9
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8
Q

what is the proper nomenclature for the philadelphia chromosome?

A

t(9;22)

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9
Q

is t(9;22) 100% specific to CML?

A
  • no

- also seen in some cases of acute leukemia

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10
Q

explain what a somatic mutational event means in relation to the philadelphia chromosome

A

genetic mutation does not exist in all cells of the CML patient - only in the leukemia cells

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11
Q

how does a somatic mutation differ from a germline/constitutional mutation?

A

somatic mutations only present in cancer cells, where germline/constitutional mutations would be present in all cells

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12
Q

explain preferential clonal expansion in relation to CML

A

the genetic changes associated with CML give rise to a fusion protein that provides descendents a competitive advantave over normal cells

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13
Q

how does preferential colonal expansion lead to CML?

A

allows their progeny to take over the bone marrow and potentially overflow out of the marrow into the peripheral blood, resulting in CML

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14
Q

what is the BCR? where is it found?

A
  • found on chromosome 22 next to the breakpoint

- coiled-coil region typically involved in homotypic protein-protein interactions

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15
Q

what is the ABL gene? where is it found?

A
  • 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
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16
Q

what happens to exons from the BCR and ABL genes during translocation?

A

-they become juxtaposed on the philadelphia chromosome forming a new gene called the BCR-ABL gene

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17
Q

is the proccess of translocation completely conservative? why or why not?

A
  • not completely conservative but the same every time

- C-term of wild type BCR and N-term of ABL are lost

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18
Q

what is contained by the N-term after translocation?

A

coiled-coil region of the BCR protein

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19
Q

what is contained by the C-term after translocation?

A

the catalytic tyrosine kinase domain from the ABL protein

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20
Q

what are the functional consequences of the lost/kept domains for the BCR-ABL protein? (2)

A

(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)

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21
Q

what are the benefits of karyotyping for CML diagnosis? (3)

A
  • gold standard
  • can identify variations of the translocation
  • can quantify how many cells are carrying the translocation
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22
Q

what are the limitations of karytoping for CML diagnosis? (5)

A
  • requires viable dividing cells
  • labour intensive/expensive
  • requires expertise in karyotyping analysis
  • low-ish sensitivity
  • requires invasive sampling procedure
23
Q

explain how CML is a neoplasm (5)

A

(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)

24
Q

what are three diagnostic testing methods for CML?

A

(1) FISH
(2) PCR
(3) nested PCR

25
Q

explain how FISH is used for CML diagnosis

A
  • 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)
26
Q

what are the benefits of FISH? (4)

A
  • easy to interpret
  • quantitative (can determine how often signal is observed)
  • more sensitive than karyotyping
  • can use peripheral blood (less invasive than bone aspirate)
27
Q

what are the limitations of FISH? (3)

A
  • expensive
  • time consuming
  • need to find enough cancer cells to analyze (since they carry the translocation not normal cells)
28
Q

give a general explanation for how PCR is used for CML diagnosis

A
  • 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
29
Q

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?

A
  • 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
30
Q

at what exon will processed mRNA transcripts start for ABL? for BCR?

A

ABL: exon 2
BCR: exon 1, 13, 14 or 19

31
Q

when using PCR for CML diagnosis, is RNA or DNA being extracted from patient samples?

A

RNA

32
Q

how to we comapre samples once they have been amplified using PCR?

A

electrophoresis agarose gel

33
Q

what should each sample show on agarose gel?

where do proteins of interest appear in relation to this?

A
  • a PCR internal control fragment at at the same molecular weight across each protein
  • protein of interest is shown below this fragment
34
Q

what are the benefits of using PCR to diagnose CML? (5)

A

(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

35
Q

what are the limitations of using PCR to diagnose CML? (2)

A

(1) requires isolation of RNA (which is an unstable substance)
(2) may miss some rare breakpoints (selective for well-known ones)

36
Q

explain how Nested PCR is used to diagnose CML

A
  • 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
37
Q

list the following diagnostic techniques in order of increasing sensitivity:
FISH, Nested PCR, PCR, karyotyping

A

karyotyping, FISH, PCR, Nested PCR

38
Q

what is the main goal of CML treatment?

A

to acheive major molecular remission (significantly lower BCR-ABL levels in the blood)

39
Q

what is the gold standard of treatment for CML?

A
  • imatinib (drug)

- small molecular compound that acts as a selective tyrosine kinase inhibitor

40
Q

how does imatinib function?

A
  • 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
41
Q

how toxic is imatinib? does it produce many adverse effects?

A
  • relatively non-toxic

- produces few adverse effects

42
Q

can imatinib reduce the rate of progression to blast crisis by several years and prevent other patients from entering blast crisis at all?

A

yes

43
Q

does histology of blood smears remain useful the longer a patient remains on imatinib?
why or why not?

A
  • no

- as the number of CML cells decreases, they become restricted to bone marrow (rather than escape into blood)

44
Q

what are two tecniques used for monitoring treatment progress? why?

A
  • PCR and karyotyping

- only two techniques that can detect the presence of a mutations rather than the amount of mutated cells

45
Q

are FISH, karyotyping and PCR qualitative or quantitative tests?
what information do they provide?
how can they do this?

A
  • qualitative tests
  • detect the presence or absence of CML
  • can do so bc CML has one specific biomarker that is sufficient for diagnosis
46
Q

do qualitative tests (FISH, PCR, karyotyping) provde information about the amount of a biomarker?

A

no

47
Q

give an example of a quantitative assay.
what is this used for?
what does this provide information about?

A
  • 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
48
Q

can CML recurrence happen after treatment?

why or why not?

A
  • yes

- philadelphia chromosome-positive cells can repopulate the blood upon withdrawl of inhibin

49
Q

is imatinib a full cure?

why or why not?

A
  • no

- imatinib can reduce the number of philadelphia chromosome-positive cells but cannot eliminate all affected cells

50
Q

why can’t imatinib eliminate all affected CML cells?

A
  • 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
51
Q

what are the mechanisms by which patients develop imatinib resistance?

A
  • 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
52
Q

why do resistant sub-clones of CML cells become more prevalent in the CML populaiton?
what does this lead to?

A
  • because they possess a selective advantage in the context of imatinib treatment
  • results in imatinib resistance
53
Q

which testing method is most effective for measuring and analyzing recurrence?
why?

A
  • 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

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