Week 2B - minimal residual disease Flashcards
what does early detection of residual cells allow
clinical intervention with the aim of reversing the proliferation of resistant leukaemic cells
what is minimal residual disease
the name given to small numbers of leukaemic cells that remain in the patient during treatment
what is the sensitivity of MRD assays
> 1 x 10^4
i.e. detection of 1 malignant cell in 10,000 normal ones
what can chromosomal translocation result in
leukaemia
what does chromosomal translocation generate
fusion gene coding for an oncoprotein
what does chromosomal translocation target
junctional breakpoints
what does RT-PCR detect
RNA from viable cells - target genes expressed that are likely to have a functional role in cellular prolifertaion
what is BCR-ABL1
the product of the chromosome translocation associated of Chronic Myeloid Leukaemia
what do BCR-ABL1 RNA transcripts remain
detectable after disease cells can no longer be seen down the microscope - complete cytogenetic remission - CCyR
what is the only tool capable of monitoring responses after patients reach CCyR
RT-qPCR
how are low levels of minimal residual diseased achieved
during therapy call for a sensitive monitoring assay for reliable detection and quantitation
what does the amount of specific mRNA reflect
the amount of protein being expressed
what is the methodology for BCR-ABL-1 transcript quantification
- EDTA peripheral blood
- mRNA extraction
- reverse transcription (cDNA synthesis)
- TaqMan real time quantitative PCR (qPCR) for ABL and BCR-ABL1
how can RNA -> DNA be amplified
polymerae chainreaction
how does PCR work
- RNA is extracted from patients blood and converted to double stranded cDNA
- cDNA can be used in conventional PCR
- Many copies of gene-specific template made – giving great sensitivity
how does TaqMan real time qPCR work
- probe binds to target cDNA of interest
- quencher close to dye, stopping flourescnece
- generation of complementary strand releases dye from its quencher
- amount of fluorescence is proportional to amount of template
how can amount of cDNA be measured
through titration against a standard curve
what is ABL gene fragment used for
a house keeping gene
so we can assess the amount and quality of RNA derived from the sample
what does the BCR-ABL1 fusion gene provide
a unique biomarker for diagnosis and monitoring response to treatment
how can continued molecular monitoring be used
- to assess whether a patient with CML is making progress toward a desired response to therapy.
- Ensure that a patient’s treatment is optimized over time
what do B and T-clonal recombinations in leukaemia generate
patient-specific DNA length and sequences - potential molecular markers for detection and quantification of leukemic cells among normal lymphocytes in remission samples
what do primers do in Allele Specific Oligonucleotide PCR (ASO-PCR)
anneal to a unique patient specific Ig sequence.
is B and T cell rearrangements specific
yes - 1x10^-4 and -5
fully quantitative
what is high levels of MRD at end of induction therapy associated with
high risk of relapse
what is considered the most widely applicable and sensitive methodology for MRD
qPCR using allele specific oligonucleotide PCR
how is MRD results categorised
positive negative intermediate
what is MRD positive
MRD detectable ≥ 1 × 10-4 with either of 2 ASO primers (level of disease corrected to control gene will be reported)
what is MRD negative
no MRD detectable by 2 markers, one with a quantitative range of 10-4.
what is MRD intermediate
MRD detectable but <10-4.
– MRD not detected but sensitivity of assay is low (<10-3).
– MRD not detected by one available marker
what happens in single nucleotide polymorphism (SNP)
we count PCR amplicons in a binary fashion, so can be used a quantitatively
what is the droplet system
routinely count 20,000 droplets, giving high enough sensitivity for MRD analysis
what do STR markers track
low level
disease in haematological stem cell transplant patients
what is next generation sequencing’s potential in MRD
Over 10 years it has become cost effective to use NGS in NHS Laboratory services.
It offers a chance to get much more out of every pathological samples.
what does the NGS methodology detect
variants at very low prevalence – the ‘variant allele frequency’(VAF) - so can be very sensitive
how is targeted NGS panels good
because several different key genes can be effectively covered for MRD
what else can occur if targeted NGS is performed
identification of new clones
treatment can be modified
clinicians can be one step ahead of cancer
