20.06.25 MRD monitoring strategies Flashcards
1
Q
What is minimal residual disease (MRD)
A
Low level cancerous cells that remain following treatment and are only detectable by highly sensitive techniques
2
Q
Examples of the clinical uses of MRD
A
- Determination of efficacy of treatment
- Allows target-driven titration of dose and duration of treatment
- Relapse risk stratification , to allow triage for optimal consolidation therapy.
- Determine prognosis after completion of standard treatment, enabling early recognition of impending relapse
- Monitor disease burden in the setting of stem cell transplantation (SCT)
- To spare toxicity and cost of SCT in those with low risk of relapse
3
Q
What is the best method to detect MRD
A
- Depends on the disease and its pathogenesis.
- PCR-based for AML
- Flow for AML, ALL
4
Q
Review of FISH for MRD
A
- Used in cases with cytogenetically defined rearrangements
- Sensitivity is low
- Fusion and beakapart probes can detect lower levels of MRD the single copy probes used to detect gains/losses
5
Q
Review of quantitative RT-PCR for MRD
A
- Accurate quantitiation of PCR products during exponential PCR phase.
- Used to measure RNA expression of translocation product, e.g. t(9;22) BCR-ABL1
- Molecular break points need to be assessed. e.g. t(16;16) or inv(16) in AML, there are 3 major fusion subtypes.
- ABL is a reliable control gene.
6
Q
Review of quantitative PCR for MRD
A
- Uses DNA
- Less popular as varying translocation break points make assay design difficult.
7
Q
Review of tandem duplication PCR for MRD
A
- Pair of primers used for amplification are orientated in the opposite direction therefore only allowing amplification when a duplication is present.
- Used for FLT3-ITD detection, in AML
8
Q
Review of flow cytometry for MRD
A
- Identifies aberrant cell surface marker expression, which is not seen in normal bone marrow or blood.
- However, due to evolution, a patient’s immunophenotype may be different when they relapse
- Cheap, rapid. Not as specific as PCR
9
Q
MRD monitoring in ALL
A
- Generally done by flow cytometry (applicable to 95% of ALL). B and T done differently. Looks at leukemia associated immunophenotypes
- RT-qPCR for fusion transcripts and Ig/TCR rearrangements. Rapid and sensitive.
10
Q
MRD monitoring in AML
A
- Fusion gene monitoring: t(8;21) RUNX1-RUNXT1.
- FLT3-ITD status: tandem duplication PCR.
- WT1 monitoring: overexpressed in AML cases at diagnosis. Low sensitivity and specificity.
- Immunophenotyping: applicable to 94% cases.
11
Q
MRD monitoring in CML
A
- Karyotype: to detect Ph chr t(9;22)(q34;q11), and other cytogenetic aberrations. Sensitivity >5%
- Interphase FISH: sensitivity ~0.5%
- qRT-PCR: Sensitivity ~1x10-5
12
Q
MRD monitoring in Lymphoma
A
-All non-Hodgkin lymphomas can be monitored using RT-PCR for Ig/TCR rearrangements
13
Q
Monitoring of bone marrow transplants
A
- Chimerism analysis bost BMT is used to determine the success of the engraftment by characterising the origin of post transplant haematopoiesis.
- Reappearing recipient cells indicate reappearing leukemic cells or normal host cells or both.
- Chimerism analysis has low sensitivity so not a true MRD. Useful when no disease specific marker is available.
- Sex mismatched BMT: XY FISH, Amelogenin on PCR.
- Sex matched BMT: PCR of microsatellite markers.
14
Q
Review of NGS technologies in MRD
A
- Independent of the specific leukemic clone present and can cope with disease progression and transformation.
- Studies have shown NGS has similar level of sensitivity to qPCR and doesn’t need patient-specific reagents.
- Standards need further defining, i.e., timepoint NGS is used, which genes are included in panel, what sequencing coverage should be used.
15
Q
Review of ddPCR in MRD
A
- High sensitivity and specificity.
- Low error rate, faster and does not require bioinformatic expertise as in NGS.
- Disadvantage: assay needs to be developed for specific base changes in the same gene.
