MRD assessment Mani Yegappan Lab for Clinical Hematology Dept of Hematology, Hosp Ampang What is MRD? • Minimal Residual Disease • Disease beyond ‘traditional’ limits of laboratory.
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MRD assessment
Mani Yegappan Lab for Clinical Hematology Dept of Hematology, Hosp Ampang
What is MRD?
• Minimal Residual Disease • Disease beyond ‘traditional’ limits of laboratory resolution/sensitivity – Morphology 500 cell count – Karyotyping 20-30 cells – FISH 500 cells
Tumor load
• At diagnosis ~1x10 12 cells • MRD <1x10 6 cells ≥6 log reduction is the target
Sensitivity
• Needle in a haystack, or • Looking for a non existent black cat in a dark room
Why MRD?
• Milestones • Monitor & assess response to therapy • Decide on therapeutic options-optimal therapy vs under/over treatment • Identify early relapse
Optimal MRD
• Sample type- blood or marrow [?plasma] • Timing- phase of treatment • Frequency of monitoring • Target- aberrancy – phenotype – gene expression
MRD techniques
• 1 in 10 5 or 10 6 cells • Flow cytometry • PCR
Flow cytometry
• Phenotype-surface and intracellular • T cells CD3 • B cells 19, 20, 22, cytoplasmic 79a • Myeloid cells 117, 13, 33, MPO • Fluorescently labelled antibodies • Diagnosis 20,000 events • MRD 500,000-1,000,000 events
Aberrant phenotype
• Cross lineage expression • Asynchronous/mutually exclusive expression • Loss of expression • ‘Immature’ markers • Knowledge of diagnostic phenotype
…but
• Antigenic variation-loss, gain • Therapy, progression
MRD Flow
• 0.5 to 1 million cells/events • More the colors, better the definition – Currently 8, but this will expand in future • Panels are personal, subjective; lack standardisation • Noise vs true event
PCR
• Standard qualitative – Easier, cheaper, less sensitive • Nested – Laborious, more sensitive, ?linear
• Real time quantitative – Laborious, more sensitive, linear within limits
PCR Targets
• • Fusion genes –
BCR-ABL1, PML-RARA, RUNX1-RUNX1 T1, CBFB-MYH1 1
• ‘normal’ gene increased expression –
WT1, MN1, BAALC IGH, IGL, TCR
rearrangements – Unique sequences • Mutations
FLT3, NPM1, CEBPA, WT1
Fusion genes
• Aberrant/pathologic • Real time quantitative PCR • Normalised to a housekeeping gene • Monitor trend-rapidity of decline/increase • Limited utility-not all leukemias • Labile RNA- sample integrity is vital
‘normal’ genes
• Expression is aberrant, but genes are not • Same limitations as fusion genes • But can be used in all leukemias • Trend analysis- rate of decline mirrors chemosensitivity, while an increasing level portends relapse, loss of response
BCR, TCR genes
• Unique clonal rearrangements • Defining feature of lymphocytes-useful in ALL & NHL • DNA based • Sequence unique junction regions • Develop specific primers-laborious
Mutations
• • • • • Of increasing relevance in CN-AML
FLT3
- can be progressively lost or gained
NPM1
- stable
CEBPA WT1
-difficult target
Utility of MRD
• Largely based on pediatric ALL experience • Pre remission-MRD levels proportional to risk of relapse • MRD+ dismal outcome-intensify • MRD- early clearance of blasts, tend to remain MRD- ?deintensify
– Avoid toxicities, older patients
Utility of MRD
• Post remission: timing of HSCT • If MRD+ end of remission, conversion to MRD- associated with favorable outcome • Intensify to achieve MRD • Post HSCT emergence of MRD+ reduction in immunosuppression, DLI etc
Utility of MRD
• CML in TKI era- poster child for MRD • <1 log reduction at 3 mo-13% chance of achieving MMR [3 log reduction] • >1 log reduction at 3 mo- 70% chance of achieving MMR • >2 fold [≥0.5 log] ?TKD mutation
Utility of MRD
• APML different strategy- still evolving • Log reduction post induction does not predict relapse risk • Detection in remission predicts relapse risk • Preemptive therapy rather than treat relapse
And the winner is… Molecular or Flow cytometry?
Molecular Lab
• Phan Chin Lee • Cleria Cheng • Citra • Tan • Prayma
Flow Cytometry Lab
• Saras • Nurul • Farouk • Angeli
If you ask me anything I don’t know, I won’t answer I never said most of the things I said Yogi Berra