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