MRD

Question 1

What molecular techniques are used for MRD?

Answer 1

1. qPCR (cDNA)
2. ddPCR (cDNA)
3. NGS MRD with error correction using UMIs (gDNA)

Question 2

Why is RNA/cDNA used for molecular MRD monitoring using NPM1, RUNX1::RUNX1T1, CBFB::MYH11, PML::RARA

Answer 2

There is high expression of these genes and thus better sensitivity

Question 3

What are the sample requirements for molecular MRD?

Answer 3

1. First pull BMA (or re-site and first pull)
2. 5ml or less BMA to avoid haemodilution
3. If using PB then 10mL or more is required
4. Use the same method of cell isolation as it may alter the leukaemic percentage

Question 4

Which molecular MRD markers should be assessed?

Answer 4

1. Leukaemia specific e.g. NPM1, CBF leuk, PML::RARA
2. Agnostic NGS panel - all markers can be considered
3. DTA not good choice as pre-leukaemic
4. Germline genes not good
5. Signally genes such as FLT3, KIT, RAS are likely to represent residual leukaemia when detected but they have low negative predictive value so should be used in combination with other markers

Question 5

What is the definition of MRD positive by qPCR and NGS?

Answer 5

qPCR: Ct < 40 in at least two out of three reps
gDNA: ≥0.1% VAF

Question 6

What is the definition of MRD relapse?

Answer 6

Negative to positive, or
1 log increase or more if MRD LL

**should confirm with repeat testing within 4 weeks, preferentially in BM sample

Question 7

What is the definition of MRD LL in NPM1+ AML

Answer 7

MRD-LL detection using cDNA in NPM1-mutated AML is provisionally defined as <2%

Question 8

What are the recommended MRD measurement timepoints for NPM1, RUNX1-RUNX1T1, and CBFB-MYH11 mutated AML?

Answer 8

1. PB after 2 cycles chemo
2. BM after consolidation
3. BM q3monthly or PB q6weekly

Question 9

What is the definition of MRD negative by qPCR?

Answer 9

MRD test negativity by qPCR is defined as Ct >40 in at least 2 of 3 replicates, when ≥10 000 copies (optimally, ≥30 000 copies) of the housekeeping gene were measured.