25. Strategies of MRD monitoring

Question 1

What is MRD?

Answer 1

Minimal residual disease - low level cancerous cells that remain after treatment; only detectable using highly sensitive techniques

Question 2

What is the purpose of MRD?

Answer 2

Determine efficacy of therapy

Enable early recognition of relapse - change in genetic profile often precedes any physical symptoms

Question 3

How is MRD monitored in AML?

Answer 3

RT-qPCR for fusions and NPM1 mutations

Also flow cytometry

Question 4

In AML, which fusions require a diagnostic sample?

Answer 4

Required for CBFB:MYH11 and PML:RARA as several different fusion transcripts possible

Not mandatory for RUNX1:RUNT1 - one transcript only

Question 5

What is commonly seen in MRD monitoring in CBF AML?

Answer 5

t(8;21) and inv(16) frequently test MRD positive at low levels for many years after completion of treatment and as long as they remain below the defined thresholds are not at risk of relapse

Question 6

What gene is usually used as a control in RT-qPCR?

Answer 6

ABL1 - constitutively expressed in all cells

Question 7

How is MRD monitored in ALL?

Answer 7

Often flow cytometry as no specific chromosomal aberration

Identify cells with immunophenotypic features of immature T-cells

RT-qPCR if known fusion transcript

Question 8

How are bone marrow transplants monitored?

Answer 8

Chimerism analysis - determine success of engraftment

PCR of STRs and X-Y markers

Question 9

What are the pros and cons of using ddPCR for MRD?

Answer 9

High sensitivity and specificity, fast, low error rate

New assay needs to be designed for specific base changes

Question 10

What are the two common transcripts in BCR:ABL?

Answer 10

e13a2 and e14a2 - found in 98% of cases

Question 11

How does RT-PCR determine the level of fusion transcripts?

Answer 11

1. Run plasmid standards with known quantities of BCR:ABL and ABL1 to create standard curves. Controls repeated in triplicate
2. Compare patient’s BCR:ABL and ABL1 levels to standard curve to quantify each. Patient samples tested in triplicate and averaged
3. Calculate ratio of BCR:ABL to ABL1
4. Multiplied by an international conversion factor to produce a result on the ‘International Scale’

Question 12

How does RT-qPCR work?

Answer 12

RNA reverse transcribed into cDNA

Taqman probe within ROI, flanked by primers

Fluorescence when probe is degraded

Question 13

How is absolute quantification obtained in RT-qPCR?

Answer 13

Obtained from exponential phase of a PCR - when product is doubling with every cycle & is proportional to the amount of initial product

Measurement must be above CT

Question 14

Why is the International Scale important in BCR:ABL MRD?

Answer 14

Allows accurate comparison between labs for clinical trials

Allows accurate determination of response levels

Question 15

What does low ABL1 signify? How is it reported?

Answer 15

Low ABL1 - amplification hasn’t worked well, RNA degraded

<10,000 copies - qualitative report only

<5000 copies - fail

Question 16

What are the 3 measures of response to treatment in CML?

Answer 16

1. Haematological response (HR) – normalisation of blood count and splenomegaly
2. Complete cytogenetic response (CCR) – No Ph chr in 20 metaphases
3. Major molecular response (MMR) - BCR-ABL1:ABL1 ratio of >0.1%

Deep MR - ratio of >0.01%

Question 17

What are the major molecular response milestones in CML?

Answer 17

At diagnosis - 100% BCR:ABL transcript levels

3 months of therapy - 10%

6 months - 1%

Major molecular response - 0.1%