Monitoring residual disease: FACS

Question 1

What is leukemogenesis and what are patients with leukemogenesis diagnosed with?

Answer 1

With leukemogenesis, there is a block in differentation at different stages of myeloid cells and uncontrolled proliferation.
Patients are diagnosed with extremely high levels of white blood cells that overgrow the normal bone marrow.

Question 2

What is the morphology of AML?

Answer 2

20-100% primitive blood cells present instead of the maximum of 5% in normal bone marrow

Question 3

What is the two hit model for AML onset?

Answer 3

Class I mutations that confer proliferative and/or survival advantage (BCR/ABL, cKIT, N-Ras, K-Ras, FLT3 activating mutations). If these mutations occur alone, there is a Chronic Myeloid Leukemia (CML)-like disease.

Class II mutations:
Impair hematopoietic differentiation and subsequent apoptosis. (translocation: PML/RAR, AML/ETO. Point mutations: C/EBPalpha and AML1). If these mutations occur alone, there is Myeloid Dyplastic Syndrome (MDS)-like disease.

If there are class I mutations, class II mutations and epigenetics, there will be AML(Acute Myeloid Leukemia).

Question 4

How is (a certain type of) leukemia diagnosed?

Answer 4

1. Bone marrow morphology
2. Look at the type of leukemia that the patient has:
   a. Cytogenetics
   b. Multicolour flow cytometry
   c. Molecular aberratons

Question 5

What is leukemia prognosis dependent on?

Answer 5

Prognosis depends on:
•Clinical characteristics:
o Age
o White cell blood count
• Cytogenetic subgroups
o (Poor: complex karyotype, monosomies of chromosomes 5 or 7)
o (Good: t(8;21), t(15;17), Inv(16)/t(16;16))
• Mutations:
o Poor: FLT3/ITD
o Good: NPM1
Treatment is done according to these diagnosis related risk factors, but still 30-40% of patients relapse

Question 6

How does relapse develop?

Answer 6

Relapse development:
1. Cancer blast cells are reduced to <5% of blood cells after treatment
2. Drug resistant cells that look healthy in microscopy are still present
o These cells induce relapse
3. Leukemia stem cells have a high self-renewal capacity, are good at DNA repair, quiescent and undifferentiated
o They are very drug resistant and therefore capable of inducing a relapse

Question 7

How can you predict the risk of relapse?

Answer 7

When you look at the bone marrow, you can see leukemia stem cells (minimal risidual disease) and predict the risk of relapse

Question 8

What is the advantage of using minimal residual disease to predict the risk of relapse?

Answer 8

MRD is the resultant of involvement of many different features
o At diagnosis: cellular resistance:
 Cytogenetic and molecular aberanties, apoptotic potential, etc
o During therapy:
 Pharmacokinetic resistance (rapid clearance of chemotherapeutic drugs)
 Therapy faithfulness
 Immune system of the patient
 Other patient and external factors

Question 9

Which detection methods are there and which is the best?

Answer 9

Morphology, cytogenetics, FISH, (real-time) RT-PCR, flowcytometry (immunophenotyping). Flowcytometry is the best if you look at the combination of availability and sensitivity.

Question 10

Which markers are used for assessment of residual disease using flowcytometry?

Answer 10

Markers used for assessment of residual disease using flowcytometry:
Myeloid: CD13, CD14, CD15, CD33, CD117

No lymphoid markers present in healthy situation: CD2, CD3, CD7, CD19, CD22, CD56

Question 11

What are the different immunophenotypic aberrancies?

Answer 11

Cross-lineage: myeloid markers on a lympoid cell
o E.g. CD13: CD2 (T-cell), CD19 (B-cell), CD56 (NK cell)

Asynchronous: Aberrant expression of “mature” markers on immature populations
o E.g. CD11b (monocyte marker) on primitive CD34+ cells

Over-expression or underexpression of markers
o E.g. CD33, CD34

Question 12

How many patients have leukemia associated immunophenotypes?

Answer 12

• Roughly 90% of patients have Leukaemia Associated ImmunoPhenotypes (LAIP)

Question 13

What % of MRD (cells) means a higher chance of relapse?

Answer 13

> 0.1% MRD (cells) means a higher chance of relapse. Prognostic relevance confirmed in clinical trials.

Question 14

When is MRD used?

Answer 14

MRD is both used in clinic and in clinical trials as a guide

Question 15

Is MRD 100% predictive of relapse?

Answer 15

No. ~30% of MRD negative patients still relapse

Question 16

How can monitoring of the relapse risk be improved?

Answer 16

1. More sensitive detection
   a. Molecular MRD
2. Sequential monitoring to identify quick relapses
   a. Relevance of monitoring in peripheral blood
3. Take clonal evolution into account
   a. Circumvent false negatives by monitoring only the dominant clone assessed at diagnosis
4. Determine presence of Leukemic Stem Cells (LSC)

Question 17

Is molecular MRD an option to determine risk of relapse?

Answer 17

It can be highly sensitive in case of patient/mutation specific PCR. However, only for ~60% of patients the mutation is known
It can be positive for a long time, also in long-term complete remission –> pre-leukemic clones

Question 18

Can NGS be used to determine the risk of relapse?

Answer 18

Next generation sequencing can be used to look at multiple mutations at a time, which can be useful because for ~40% of patients the mutation is unknown.

During remission, there is still a high proportion of certain mutations, this means they may be from pre-leukemic cells. Mutations can also be present in non-cancer population and increase with age. When these “normal” mutations are discarded, NGS can better be used as prognosis

Question 19

Can NGS and MRD flowcytometry be used together?

Answer 19

The techniques are complimentary. Double positives have very poor prognosis. Combining both techniques may lead to a more accurate risk assessment.

Question 20

What is important to keep in mind with sequential monitoring?

Answer 20

The timepoint when you look at a patient is very important. Kinetics of MRD may differ by AML subtype or treatment. MRD can be measured in peripheral blood.

Question 21

What are disadvantages of measuring MRD in bone marrow?

Answer 21

• Burden to patient: especially those in continuous remission: (unnecessarily) painful.
• Burden to doctors and laboratory: expensive
• What to do if the patient becomes MRD positive

Question 22

Can you measure MRD in blood?

Answer 22

Good correlation between MRD in bone marrow and MRD in peripheral blood. Peripheral blood is 1 log less sensitive compared to bone marrow. Some patients don’t have blasts and thus no MRD in the peripheral blood because those cells mostly reside in the bone marrow. Maybe look into bone marrow, when patient doesn’t have any MRD in peripheral blood

Question 23

Why is it important to look at clonal evolution when looking at relapse risk?

Answer 23

There can be shifts in population between diagnosis and relapse. A very small population at diagnosis can be the main population at relapse. This is how how you can miss it. In most cases it has an immature (stem cell like) phenotype. This small population can be found using a very sensitive method.

Question 24

What are challenges of leukemia stem cell detection by FACS?

Answer 24

o Very low frequency requires a large amount of cells (4*10^6) to acquire enough events
o Heterogenous LSC specific marker expression between and within bone marrow samples of AML patients

Question 25

What is the one tube assay?

Answer 25

An assay that takes clonal evolution into account. One tube for LSC detection at diagnosis and follow-up.
• Identifies almost all CD34+CD38- LSC
• Needs no extensive experience in BM immunophenotyping
• Multi-institutional approach possible
• Can be extended with other markers in the PE-channel
• Takes into account immunophenotype shifts
• Requires 8 million cells since stem cell frequency is low

Question 26

Which two factors can point to patients with a high risk of relapse?

Answer 26

Patients with leukemic stem cells and minimal residual disease

Question 27

What is the clinical relevance of MRD?

Answer 27

Clinical relevance of MRD:
• Prognositic factor:
o Established in retrospective and prospective studies
• Predictive factor:
o Use for individual patient needs additional fine tuning
o Optimization of the assay
o Prediction models including several risk parameters
• Disease monitoring
o During therapy to tailor treatment strategies
• Surrogate endpoint:
o Early efficacy assessment in trials involving novel therapies
o Needs validation in a positive clinical trial (significantly effective drug) which has MRD measurements included

Question 28

What are remaining questions in monitoring residual disease?

Answer 28

Are all aberrant CD markers present on the same cell or are these different clones? Should we add up all CD markers to accurately estimate the residual burden?
Is there a specific characteristic defining relapse initiating cells that we can target?