Minimal Residual Disease in Acute lymphoblastic leukemia

1. Minimal Residual Diseases in Acute
Lymphoblastic Leukemia
Dr. Liza Bulsara

2. • Minimal residual disease (MRD)/ Measurable Residual Disease describes
disease that is detected only by laboratory techniques more sensitive than
morphology, such as flow cytometry (immunologic MRD) or polymerase
chain reaction (PCR) (molecular MRD).
• As it is found in the absence of clinical signs or symptoms, it is termed as
“ Minimal”.
• Specific markers are required for detecting 1 cell in 10,000 normal cells to
distinguish them from malignant cells and lymphocytes.
E Paietta , Assessing minimal residual disease (MRD) in Leukemia: a changing definition and concept, A review, Bone Marrow Transplantation (2002) 29, 459–465

3. Timeline:
• 1980, Janossy and colleagues reported the use of antibody staining of terminal
deoxynucleotidil transferase (TdT) to differentiate leukemic lymphoblasts from normal
lymphocytes in the cerebrospinal fluid of patients with ALL
• A year later, they reported that anti-TdT in combination with an anti-T cell antibody
could identify MRD in the bone marrow of patients with T-ALL in morphologic
remission
Mel Greaves, Leukaemia ‘firsts’ in cancer research and treatment, Nature review: Cancer, VOLUME 16, O 162-166, march 2016.

4. Learning Objectives:
MRD detection as a clinical
diagnostic tool.
To determine the properties
of different methods for
MRD detection.
To understand the issue of
assay sensitivity in the
context of treatment
reduction.
To determine clinically
relevant time points in
treatment for informative
MRD detection.
To understand that the
prognostic impact of MRD
positivity may vary between
different ALL subsets.
To recognize that MRD is an
important tool to fine-tune
allogeneic hematopoietic
stem cell transplantation.
To determine whether MRD
detection may change the
classical definition of
relapse.
To understand the role of
MRD as an endpoint in
clinical trials.

5. MRD detection: Clinical diagnostic tool.
• MRD detection is part of state-of-the-art diagnostics and is
needed in the management of ALL
• To risk-stratify patients with ALL because response is of high
prognostic relevance.
• The prospective analysis with the MRD method of choice to
determine the prognostic significance of certain MRD levels
on the background of a predefined uniform chemotherapy
regimen.

6. Techniques of MRD assessment:
• Morphology
• Clonogenic Assays
• Immunophenotype
Analysis
• Karyotype Analysis
• FISH
• PCR
SA Buckley, FR Appelbaum, RB Walte, Prognostic and therapeutic implications of minimal residual disease
at the time of transplantation in acute leukemia, Bone Marrow Transplantation (2012), 1–12

7. Morphology
Limited by its low sensitivity.
Generally, only 1 of 100 cells can be identified as
malignant.
Inability to distinguish between immature cancer
cells and early regenerating cells further restricts
the usefulness of standard morphologic methods.
Sensitivity and specificity of morphology can be
enhanced when combined with other tools such as
immunophenotyping or FISH.

8. Clonogenic Assays:
• In vitro culture techniques, bone marrow samples from patients
are grown under conditions favourable for stimulation of
leukemic cells.
• Advantages:
1. Identification populations of occult malignant cells that can
be expanded.
2. Allowing their biologic characteristics and growth
requirements for analysis by immunologic, cytogenetic, or
molecular techniques.
• Disadvantages: Difference of growth rates of leukemic
progenitor cells and the cumbersome nature of assays

9. Immunophenotypic Assays
• Use of monoclonal antibodies by means of flow cytometry or fluorescence
microscopy to detect nuclear, cytoplasmic, and surface antigens is fast and
reliable.
• Sensitivity of detection can be as high as 1 abnormal cell per 10,000 to
1,00,000 normal cells using double- or triple-color immunofluorescence
techniques or fluorescence microscopy screening multiple slides per sample.
•Disadvantages:
(1) The lack of antigen specificity for malignant cells as identical or similar
antigen profiles.
(2) several subpopulations are difficult to identify.
(3) The inability to identify phenotypic switch.

10. Leukemia associated immunophenotypes
S. Pervez, M. Khurshid, Classification and Immunophenotyping of Acute Leukemias: A Prospective Study, Journal of Pakistan
Medical Association, April 2000, P 106-110

11. KARYOTYPIC ANALYSIS
An Important Tool In Risk
Stratification .
Cytogenetic Studies Provide
Specificity Because They
Can Unambiguously Identify
Malignancy-specific Markers
And Detect Cytogenetic
Signs Of Clonal Evolution At
Relapse.
The Pitfalls Of Conventional
Cytogenetic Analysis Are Its
Labor Intensity (Because It
Requires In Vitro Cultures)
And The Dependency Of
Karyotype Identification On
Dividing Cells, Thereby
Missing Populations Of
Residual Cells With A Low
Proliferative Index.
Despite Its Specificity, The
Sensitivity Of The Method Is
Low And Does Not Exceed
That Of Morphologic
Assessment Of Marrow
Smears.

12. FISH
• FISH : A promising tool for the identification on a molecular level of both chromosomal
aberrations and malignancy-specific DNA sequences.
• The sensitivity level achieved by FISH( one cell in 10-³) is markedly below that desired for
MRD detection (one cell in 10,000)
• Advantages:
1. High specificity
2. Time-efficient manner to study larger number of cells.
3. The quantifiability of results.
4. Analysis of both metaphase and nondividing interphase cells.

13. Genetic Abnormality of ALL
Robin Foà; Sabina Chiaretti; Anna Guarini; Antonella Vitale,Adult acute lymphoblastic leukemia, Review. Rev. Bras. Hematol. Hemoter. vol.31 supl.2 São Paulo Aug. 2009

14. PCR
• It allow the detection of leukemia-specific gene rearrangements by identifying
either leukemia-specific translocations or clone-specific immunoglobulin heavy
chain (IgH) gene and T-cell receptor (TCR) gene rearrangements.
• Unparalleled sensitivity. A single malignant cell can be identified among 10,000
to 10,00000 normal cells.
• It is the preferred tools in studies addressing the detection and role of MRD.
• Disadvantages:
1. Sample contamination.
2. False-positive results can occur.
3. Measures transcript expression, a malignant clone that transiently does not
express its diagnostic transcript would evade detection by this technique.

15. Flow Cytometry
• Flow cytometry gating strategy for B- and T-ALL after 5 color
analysis on a FACSCantoII (BD Biosciences) using Diva software.
• For this example of B-ALL (top panel) the initial
immunophenotype was CD45+, CD19+, CD10+,CD38dim,
CD58++, CD123++.
• Cells were first gated on CD19 and side scatter then on CD10 and
CD45. From this whole population of CD19+ CD10+ cells, blasts
were progressively gated by their expression of CD81/CD58, dim
CD38 and CD123. Back-gated blasts are stained red. MRD level
(blasts/nucleated cells): 500/130,0000. MRD 0.0038%.
• For this example of T-ALL (lower panel) the initial
immunophenotype was cCD31, sCD3-, CD51 CD99+, TdT1/2.
• Cells were first gated on cCD3 and side scatter then on sCD3 and
CD5. From this population, blasts were progressively gated by the
expression of CD99 and low TdT. Back-gated blasts are stained
red.
• MRD level (blasts/nucleated cells): 1890/700,000 , MRD 0.0027%.

17. SA Buckley, FR Appelbaum, RB Walte, Prognostic and therapeutic implications of minimal residual disease at the time of transplantation in acute leukemia, Bone Marrow Transplantation
(2012), 1–12

18. Sensitivity of methods to detect MRD
Method Sensitivity Principle
Morphology 1:100 Morphologic differences of tumor cells
Conventional
Cytogenetics
1:100 Demonstration of Specific chromosomal Abberation.
Southern Blot 1:100 Demonstration of specific DNA sequence : Chromosomal Abberation.
FISH 1:1000 Demonstration of Specific DNA Sequence : chromosomal Abberation
Immunologic
Methods
1:10000 Demonstration of tumor specific antigen or antigen combination
PCR 1:1,00,000 Demonstration of specific DNA Sequence: chromosomal aberration or
mutation
RT-PCR 1:1,00,000 Demonstration of specific DNA Sequence: chromosomal aberration or
mutation
Eckschlager , Philip l et al, Flow cytometry in clinical practise, journal of immunohistology ,jun 1999, vol 52-2, P 32-35

19. Clinical application of
MRD diagnostics
• MRD diagnostics is strongest prognostic
factor, allowing for risk group assignment into
different treatment arms, ranging from low-
risk/standard-risk with treatment reduction to
medium-risk or high-risk with mild or strong
intensification, respectively.
• AIEOP-BFM-ALL 2000 studies have shown
that MRD-based treatment strategies further
improve outcome in the involved patients, both
in BCP-ALL and T-ALL patient.
Campana D. Minimal residual disease monitoring in childhood acute lymphoblastic
leukemia. Curr Opin Hematol. 2012;19(4):313-318.

20. . Theunissen P, Mejstrikova E, Sedek L, et al; EuroFlow Consortium. Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia. Blood.
2017;129(3):347-357

21. MRD in Special Subgroups:
• MRD at the end of induction (day 33) is more informative in pcB-ALL,
• MRD at the end of consolidation (day 78) is more informative in T-ALL
• MRD levels in pcB-ALL correlate with risk of systemic relapse, whereas MRD in
T-ALL is predictive of both systemic and extramedullary relapse.
• >10% leukemic blasts by FCM on day 15 is enrolled into the HR group; any
patient with pcB-ALL who has MRD >10-3 (0.1%) at day 33 and is still MRD+ at
day 78 is stratified into the HR group
• Any pcB-ALL patient with MRD >0.1% at day 15 is considered standard risk if
PCR results (with at least one highly sensitive molecular target, quantitative range
up to 10-4) at day 33 and day 78 are also negative
Gaipa G, Cazzaniga G, Valsecchi MG, et al. Time point-dependent concordance of flow cytometry and RQ-PCR inminimal residual disease detection in childhood acute lymphoblasticleukemia.
Haematologica. 2012;97(10):1582-1593.

22. Borowitz MJ, Devidas M, Hunger SP, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a
Children’s Oncology Group study. Blood. 2008;111(12):5477-5485.

23. Sample
collection:
Pui CH, Pei D, Coustan-Smith E, et al. Clinical utility of sequential minimal residual disease measurements in the context of risk-based therapy in
childhood acute lymphoblastic leukaemia: a prospective study. Lancet Oncol. 2015;16(4):465-474.

25. • Sensitivities of 1/1000 require sufficient numbers of BM cells.
• only the first BM aspirate & large volumes is also discouraged.
• To collect >2 mL but <5 mL of the first BM aspirate.
• RQ-PCR–based MRD studies require, for each follow-up time point, >2 x 10-6
cells, which is sufficient to extract >6ug of DNA, needed for analysis of >2 MRD-
PCR targets in triplicate and the control gene in duplicate.
• only 50% of DNA is recovered from the theoretical 13 ug of DNA, present in
2x10-6 cells.
Yeoh AE, Ariffin H, Chai EL, et al. Minimal residual disease-guided treatment deintensification for children with acute lymphoblastic leukemia: results from the Malaysia-
Singapore acute lymphoblastic leukemia 2003 study. J Clin Oncol. 2012;30(19): 2384-2392

26. • Current flow cytometric MRD studies require even more cells: >5 x 10-6 cells.
• The cell recovery is related to the time point, with low cell yields at days 15 and
33 but higher cell yields at day 79 and later time points.
• Lack of sufficient cells at day 33 is more a problem, because at that time, it is
important to identify patients with undetectable MRD levels, using MRD-PCR
targets with a quantitative range of >10-4.
• Appropriate BM sampling is a critical part of MRD based clinical studies

27. MRD negativity:
• It implies that no MRD is detected with high certainty, using an MRD technique that can truly measure low MRD
levels (quantitative range, <10-4)
• This definition is needed to identify MRD-based low-risk patients with very low chance of relapse(3-5%); to
consider therapy reduction.
• The Dutch Childhood Oncology Group(DCOG) performed a study with significant treatment reduction in the
MRD-based low-risk group, resulting in an excellent outcome with very few side effects.
• In this DCOG-ALL10 treatment protocol, the sharp criteria of the MRD-based low-risk group of the original I-
BFM-SG study have been retained to define MRD negativity, using >2 different types of sensitive IG-TR PCR
targets, thereby avoiding or reducing oligoclonality problems and related false negativity.
• This made the MRD based low-risk group one-third smaller than previously (≈28% instead of ≈43%).
• MRD technologies should aim for 10-4 to10-5 to define the MRD-based risk groups accurately
van der Velden VH, Cazzaniga G, Schrauder A, et al; European Study Group on MRD detection in ALL (ESG-MRD-ALL). Analysis of minimal residual disease by Ig/TCR gene rearrangements:
guidelines for interpretation of real-time quantitative PCR data. Leukemia. 2007;21(4):604-611.

28. MRD & SCT
MRD monitoring of pre-SCT
response demonstrated the
necessity of optimizing the
quality of remission before
transplant to prevent post-
SCT relapse.
the most important predictor
for post-SCT outcome.
MRD measurements after
SCT allows prediction of
relapse.
MRD measurements are now
guiding treatment decisions
in childhood ALL in patients
undergoing SCT.
The adverse prognostic of
MRD positivity before SCT,
but a particularly bad
prognosis was associated
with MRD reappearance
after SC

29. MRD Remission/Relapse:
Bruggemann M, Schrauder A, Raff T, et al. Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in
Kiel, Germany, 18-20 September 2008. Leukemia. 2010;24(3):521-535

30. Recent Advances

31. Dworzak MN, Gaipa G, Schumich A, et al. Modulation of antigen expression in B-cell precursor acute lymphoblastic leukemia during induction therapy is partly transient:
evidence for a drug-induced regulatory phenomenon. Results of the AIEOP-BFM-ALL-FLOW-MRD-Study Group. Cytometry B Clin Cytom. 2010;78(3): 147-153.

32. EuroFlow-based multidimensional analysis of normal and malignant BCP
cells.
Ladetto M, Bru¨ggemann M, Monitillo L, et al. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia. 2014;
28(6):1299-1307.

33. Logan AC, Vashi N, Faham M, et al. Immunoglobulin and T cell receptor gene high through put sequencing quantifies minimal residual disease in acute lymphoblastic leukemia and
predicts post-transplantation relapse and survival. Biol Blood Marrow Transplant. 2014; 20(9):1307-1313.
High-throughput sequencing of IG-TCR targets (DNA level)

34. Risk Adapted therapy based on MRD
Based on MRD level:
High MRD Level: Intensification of
Post remission treatment (ASCT in
First Remission)
Low/Undetectable MRD Level:
Decreased Duration or intensity of
treatment
COG of North America has
combined two prognostic markers
to assign post remission therapy :
1) Result of remission induction
day 14 bone marrow examination
2) A day 29 MRD Evaluation.
For Low MRD, Defined as MRD
Levels <10 -4 at post remission time
points, treatment will be stopped
after a year and maintenance
therapy will be omitted.
For High MRD, Defined as MRD
Levels >10 -4 at any post remission
time points, intenstification
treatment and focusing on Allo-
SCT.

35. Conclusion:
In the ALL field,
MRD diagnostics is
no longer A (clinical)
research tool for
evaluation of clinical
trials only but has
become part of
diagnostic patient
care.
Standardized MRD
diagnostics should be
available for
assessment of
treatment response in
each individual ALL
patient, accurate risk
group assignment
with risk-adapted
treatment.
Evaluation of new
treatment
modalities, where
MRD
measurements can
demonstrate the
effectiveness of
the novel treatment
and be used as a
surrogate end point