1. Payvand
Clinical Specialty Lab.
3rd Biennial International Pediatric Oncology Congress
In memory of Prof. Parvaneh Vossough
Evaluation of Minimal/Measurable Residual Disease, MRD in Acute Lymphoblastic Leukemia
Behzad Poopak, DCLS PhD.
Associate Professor of Hematology

2. Objectives My main Objectives are to review:
Measurable Residual Disease & its Advantages
Main techniques for MRD assay with main focus on MFC
Technical requirements
The condition in Iran

3. Detection of MRD during follow-up of
ALL patients
- Persistence of a very small number of malignant cells that cannot be detected by ‘routine’ morphology
- MRD indicates “persistent disease” despite “complete remission”
Minimal or, more appropriately,
Measurable Residual Disease (MRD)
denotes the presence of leukemia cells down to levels of 1: 𝟏𝟎 𝟒 to 1: 𝟏𝟎 𝟔 wbcs, compared to 1:20 (5%) in morphology-based assessments.
Detection of MRD during follow-up of ALL patients. (A) Schematic diagram of relative frequencies of ALL cells in BM during and after treatment. I, induction treatment; C, consolidation treatment; II, reinduction treatment. The detection limit of cytomorphology and the detection limit of immunophenotyping and PCR techniques is indicated. (B) Follow-up of a T-ALL patient with CD5/TdT double immunofluorescence microscopy.58 The frequencies of the T-ALL cells in blood and BM are very comparable in this patient. D, diagnosis; CR, complete remission; Re, relapse.
Jacques J. M. van Dongen et al. Blood 2015;125:

4. MRD, Introduction MRD diagnostics is:
MRD Monitoring is routine clinical practice in treatment of virtually all childhood & many adult ALL patients.
MRD diagnostics is:
MRD diagnostics before allogeneic SCT in childhood ALL
the most important predictor for post-SCT outcome
MRD measurements after SCT
Allows prediction of relapse
The strongest prognostic factor
The most powerful predictor of outcome
Allow risk group assignment
Highly predictive of relapse (in children, adolescents & young adults)
Guiding treatment decision in relapsed ALL
Guiding treatment decision undergoing SCT
Evaluation of treatment effectiveness in clinical trials with innovative drugs, such as antibodies
SCT:stem cell transplantation, MRD measurements might well be used as a surrogate end point, thereby shortening the clinical trials significantly.
If so, the novel drugs will become faster available for the patients at affordable prices.
identify good & poor responders
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5. Which sensitivity and which time points are required for risk group definition?
MRD levels of 10-2(1%), 10-3 (0.1%), and 10-4 (0.01%), and MRD negativity were clearly related to different outcomes at the first follow up time points.
MRD measurements at 1 (day 33) & 3 months (day 78) appeared to provide the most important prognostic information
- Low-risk patients are MRDnegative at both time points (no detectable MRD, ≤10-4 or ≤0.01% );
- High-risk patients have high MRD levels (≥5 x10-4 or ≥ 0.05% ) at the 3-month time point;
- Medium-risk patients have moderate to low MRD levels (<5 x 10-4 or < 0.05%) at the 3-month time point .
Importantly, at later time points (after consolidation, after reinduction, and during first part of maintenance treatment), any MRD positivity was related to poor outcome.
Patients who have high levels of MRD (ie, ≥1%) at the end of remission induction therapy & persistent MRD after subsequent consolidation treatment have a very high risk of relapse if treated with currently available chemotherapy.
The best treatment option for these patients at this point in time is allogeneic HSCT, particularly if levels of MRD can be reduced to undetectable status before transplant.
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6. Long-term follow-up in childhood ALL patients, classified according to MRD measurements
Long-term follow-up in childhood ALL patients, classified according to MRD measurements. (A) Disease-free survival of 129 ALL patients, classified according to 3 MRD-based risk groups in the International BFM study.1 Patients were classified as MRD-low-risk if no MRD was detected at day 33 (TP1) and at day 78 (TP2); patients with MRD ≥10−3 at TP2 were classified as MRD-high-risk; all other patients had MRD <10−3 at TP2 and were classified as MRD-intermediate-risk. (B) Disease-free survival of 54 infant ALL cases, treated according to the INTERFANT-99 treatment protocol.67 Patients were considered MRD-high-risk if the MRD level at TP3 was ≥10−4; patients were considered MRD-low-risk if MRD levels were <10−4 at both time points; all remaining patients were considered MRD-medium-risk. Only 3 of 24 MRD-low-risk patients relapsed, whereas all 14 MRD-high-risk patients relapsed. (C) Event-free survival of 3184 BCP-ALL patients of the AEIOP-BFM 2000 study (with kind permission by Dr V. Conter, Monza, Italy).63 Patients were classified as MRD-standard-risk (SR) if no MRD was detected at day 33 (TP1) and at day 78 (TP2) and as MRD-intermediate-risk (IR) when MRD was positive at 1 or both TPs but <10−3 at TP2. Patients with MRD ≥10−3 at TP2 were classified as MRD-high-risk (HR). (D) Event-free survival of 464 T-ALL patients of the AEIOP-BFM-ALL 2000 study (with kind permission by M. Schrappe, Kiel, Germany).64 The MRD-based classification is the same as for C.
Jacques J. M. van Dongen et al. Blood 2015;125:
©2015 by American Society of Hematology

7. Technical Requirements
Bone marrow sampling
Bone marrow transport
Type of Flow cytometers
Preparation of samples
Antibody panel selection
Analysis & report

8. Technical Requirements
Bone marrow sampling
Anti-coagulants: EDTA or heparin (no significant difference between them)
Heparin is the best for maintaining the characteristics of the antigens, particularly in older specimen (collection >24hrs).
Heparin, in comparison to EDTA, better keeps the leukocytes viable and intact.
EDTA may affect the expression of certain antigens such as CD10, CD11b, CD16 or CD64.
EDTA specimens are appropriate for routine use because the EDTA tubes are available, can be stored up to 24 hrs. & used for molecular tests (PCR).

9. How many cells are needed for reliable MRD measurements?
Sensitivities of ≤10-4 (0.01%) require sufficient numbers of BM cells.
To maximize assay sensitivity, it is mandatory to avoid hemodilution of BM samples, thus, submitting the first BM pull for MRD analysis is strongly recommended, at least for follow up BM samples intended for MFC MRD.
Aspiration of large volumes is also discouraged
It is advised 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 106 cells, which is sufficient to extract ≥6mg of DNA, needed for analysis of≥2 MRD-PCR targets in triplicate and the control gene in duplicate.
Please note that generally only 50% of DNA is recovered from the theoretical 13 mg of DNA, present in 2 x 106 cells.
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10. Peripheral Blood vs. Bone Marrow Aspirate
Blood MRD levels in T-ALL patients were comparable or up to 1 log lower than in BM.
In BCP-ALL patients, blood MRD levels were logs lower than in BM,
Consequently, for both BCP-ALL and T-ALL patients, BM sampling is a prerequisite.
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11. Technical requirements Bone marrow transport
In the multi-center setting, we recommend transport at controlled room temperature (18-22 °C)
If BM is stored undiluted:
There would be no need for a viability marker.
Up to 3 days storage is allowed but less than 24 hours is the choice
A few exception such as Burkitt,s type should be considered.

12. Technical Requirements
Flow cytometers
Accurate and sensitive detection of low frequencies of ALL cells,
≤1 ALL cell in normal cells (≤ 0.01% or ≤ 10-4)

13. Technical Requirements
Flow cytometers
The sensitivity of flow-cytometric method depends on:
- Number of MFC colors
- 10−3 to 10−4 MRD cells with 3–4 colors
- 10−4 to 10−5 with >6 colors.
New ways of sample preparation(bulk lysis) & improvements in analysis strategies can increase MFC sensitivity.

14. Characteristics of Next Generation Flow cytometry (≥8 colors) MRD techniques
Estimated sensitivity ( x 106 cells analyzed), comparable to the sensitivity of RQ-PCR–based MRD analysis
Applicability BCP-ALL: 95%; T-ALL: 90%
Availability Multiple laboratories in Europe, South America, Asia, South Africa, and Australia (still limited in United States) & fortunately in Iran at now.
Standardization/ assay verification
Full technical EuroFlow standardization and assay verification
QA rounds: Yearly external technical QA (will be increased to several QA rounds per year)
Clinical validation Ongoing
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15. Concordance bet. Flow cytometric & Molecular MRD data
Only samples in which MRD could clearly be detected by FCM-MRD or samples which had at least 4 x 106 cells acquired were included in the analyses. Based on an LLOQ of 40 events, this allowed a sensitivity of at least 10-5.
TP, time points.

16. Next Generation Flow Cytometry (≥8 colors) Advantages vs. Disadvantages
Rapid (within 3-4 h)
Sample cellularity & the degree of hemodilution is evaluable
Highly standardized with possibilities for automated gating (Infinicyt software)
Efficient data storage and management with easy data comparison
Accurate quantitation
Provides information on normal and malignant cells
Ready for IVD development
Disadvantages
Education and training required, high expertise to perform MRD assays & its interpretation
The similarity to normal precursor cells
The modulation of antigen expression during treatment
Costs?
- Machine & Kits or Abs Panel
- Maintenance
Many cells needed to reach the required sensitivity, eg, 5.0 x 106,
The lack of inter-laboratory standardization and quality control & immunophenotypic shifts
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17. Common modulation of antigen expression during ALL treatment
Treatment-induced immunophenotypic shifts within the ALL cell population, including lineage shifts in approx. 5% of pediatric cases, such as CD2+ BCP-ALL cases with an early switch to the monocytic lineage
The expression of CD58 is not significantly changed compared to levels at diagnosis, either in BM or in PB samples

18. How many cells are needed for reliable MRD measurements?
If a cluster of BCP-ALL cells should be present to consider a sample as positive, one should acquire at least 4 million cells (≥ 4 x 106)
Because the cellularity of BM samples obtained during the early phases of treatment is frequently low, Bulk lysis protocol recommended

19. Recovery of BM mononuclear cells at different time points during follow-up
Recovery of mononuclear cells is relatively low at days 33 and 78 (median, 5-8 x 106). Recovery at day 78 and at later time points is much higher (median, × 106)
ALL cell frequencies in blood and BM samples during follow-up. (A) Frequencies of T-cell marker+/TdT+ T-ALL cells, as detected by immunofluorescence microscopy in 321 paired blood and BM samples, obtained from 26 patients.58,60 The T-ALL cell frequencies are comparable in many pairs, but differences can occur up to 1 log. Orange, sample <3 months of follow-up; green, >3 months of follow-up. (B) (Left) Frequencies of ALL cells in 149 paired blood and BM samples from 22 T-ALL patients, analyzed by RQ-PCR of TR gene rearrangements and TAL1 deletions.60 A strong correlation was observed between the blood and BM frequencies in T-ALL. (Right) Frequencies of ALL cells in 532 paired blood and BM samples from 62 BCP-ALL patients, analyzed by RQ-PCR of IG and TR gene rearrangements.60 The MRD levels were significantly higher in BM compared with blood. Moreover, the ratio between the MRD levels in BM and blood was highly variable, ranging from 1 to 3 logs. Orange, sample <3 months of follow-up; green, >3 months of follow-up. (C) Frequencies of ALL cells in 141 paired BM samples (left-right) from 26 patients, showing a very high concordance.62 Only in case of very low MRD levels was variation seen, mainly because of levels outside the quantitative range of the RQ-PCR assay. Orange, sample <3 months of follow-up; green, >3 months of follow-up. (D) Recovery of BM mononuclear cells after ficoll density centrifugation at different time points during follow-up in the DCOG-ALL11 protocol. Recovery of mononuclear cells is relatively low at days 33 and 78 (median, 5-8 × 106). Recovery at day 78 and at later time points is much higher (median, × 106).
Jacques J. M. van Dongen et al. Blood 2015;125:
©2015 by American Society of Hematology

20. Technical Requirements: Preparation of samples Evaluation of the Bulk-Lysis Protocol
Standard protocol (FL) bulk-lysis protocol (BL)
(B) Absolute number of leukocytes acquired. Using BL, on average 12-fold more leukocytes could be acquired (P < .0001)
(A) Using the bulk-lysis method, significantly less debris and significantly more leukocytes were measured.
Evaluation of the EuroFlow bulk-lysis protocol. For reasons of comparison, each of the BM samples (day 15: n = 15; day 33: n = 15; day 78: n = 12) was processed according to the standard EuroFlow protocol (FL) and in parallel according to the EuroFlow bulk-lysis protocol (BL). (A) Number of leukocytes, debris, and doublets, calculated as percentage of acquired events. Using the bulk-lysis method, significantly less debris (P = .032 by paired Student t test) and significantly more leukocytes (P = .03 by paired Student t test) were measured. There were no significant differences between the 2 methods for the percentage of doublets. (B) Absolute number of leukocytes acquired. Using BL, on average 12-fold more leukocytes could be acquired (P < .0001). Please note that we included relatively many day 15 samples in order to be able to evaluate the impact of the 2 methods on the MRD levels as well. However, these day 15 samples generally have a very low white blood cell count; consequently, the number of leukocytes acquired after BL is still relatively low in a subset of samples. (C) Distribution of leukocyte subpopulations, defined as percentage of leukocytes. By paired Student t test (2-sided), small but statistically significant differences were observed for T/NK cells (mean: 24% vs 26%, P = .0047), granulocytes (mean: 33% vs 38%, P < .001), and monocytes (mean: 3.2% vs 4.5%, P < .001), whereas no significant differences were observed for the remaining populations. Of note, in 2 samples, MRD was only detected using the bulk-lysis method (0.013% and 0.018%) but not using the whole BM method. In the 11 samples MRD positive by both methods, MRD levels were not significantly different from each other (paired Student t test: P = .30), with mean values of 6.3% and 6.7% by whole BM and bulk-lysed BM method, respectively. Correlation analysis showed a Spearman r of (95% confidence interval: ; P < .0001). PC, plasma cells.
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21. Technical Requirements:Preparation of samples Mononuclear vs
Technical Requirements:Preparation of samples Mononuclear vs. Nucleated Cells

22. Two separate approaches have been used for assessing MFC MRD:
Technical Requirements: Antibody panel selection Approaches for MFC-MRD assessment (LAIP vs. DfN)
Two separate approaches have been used for assessing MFC MRD:
The LAIP approach, which defines Leukemia Associated Immunophenotypes at diagnosis and tracks these in subsequent samples.
The Different-from-Normal (DfN) approach, that is based on the identification of aberrant differentiation/maturation profiles at follow up.
The ELN MRD working party suggests the term: ‘LAIP-based DfN approach’ for this combined strategy.
LAIP Based on
- Cross-lineage expression
- Lack of expression of an antigen
- Overexpression
- Asynchronous expression of antigens
The difference between these two approaches is likely to disappear if an adapted, sufficiently large panel of antibodies, preferably ≥ 8 colors is utilized.

23. LAIPs and DfN-LAIPs can be further categorized as: Diagnostic
Antibody panel selection Approaches for MFC-MRD assessment (LAIP vs. DfN)
The ELN MRD working party suggests the term: ‘LAIP-based DfN approach’ for this combined strategy.
To be more specific, aberrancies may be referred to as LAIPs or DfN-LAIP, whichever is the more appropriate term.
LAIPs and DfN-LAIPs can be further categorized as:
Diagnostic
Follow-up, based on diagnosis information
Follow-up, no diagnostic information
Changed, i.e. new aberrancy compared to diagnosis LAIPs or previous follow-up LAIPs

24. Composition of BCP-ALL MRD panel
Tube PacB PacO FITC PE PerCPCy PECy7 APC APCA750
CD CD45 CD81 CD66c + CD123 CD34 CD19 CD CD38
CD CD45 CD81 CD304 + CD73 CD34 CD19 CD CD38
Good/Fair separation in 99% of cases
BCP-ALL cells vs. the nearest normal/reactive BCP subset
CD73 & CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients
Their expression profile remains relatively stable early after starting therapy,
Overexpression of CD66c/CD123 or CD73/CD304 was observed in 45% and 46% of cases, respectively;
Lower limit of detection, LOD A minimum of 10 clustered events to consider a sample as MRD positive
Lower limit of quantitation, LLOQ a minimum of 40 clustered events for accurate quantitation of the MRD level

25. MRD analysis in Iran
4-colour MFC for MRD assay has been available since at least 4 years ago in Iran but the sensitivity was only 10-3 to 10-4
We were not completely confident on low positive MRD level (eg., %)
Interference by Hematogones is a main problem
8-10 color IVD flow cytometer machine is available since 3 months ago in our center
Euroflow protocol for MRD assay is established by kit and is going to be set it up by home brew cocktail prep according to Euroflow
Infinicyt Flow Cytometry Software is available at now in IRAN
Euroflow MRD assay are running at now but with challenges which are related to sanction and imbalance in money exchange rate

26. Total cells acquired: 4,554,381; CD45 positive cells acquired: 4,491,422
MRD Tube 1: %
8-color tube 1: CD45, CD10, CD19, CD20, CD34, CD38, CD81 and CD66c/CD123 evaluated.
MRD Tube 2: %
8-color tube 2: CD45, CD10, CD19, CD20, CD34, CD38, CD81 and CD304/CD73 evaluated
Cytomorphology Description:
BM smear: Blast: 1%
MRD cells
Red color

27. Conclusion
8-colour MFC for MRD assay with specific software are now available & the sensitivity is up to 10-5 (0.001%)
mAbs selection & order at Dx. & request for MRD assay should be harmonized
It is very helpful for analysis of MRD to know the immunophenotype of leukemic cells at diagnosis & the time point which specimen collected.
Coverage by insurance organizations should be defined
Specific consideration of governmental organization to MRD assay and services support needed.

28. Thank you