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1 Clinical diagnostic cytometry Gerrit J Schuurhuis Dept of Hematology VU University Medical Center Amsterdam, Netherlands Use of immunophenotyping at diagnosis to trace residual disease after therapy 1. MRD 2. Leukemia stem cells (LSC) Example of 8 color diagnosis LAIP panel Types of LAIPs LAP Examples Cross-lineage : expression of lymphoid-associated markers CD2, CD4, CD5, CD7, on myeloblasts CD10, CD19, CD56 Asynchronous : expression of antigens not normally seen in a / a normal myeloid development or aberrant co-expression of antigens seen in a / a early and late stages of maturation b, HLA DR /d, Antigen over-expression/under-expression: increased or diminished intensity of /d/b, /d/b, to a degree not seen in normal developing myeloblasts CD38 /d/b tube FITC PE PerCP- CY5.5 PC7 APC APC-H7 BV421 V500c 1 CD7 CD56 CD45 2 CD2 CD22 CD19 HLADR CD45 3 CD36 CD14 HLADR CD HLADR CD45 Aberrant light scatter: normal intensity of in combination with abnormally low or high forward or side scatter a /SSb a positive ;, negative ; b, bright ; d, dim ; SS, side scatter >100 different LAIPs Terwijn et al J Clin Oncol 2013 Jaso et al., BMT 2014, 49: 1129 How to form a leukemia associated immuno phenotype (LAIP) at AML diagnosis How to form a LAIP at AML diagnosis Combination of markers o Blast (CD45/low) o Primitive markers (,, 3) Primitive parts of a leukemia may be most therapy resistant and best suitable/most relevant for MRD o Myeloid markers (, ) o Aberrant markers - Cross-lineage (e.g. CD2, CD7, CD19) - Asynchronous (e.g., CD56, CD65) - Over-expression (e.g., ) - Under-expression (e.g. HLADR-) oaberrant combinations of primitive markers oaberrant combinations of myeloid markers oaberrant expression of other antigens o cross-lineage o asynchronous o over-expression o under-expression 1

2 2 How to form a LAIP at AML diagnosis Blasts in NBM oaberrant combinations of primitive markers oaberrant combinations of myeloid markers oaberrant expression of other antigens o cross-lineage o Asynchronous o over-expression o under-expression oblast (CD45dim/low) Gating of in NBM Primitive markers in normal bone marrow Blasts 1.95% 0.83% 0.83% 0.98% % 9 10 MYELOBLAST PRO NEUTROPHIL MYELOBLAST PRO NEUTROPHIL 3?

3 3 and 3 in NBM MYELOBLAST PRO NEUTROPHIL 3 NBM0.61% Facts in NBM: 1. About 50% of + cells are Almost 100% of 3+ cells are + 3? NBM: all 3+ are + ( ) Which population is usable as LAIP in AML 0.01% a. +3+ b. +3- c. -3+ d. -3- /3 defined sub-populations in NBM 3+ Facts in NBM: 1. About 50% of + cells are 3+ So: about 50% of + is 3-2. Almost 100% of 3+ cells are + So: there are almost no 3+ - cells Potential LAIP in a negative AML (10-20% of AML cases) 3 NBM0.61% NBM: all 3+ are + ( ) 0.01% Facts in NBM: 1. About 50% of + cells are 3+ So: about 50% of + is 3-2. Almost 100% of 3+ cells are + So: there are no 3+ - cells Which population is usuable as LAIP in AML 3+- LAIP in negative AML 3 NBM0.61% AML 3 42% Can you define / aberrancies: a. +/+ b. +/- NBM: all 3+ are + ( ) AML: large part of 3+ are - ( ) ) % c. -/+ d. -/- 0.01% is neg a. +3+ b. +3- c. -3+ d is aberrant APC

4 4 Can you define / aberrancies: MYELOBLAST PRO NEUTROPHIL Can you define / aberrancies: a. +/+ b. +/- c. -/+ depending on maturation stage d. -/- depending on maturation stage 3? Can you define 3/ aberrancies: what about 3/ combination MYELOBLAST PRO NEUTROPHIL 3? How to form a LAIP at AML diagnosis oaberrant combinations of primitive markers MYELOBLAST PRO NEUTROPHIL oaberrant combinations of myeloid markers oaberrant expression of other antigens o cross-lineage o Asynchronous o over-expression o under-expression

5 5 Myeloid markers in normal bone marrow 0.83% MYELOBLAST PRO NEUTROPHIL 26 Myeloid markers in AML Normal BM CD45dim/+ 0.00% 0.02% MYELOBLAST PRO NEUTROPHIL AML follow-up:wbc Pre-B-cells No MRD 27 Quality of / LAIPs. LAIPs all CD45dim Quality of / LAIPs. LAIPs all CD45dim What is the best LAIP: a. +/+/- b. +/-/+ c. -/+/+/- d. -/+/-/+ e. -/-/+/- f. -/-/-+ What is the best LAIP: a. +/+/- 1. About equally aberrant b. +/-/+ 2. c. -/+/+/- 4. d. -/+/-/+ 3. e. -/-/+/- f. -/-/-+ Depending on the stage (>myelocytes) Keep in mind: Primitive parts of a leukemia may be most therapy resistant and best suitable/most relevant for MRD Overall: a,b > c,d > e,f seen increasing maturation from a to f

6 6 Myeloid markers in AML How to form a LAIP at AML diagnosis Normal BM 0.00% CD45dim/+ AML +- (84%) oaberrant combinations of primitive markers 0.02% oaberrant combinations of myeloid markers AML follow-up:wbc No MRD Pre-B-cells oaberrant expression of other antigens o cross-lineage (CD2, CD7, CD19, CD22, CD56) o Asynchronous o over-expression o under-expression +/- and -/+ are aberrant 31 Lymphoid markers in normal bone marrow Aberrant immunophenotypes in AML 0.83% 0.01% 0.02% Cross-lineage 0.03% 0.05% 0.01% How to form a LAIP at AML diagnosis oaberrant combinations of primitive markers MYELOBLAST PRO NEUTROPHIL oaberrant combinations of myeloid markers oaberrant expression of other antigens o cross-lineage (CD2, CD7, CD19, CD22, CD56) o Asynchronous (, CD14, ) o over-expression o under-expression

7 7 MYELOBLAST PRO NEUTROPHIL Maturation markers in normal bone marrow 0.83% +LAIPS: 0.01% 0.03% 0.02% 38 Aberrant immunophenotypes in AML Asynchronous MYELOBLAST PRO NEUTROPHIL Maturation markers in normal bone marrow 0.83% +LAIPS: 0.01% Aberrant immunophenotype: asynchronous expression Normal BM (0,02%) 0.03% 0.02% AML (49%) 41

8 8 How to form a LAIP at AML diagnosis 3 NBM0.61% AML 3 42% + oaberrant combinations of primitive markers oaberrant combinations of myeloid markers NBM: all 3+ are + ( ) AML: large part of 3+ are - ( ) ) 0.01% Special case of asynchronous expression: is aberrant % oaberrant expression of other antigens o cross-lineage (CD2, CD7, CD19, CD22, CD56) o Asynchronous (, CD14, ) o over-expression (,, ) o under-expression APC Aberrant immunophenotypes in AML How to form a LAIP at AML diagnosis Antigen over-expression oaberrant combinations of primitive markers oaberrant combinations of myeloid markers oaberrant expression of other antigens o cross-lineage (CD2, CD7, CD19, CD22, CD56) o Asynchronous (, CD14, ) o over-expression (,, ) o under-expression (--, -) 45 Aberrant immunophenotypes in AML Aberrant immunophenotypes in a positive AML white blood cells blasts lymfo s primitive cells LAIP cells Lack of Antigen expression Cross-lineage Asynchronous Antigen over-expression Lack of Antigen expression AML NBM BV421 : BV421 : BV421 : BV421 : BV421

9 9 Expression levels of maturing monocytes 49 Monocytic markers in normal bone marrow Monocytes 3.41% Expression levels of maturing monocytes 0.01% 51 LAIP using the maturation of monocytes Frequency of LAIP types in HOVON 42A LAP type N o at diagnosis (N o of patients) N o after 1st cycle N o after 2nd cycle N o after consolidation Asynchronous 157 (131) 11 (7%) 13 (7%) 10 (8%) Cross-lineage 469 (308) 74 (45%) 74 (41%) 58 (48%) Lack of 389 (283) 51 (31%) 72 (40%) 40 (33%) Antigen overexpression 26 (23) 7 (4%) 7 (4%) 3 (2%) LAPs on mature blasts 142 (80) 21 (13%) 17 (9%) 10 (8%) Total LAPs Terwijn et al., J Clin Oncol 2013

10 10 MRD panel 8 colors How to process samples At diagnosis tube FITC PE PerCP- CY5.5 PC7 APC APC-H7 BV421 V500c 1 CD7 CD56 CD45 2 CD2 CD22 CD19 HLADR CD45 3 CD36 CD14 HLADR CD HLADR CD45 o Stain the lysed cells with the monoclonal antibodies o Achieve a minimum of 100,000 CD45+ events At follow-up 5 common markers A second run only needed in exceptional cases o o Stain the lysed cells with the monoclonal antibodies Achieve a minimum of 1 x 10e6 CD45+ events (The minimal number for reliable detection of MRD) Gating strategy WBC Gating strategy blasts 58 Gating strategy blasts follow-up Difficult to find the blasts Gating strategy lymphocytes 59 60

11 11 Case MRD (351) Case MRD (351) Primitive markers: +, +, 3+ Case MRD (351) Case MRD (351) Myeloid markers: +, but - (aberrant) Aberrant markers: CD22(low) Small populations with CD19, CD7 Case MRD (351) LAIPs confirmed with second run 85% Be LAP aware +- of polymorphism 84% granulocytes +CD22+ 63% Aberrant markers: small population with CD36

12 12 LAPs confirmed with second run Follow-up gating (MRD 351) +CD7+ 9,6% +CD19+ 12% Follow-up gating (MRD 351): LAIP: +/-/+ Follow-up % Diagnosis Follow-up gating (MRD 351): LAIPs: +/+/CD19+ +CD7+/CD7+ CD19 Gate set based on lymphocytes Higher expression of CD7 Gate set at diagnosis Follow-up % % of WBC: CD45dim 5.9 : 1.29 CD22: 0.14 CD19: 0.08 CD7: 0.10 CD22 Follow-up gating (MRD 351) Follow-up % Follow-up %

13 13 LSC panel Tube FITC PE PerCP-CY5.5 PC7 APC APC-H7 HV450 KO LSC 1 PBS CD38 PBS CD45 CD44 Clec12a CD56 CD38 CD45 2 CD7 TIM-3 CD38 CD19 CD CD2 3 CD38 CD19 CD45 CD36 CD123 CD38 CD14 CD45 CD96 CD38 CD14 CD45 CD22 CD38 CD45 74 P r e s e n t a t i o n C o n f i d e n t i a l AML- Leukemic Stem Cell MRD Contents Slide(s) Content of LSC tube 3 How to identify LSCs in a diagnosis/f-up AML. 4 Analyzing samples (WBC, lymphocytes and blast cells) 5 Gating + blasts.. 6 Gating CD38 low progenitors.. 7 Gating CD38 verylow stem cells 8 Determining LSC/HSC 9-21 How to gate positivity. 10,11 CD44 How to gate CD44 positivity CD45RA How to gate CD45RA positivity. 15,16 CD123 How to gate CD123 positivity. 17,18 PE-Combi How to gate Combi positivity Back gating Department of Hematology VU University Medical Center, Cancer Center Amsterdam Amsterdam, The Netherlands Gating summary 25 How to report results 26 Examples 27,28 Click to be directed to slide of interest Content of LSC tube FITC PE PerCP-CY5.5 PE-Cy7 APC APC-H7 BV421 V500c CD45RA CLL-1 CD123 CD38 CD44 CD45 TIM-3 CD7 How to identify LSCs in a diagnosis/f-up AML Combination of markers: Blasts (CD45 dim / low ) Primitive population ( + ) Stem cell compartment (CD38 - ) Aberrant marker: - CD45RA - CLL-1 / TIM-3 / CD7 / / CD22 and/or CD56 (=Combi) - - CD123 - CD44 CD22 CD56 LSC, leukemic stem cell

14 14 Analyzing samples Gating + blasts 1. Gate white blood cells (WBC) - step 1: exclude low cells - step 2: exclude CD45 low cells Specific expression level for appropriate cut-off differs per sample. A histogram may help. Never gate through a population with a homogeneous distribution of marker of interest. Important: expression may be higher or lower than normal + cells 2. Gate lymphocytes - step 3: gate CD45 high /SCC low population - step 4: gate low / low population CD45 HV500C 3. Gate blast cells - step 5: gate CD45 dim / low population - step 6: extra gate around low cells BV421 BV421 BV421 As a guideline, not per se for gating purposes WBC lymphocytes blast cells Blast cells #2813 Gating CD38 low progenitors Both cut-off of CD38 low (progenitors: this slide) and CD38 verylow (stem cells: next slide) may differ between samples. If possible use upper limit of spherotech beads, if not possible use red fraction or choose as cut-off point Gating CD38 verylow stem cells Preferably use median of red fraction (make sure this fraction has no CD38 high suppopulation). If not possible, gate below the bulk of spherotech beads or choose as cut-off point (2) Cut-off for CD38 low above red fraction, above 99% of beads, often close to Cut-off for CD38 low above red fraction, above 99% of beads, often close to BV421 BV421 Horizon V450 Cut-off for CD38 verylow mean of the red fraction, below the beads, often close to BV421 BV421 BV421 Cut-off for CD38 verylow median of the red fraction, below the beads, often close to Blast cells + blast cells red fraction Spherotech beads +CD38low #2622 Blast cells + blast cells red fraction Spherotech beads +CD38low +CD38verylow #2622 Determining LSC/HSC After defining CD38 low and CD38 verylow population, the other makers in the LSC tube (, CD44, CD123, CD45RA and Combi) are used to define normal hematopoietic stem cells/progenitor cells versus leukemic stem cells/progenitors cells. is especially useful at time of diagnosis. is usually weakly expressed on HSCs at time of diagnosis but can be up-regulated on HSC after treatment. Diagnosis examples: Specific features on expression of these markers are described in the next slides. Positivity/negativity of marker expression is preferably defined by fluorescence of spherotech beads or of a negative lymphocyte (sub)population. Marker negative or Marker positive PC7 PC7 PC7 PC7 BV421 + blast cells red fraction +CD38low +CD38verylow lymphocytes +CD38+ blasts + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos

15 CD38 APC Red fraction 15 How to gate positivity When gating for marker positivity/negativity, both lymphocytes, red fraction and/or beads can be used as these are all - as shown here. overlay lymphocytes red fraction spherotech beads CD44 CD44 is highly expressed on HSCs (CD44 ++ ) at time of diagnosis and F-up AML. LSCs are CD CD44 -/dim are indicative for a-specific events. CD44 expression is only used as discriminative marker when there is unambiguous overexpression. examples: a-specific HSCs LSCs HSCs LSCs Not clear: HSCs or LSCs Not clear: HSCs or LSCs not used as LSC marker in this patient not used as LSC marker in this patient PC7 PC7 PC7 PC7 lymphocytes beads red fraction PC7 PC7 PC7 PC7 negative population +CD38+ blasts + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos lymphocytes +CD38+ blasts + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos How to gate CD44 positivity Gating CD44 overexpression is relatively difficult. However, looking at the pattern of the total CD38 low and CD38 verylow together with lymphocytes ( CD44 ++ ) might help. Relatively easy example: How to gate CD44 positivity Difficult example: histogram distinguishes two populations in + CD38 verylow. Red fraction and spherotech beads support this cut-off point. beads Expression of CD44 on LSCs is often higher than that of the lymphocytes, while HSCs have less expression. Overexpression or normal CD44 expression which could be found on HSCs? Not clearly distinctive + blast + CD38 low + CD38 verylow + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos + blast + CD38 low + CD38 verylow + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos CD45RA CD45RA is not expressed on HSCs and is a stable marker in both diagnosis and f-up. Mostly seen with clear separation between HSCs and LSCs. examples: How to gate CD45RA positivity When gating the CD45RA negative fraction, the lymphocytes or spherotech beads can be used as negative control. Lymphocytes are clearly divided in two populations in a histogram Reference populations Example 1 Example 2 beads Negative lymphocytes positive lymphocytes beads +CD38+ blasts + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos reference populations +CD38+ blasts + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos

16 16 CD123 CD123 is similar to ; weakly expressed on HSCs in AML at diagnosis. CD123 can be up-regulated on HSCs after treatment. examples: How to gate CD123 positivity When gating for marker positivity/negativity both lymphocytes, red fraction and beads can be used as these are all CD123 - as shown here. overlay lymphocytes red fraction spherotech beads lymphocytes beads red fraction lymphocytes +CD38+ blasts + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos negative population +CD38+ blasts + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos PE-Combi Positivity of at least one of the six markers in the combi-pe channel is needed to discriminate LSCs from HSCs. examples: How to gate PE-Combi positivity Since the PE channel contains multiple markers, there is no negative samplepopulation which could be used as reference. In this case are beads the best discriminator (red fraction in case of retrospective analyses). Example with lots of stem cells: beads red fraction lymphocytes +CD38+ blasts + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos + blast + CD38 low + CD38 verylow + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos How to gate PE-Combi positivity Apart from the reference fraction; the combi expression pattern of the + CD38 - blasts help to set the gates Example with few stem cells: Back-gating (1) For fine-tuning, we recommend back-gating after gating marker positive LSC/marker negative HSC. Back-gating serves two aims: 1) in cases of overlapping LSC and HSC population (e.g. slide 18), LSC and HSC may be purified by backward/forward gating in /, /CD38 and /CD45 bivarial plots. -A BV421 BV421 red fraction BEFORE -A BV421 BV421 The circled HSC population (green) is likely part of the neoplastic population + blast + CD38 low + CD38 verylow + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos + blast + CD38 low + CD38 verylow + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos

17 CD45 V500c CD45 V500c -A CD45 HV Back-gating (2) Differences in scatter properties between LSC and HSC are further elucidated in Terwijn M et al. Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia. PLoS One 2014; 9: e Back-gating (3) 2) To exclude a-specific events after cells are gated for abberant marker expression (a clean example see below, first gated on CD45RA expression) -A BV421 BV421 -A BV421 BV Are the CD38 low /CD38verylow 2. Is there a-specificity in lower 3. Are there scattered events in a 4. Does CD44 expression gives any events clustered in the lower? In most cases, stem CD45/ plot? advice in this case? CD44 low /CD44 neg /? Scatter may be seen cells and progenitors will be can be associated with a-specificity higher in the and is likely a-specific seen as one tail AFTER Gated on CD123 expression together with scatter properties -A BV421 BV421 + blast + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos + blast + CD38 low + CD38 verylow + /CD38 low /marker neg, + /CD38 low /marker pos, + /CD38 verylow /marker neg, + /CD38 verylow /marker pos Gating summary WBC, beads (when included), red/dead (CD45 negative ) population lymphocytes blast cells How to report results 1. Upload the FCS files onto in order to let us check 2. Analyze the data on your own and sent us the following information/pictures - (1) /, (2) /CD45, (3) / plots To show location of populations lymphocytes, blasts, + blasts, stem cells - (4) CD38/ plot with complete + fraction and reference population To show gating of + CD38 low and + CD38 verylow + blast cells use lymphocytes as reference - (5) CD38/stem cell marker plot with complete + fraction and reference population To show gating of marker positivity/negativity CD38 low CD38 verylow use beads as reference - CD45RA + vs. - - CD45RA + vs. - For example + versus : expression in LSC versus HSC - +/(+) vs. +/- - +/(+) vs. +/- - CD vs CD vs CD123 +/(+) vs. +/- - CD123 +/(+) vs. +/- - LSC-combi + vs. - - LSC-combi + vs. Back gating - Do this for all markers - (optional 6,7,8,9) stem cell marker of interest versus other stem cell marker To compare positivity/negativity with positivity/negativity of other markers and to fine-tune LSC versus HSC frequency. - define LSC frequency with the different markers and assess final LSC frequency - See following slides as template/example, both starting with CD45RA expression (1) (2) (3) Stem cells gated on CD45RA negativity/positivity - (1) / - (2) /CD45 - (3) / - (4) CD38/ - (5) CD38/stem cell marker - (5b) gating strategy (negative lymphocytes) - (optional 6,7,8,9) stem cell marker of interest versus other stem cell marker (1) (2) (3) Stem cells gated on CD45RA negativity/positivity - (1) / - (2) /CD45 - (3) / - (4) CD38/ - (5) CD38/stem cell marker - (5b) gating strategy (negative lymphocytes) - (optional 6,7,8,9) stem cell marker of interest versus other stem cell marker Population # of events Population # of events white blood cells white blood cells Lymphocytes Lymphocytes (4) (5) Percentages of WBC (5b) Blast cells (4) (5) (5b) Blast cells blasts blasts CD38 low blasts CD38 low blasts /CD38 low /marker neg /CD38 low /marker neg /CD38 low /marker pos /CD38 low /marker pos % 0.067% + CD38 verylow blasts /CD38 verylow /marker neg % 0.023% + CD38 verylow blasts 25 + /CD38 verylow /marker neg % 0.013% + /CD38 verylow /marker pos 64 Verification of leukemic Beads phenotype with other Not markers in sample (graph 6-9). Starting with CD45RA, the combi results (6) and CD44 (7) match with CD45RA, while CD123 (7) and (9) show overlap between LSC and HSC % % (6) (7) (8) + /CD38 verylow /marker pos 7 Beads Not in sample Verification of leukemic phenotype with other markers (graph 6-8). Starting with CD45RA, the combi results (6) match with CD45RA, while CD123 (7) and (8) show overlap between LSC and HSC (6) (7) (8) (9)

18 18 AML case with small aberrant population at diagnosis How to form a LAIP at AML diagnosis Diagnosis Follow-up 1 9% 70% oprimitive markers (,, 3) Sequential MRD monitoring in an allogeneically transplanted patient MRD in Peripheral Blood vs BM LAP: +/CD45 dim /CD7+/+ Diagnosis AML A B BM: 0.04% CD56 C 3-10 fold difference Time after : Tx -3 days 38 days 74 days 108 days 131 days 203 days MRD frequency : 0.70% 0.01% 0.48% 2.30% 0.04% 0.04% Kelder et al, unpublished PB: 0.01% CD56 Future (?): omitting BM-MRD. Benefit for physician, patient and technician Immune suppression Stop of immune suppressing Similar to : * Maurillo et al., Haematologica 2007, 92: 605 * M-C Béné, unpublished MRD in PB is more specific Diagnosis MRD detection in follow-up AML BM MRD in relapsing patient Aberrancy: CD45dim/+/CD7+/+ MRD in relapsing patient Blasts: 34% : 93% Diagnosis: 30% LAIP op blasts Follow-up MRD >0.1% Follow up 1: 0.29% Follow up 2: 0.13% Relapse: 49% LAIP on blasts Cut-off point to define MRD+ and MRD- in most studies is 0.1% (of WBC) W. Zeijlemaker and GJ. Schuurhuis (2013). Minimal Residual Disease and Leukemic Stem Cells in AML, ISBN:

19 19 MRD in remission patient Aberrancy: CD45dim/+/+/- MRD in patient in remission Blasts: 98% : 98% Diagnosis: 74% LAIP on blasts Follow up 1: 0.01% Follow up 2: 0.01% 7 months post-diagnosis: <0.01%