28-Sep-12

1

Flow Cytometric Findings in Acute Myeloid Leukemia

Prashant SharmaHematology Department,

PGIMER, Chandigarh

FCM in AML

• Role: Not just “optional” for diagnosis

• Definitive roles in:– AML-M0

M7– M7

– Mixed phenotype acute leukemias

– Rapid identification of M3

• Emerging role in MRD detection (still primarily molecular-based)

Aims

• To familiarize the participants with the FCM appearances of the common AML subtypes.

• To introduce them to the core• To introduce them to the core interpretative strategies in FCM analysis of AML.

• To highlight some commonly encountered issues in the FCM analysis of AML.

Cases

1. AML-M0

2. AML-M2

3. AML-M3

4. AML-M4

5. AML-M7

6. Mixed phenotype acute leukemia (Bi-lineal, T-myeloid)

Case 1: Background

• 36 yr/M with fever, pallor

• O/E: Splenomegaly 2 cm BCM

• Hb 9.7 g/dl; TLC 43,200/µl and platelets 2 45 000/µl2,45,000/µl

• Peripheral smear shows ~96% blasts, negative for myeloperoxidase

• Specimen: EDTA blood

Case 1: CD45-SSC

• A prominent blast cluster and with marked depletion of other cell populations

• Blasts are CD45dim / SSClow

28-Sep-12

2

CD45-SSC

P1: 92% of all events

CD45-SSC

P1: 92% of all events P2: 7% of all events

CD45-SSC

P1: 92% of all events P2: 7% of all eventsP3: 1% of all events

CD34+ CD117+ (heterogeneous)

Neg for CD64 & CD14

Co-express CD33 & CD34

Neg for CD13 & CD15

HLA-DR+

CD13neg CD11b- CD16-

28-Sep-12

3

Neg for MPO & CD4/8/TdT CD7+ CD2- CD5-

CD38+ CD58+

CD10neg CD20neg CD19neg

Summary of markers

• 92% blasts expressing: – Myeloid antigens: CD117 (~77%) and CD33 (~96%)– Markers of immaturity: CD34 (~96%), HLA-DR

(~98%), CD58 (~100%) and CD38 (~98%) – Aberrantly express CD7 (~98%)

• Blasts are negative for myeloid and monocytic antigens (cMPO, CD14, CD64, CD11b, CD13, CD15, CD16), B-cell antigens (cCD79a, CD19, CD20), T-cell antigens (cCD3, CD5, CD4, CD8, CD2) and CD10, TdT.

• Impression: Acute Myeloid Leukemia with minimal differentiation and aberrant CD7 expression.

Learning points from case 1

• AML with minimal differentiation by FCM correlates with FAB M0/M1. Distinction between the 2 requires MPO+/Auer rods.

• AML M0 requires flow cytometry (or IHC• AML-M0 requires flow cytometry (or IHC or EM) for diagnosis.

• CD7 in AML: adverse prognostic marker, correlates with loss of CEBPA, early relapse or residual disease.

Röhrs S et al. CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequence of epigenetic regulation. J Hematol Oncol. 2010;3:15.

• Morphological correlation: At the very least, a smear prepared from the flow sample should always be examined.

• Cytochemical (slide) MPO correlates

Learning points from case 1

• Cytochemical (slide) MPO correlates extremely well with the FCM MPO.

(The latter is purported to be more sensitive, but in borderline +ve cases it may be difficult to interpret.)

Nakase K et al. Detection of MPO by flow cytometry in acute leukemia. Cytometry. 1998 ;34:198-202.

28-Sep-12

4

Case 2: Background

• 34-yr/M with bone pain, petechiae x 1m

• Hb 9.8 g/dl; TLC 43,000/µl and platelets1,11,000/µl.

• DLC: Blasts 35%, neutrophils 31%, lymphocytesDLC: Blasts 35%, neutrophils 31%, lymphocytes24% & monocytes 10%.

• Auer rods +

• Blasts are MPO +ve.

CD45-SSC

Blast cluster shows continuity with the maturing granulocytic population.

No significant monocytic region population.

Residual populations

Blasts (P1) 30%, lymphocytes (P2) 12%, granulocytic cells (P3) 55%

CD34+ CD33heterogeneous

CD14- CD64-

CD13+, ~12% are CD15+

CD117- CD64-

HLA-DR+

CD11b- CD16-

28-Sep-12

5

cMPO+

cCD79a- cCD3- TdT-

Dim CD19 in ~20% blasts

Negative for CD2, CD5,

CD8, CD10, CD20

Summary of markers

• ~28% blasts expressing CD34 (~92%), HLA-DR, cMPO (~96%), heterogeneous CD33 (~61%) and moderate CD13 (~73%) with aberrant CD19 (20%).

• Negative for cCD79a, cCD3, TdT, CD10, CD20, CD5, CD7, CD2, CD14, CD64, CD 11b and CD16

• Impression: AML with maturation (FAB AML M2) with low level expression of CD19.

• Simply tracking the blast population (labelled in the appropriate plot) for every marker yields the diagnosis in uncomplicated cases

Learning points from case 2

uncomplicated cases.

• The aberrant coexpression of CD19 (or CD22, CD56) is highly associated with t(8;21), a favorable prognostic indicator.

Ferrara F et al. Immunophenotypic analysis enables the correct prediction of t(8;21) in AML. Br J Haematol. 1998;102:444-8.

Case 3• 19-yr, male in Emergency with altered

sensorium

• Bleeding gums, fever x 3 weeks

• O/E: Ecchymosis +, No organomegaly.O/E: Ecchymosis , No organomegaly.

• Hb 7.1 g%, TLC 2,000/μl, Plt 76,000/ μl

• PT 33” / control 12”

• Clinical ∆: Pancytopenia, coagulopathy: – ? Acute promyelocytic leukemia

– ? Meningococcal meningitis

Sample for FCM

Blasts 14%, abnormal pro-myelocytes 69%, Auer rods +

28-Sep-12

6

CD45-SSC CD45-SSC: Gated

Markers of immaturity

Heterogeneous CD13

Negative for CD34 & HLA-DR, heterogeneous (negative to dim +ve) CD13 & CD64)

Other myeloid markers

Compact CD33

Negative for CD14, heterogeneous (negative to dim +ve) CD117 & compact moderate CD33)

CD117: 24%

Bright cMPO, low level CD15 (~15%), CD4+

Negative for TdT, CD16, CD5.

Moderate CD58+, neg for B- and T-

markers CD19, CD20, CD19, CD20,

CD10, cyCD79a, CD5, CD7, CD8 and cyCD3

28-Sep-12

7

Summary of markers

• An abnormal blast region cell cluster extending to granulocytic region on SSC/CD45 plot (~92%)

• The blasts express cMPO (~59%), CD33 (~94%), CD15 (~15%), CD13 (~44%), CD38 (~65%) and CD58 (~95%). They lack ( ) ( ) yexpression of CD34 and HLA-DR. and CD64.aberrant expression of CD4 (~32%).

• Negative for CD19, CD10, CD20, CD5, CD7, CD8, CD2, CD14, CD11b, TdT and CD16

• Impression: Consistent with acute promyelocytic leukemia with aberrant CD4 expression.

• “Just in case…” samples: In suspected or even remotely possible acute leukemia, keep BM for FCM at the time of aspiration

• M3 on FCM: A virtually single major

Learning points from case 3

• M3 on FCM: A virtually single major population with weak to moderate CD45 and wide ranging SSC signals (moderate to high) is strongly suggestive of AML-M3.

• Other corroborating features of AML-M3:– Increased baseline auto-fluorescence even on

unstained / isotype controls

– Absence of CD34 & HLA-DR reactivity

Lessons from Case 3

y

– Heterogeneous CD13 & compact CD33 clusters

• M3 is sometimes equivocal on morphology alone while RT-PCR takes time. FCM can corroborate a BMA / PS report.

Mimics of M3

• G-CSF therapy: A pitfall in both morphology and in FCM

• Myeloproliferative neoplasms (MPN) and MDS MPNs: The clinical backgroundMDS-MPNs: The clinical background helps.

• On FCM: CD11b & CD16 expression favor non-APML disorders.

Case 4: Background

• 54/M, fatigue and bleeding gums

• Hepatosplenomegaly, skin rash

• Hb 8.8 g/dl; platelets 39,000/ml, TLC 4 300/ l4,300/ml

• BM: 23% blasts, 30% promonocytes (total 53%), MPO negative, NSE intense positive.

Case 4: Morphology

28-Sep-12

8

CD45-SSC

A large cluster is seen shifted upward and to the right (almost merging with the sizeable monocytic region).

Gating monocytic AMLs

The neoplastic cluster displays bimodal CD45 with merging of cellular events from blast region and monocytic regions.

Ensure that the tumor is not actually composed of 2 separate populations.

Red: Blasts; Blue: Monos

Blasts: CD34+, CD117+, CD64+ (25%) CD14-, Monos: CD34-, CD117-, CD64+ CD14+

Red: Blasts; Blue: Monos

Blasts: CD33+, CD15+, CD64+ (25%) CD14-,

Monos: CD33bright, CD15-, CD64+ CD14+

(admixed granulocytic population is –ve for CD14, CD64, dimmer CD33)

Red: Blasts; Blue: Monos

Blasts: CD13+, CD11b-, HLA-DR+/-, CD4-, Monos: CD13+, CD11b+, CD16+, CD4+

Red: Blasts; Blue: Monos

Blasts: TdT+, CD79a-, CD3-, CD10-, CD20-, CD2-, CD8-

Monos: TdT-, CD79a-, CD3-, CD10-, CD20-, CD2-, CD8-

28-Sep-12

9

Summary of findings

• A prominent CD45dim cluster (blast region, ~31%) merging with the mono region (12%, CD45 moderately bright, higher SSC).

• Blasts: CD34 (89%), CD13 (94%), CD33 (84%), CD117 (50%), CD38 (85%) and TdT (60%).( ) ( ) ( )

• Maturing monocytes: CD16, CD64, CD11b, CD4, CD13, CD33.

• Blasts neg: cMPO, cCD79a, cCD3, CD19, CD10, CD20, CD5, CD8, CD2, CD14, CD64, CD11b, CD16.

• Impression: AML with monocytic differentiation {c/w BMA AML-M5b}.

• Distinction between M4 / M5a / M5b by FCM alone is difficult. Require correlation with morphology.

• Look for the monocytic patterns

Learning points from case 4

• Look for the monocytic patterns.

• Identify and track all discrete cell populations separately to provide information about differentiation patterns of blasts.

Case 5

• 3 yr, F

• Down’s syndrome, now with fever and pallor x2 weeks

N li l LN• No liver, spleen, LN

• PS: 14% abnormal cells

• BMA: 40% abnormal cells (?blasts) negative for MPO.

FSC-SSC, CD45-SSC

Multiple populations: lymphocytes, blasts, blasts merging with mono, nRBC/debris

Gating on CD45-SSC

P1 (red): Hematogones, P3 (blue): negative for myeloid, B- & T- markers. P2 (green): showed myeloid antigens. Was this the neoplastic clone?

28-Sep-12

10

CD34 back-gating

On back gating from CD34, the violet cells (~30%) localized to P2, thus confirming their immature nature.

Tracking P2 (CD34+, blast region)

Expressed CD34, CD33, CD14, CD11b, CD15, HLA-DR

CD7+, cMPOneg Add-on testing: CD41, CD61

Summary of markers

• Cell cluster in the blast region extending to monocytic region (~40%)

• Express CD41, dim CD61, CD33, CD13, CD11b, CD15, HLA-DR, CD34, aberrant CD7(~93%).( )

• Negative for cMPO, cCD3, CD19, CD117, CD10, CD2, CD5, TdT, CD14 & CD 64.

• Impression: AML-M7 in Down syndrome• Transient abnormal myelopoiesis unlikely:

– Patient’s age – CD13, CD11b +ve blasts .

In Down syndrome, AML blasts are express myeloid associated antigens CD13 and CD 11b as compared to blasts in most cases of TMD; Am J Clin Pathol 2001;116:204-210.

• CD34 back-gate in cases with multiple populations (also CD117, HLA-DR back gate).

• A primary acute leukemia panel with add-

Learning points from case 5

p y pon testing as indicate clinically or by the initial results.

• TAM vs. AML-M7 in DS• Hematogones may lie in the blast region

esp. in children.

28-Sep-12

11

Case 6: Background

• 4-year-old boy

• Pallor, hepatosplenomegaly, pleural effusion, blasts in PS. Clinical ∆: ALL.

Hb 8 1 /dl l t l t 106 000/ l d• Hb: 8.1 g/dl; platelets: 106,000/μl and TLC: 21,400/μl (Neutrophils 15%, lymphocytes 49%, monocytes 13%, myelocytes 02%, metamyelocytes 02% and blasts 21%).

BM

• BMA- Diluted

• BM touch showed 70% blasts of variable morphology (small lymphoid with cleavage and larger undifferentiated to myeloid)and larger undifferentiated to myeloid)

• The larger blasts showed ~10% MPO positivity.

• FCM on peripheral blood.

Morphology CD45-SSC

Multiple populations in the blast window.

CD45-SSC

P1: CD45 dim, higher SSC, P2: CD45 brighter, lower SSC

Establishing the nature of the 2 populations

P1: CD34+, CD33+, CD64-, CD14-, P2: CD34+, CD33-, CD14-, CD64-

28-Sep-12

12

Myeloid & mono markers

P1: CD13+, CD15+, CD11b-, CD16-, P2: CD13-, CD15-, CD11b-, CD16- P1: HLA-DR+, cCD3-, CD11b-, CD11b-, P2: HLA-DR-, CD11b-, cCD3+, TdT-

P1: CD7-, CD4-, cMPO+, CD79a-, P2: CD7+, CD4+, cMPO-, aCD79a-

T-cell markers

P1: CD8-, CD4-/+, CD5-, CD2-, P2: CD8-, CD4+, CD2+, CD5+

Summary of markers

• P1: ~18% of all acquired events. Dimmer CD45 with higher side scatter. Express CD117, CD33, CD13, HLA-DR, CD38 and CD34. MPO is expressed immunophenotypically by ~7% blasts.

• P2: ~14% of all cells. Moderate CD45, lower SSC. Co-CD3 (81%) CD2 CD4 CD5 CD7 CD10express cCD3 (81%), sCD2, CD4, CD5, CD7, CD10

and CD34 along with CD1a. CD8, CD10 and TdT are negative in this population.

• Both the types of blasts are negative for CD19, cyCD79a, CD20, CD11b, CD16, CD14 and CD64.

• Diagnosis: Mixed Phenotype Acute Leukemia (bi-lineal, T/Myeloid).

• FCM is mandatory for the diagnosis of mixed phenotype acute leukemia.

• Pattern-recognition and tracing the immuno phenotype of each and every

Learning points from case 6

immuno-phenotype of each and every population in the blast region and nearby can yield significant information.

28-Sep-12

13

Take home message: Basics

• Use all the information available: clinical, blood counts, morphology, cytochemistry.

• Apply a consistent, logical approach to analysisanalysis.

• Know the biological basics.

• Know the artifacts: sample-related, processing-related, machine-related.

• Get familiar with the software.

Take home message: Analysis

• Apply a variety of gating strategies to the same population of cells to obtain the maximum information.

• Use consistent gating strategies to avoid• Use consistent gating strategies to avoid missing subtle alterations / populations.

• Use cell numbers / percentages as adjunctive information, not as the sole means to evaluate a population.

Take home message: Troubleshooting

• If faced with a pattern one does not understand:– Re-check the technique, the reagents and the

machinemachine,

– Call the clinician for more information,

– Read, search the internet, email the *.fcs files to someone else to take a look.

sharma.prashant@pgimer.edu.in