The Diagnosis of Myeloid Neoplasia by Flow Cytometry

Brent L. Wood MD PhDDept. of Laboratory Medicine

University of Washington, Seattle

Basic Principles

Abnormal population identification

• Normal– Antigens expressed in consistent and

reproducible patterns with maturation• Neoplastic

– Increased or decreased normal antigens– Asynchronous maturational expression– Aberrant antigen expression– Homogeneous expression

Normal B cell Maturation

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Normal B cell Maturation

Wood (2004) Methods Cell Biology 75:559-576

0.1% abnormal immature B cells

ALL MRD

06-01469

Myeloid Stem Cell Disorders

• Similar immunophenotypic abnormalities– Myelodysplasia– Myeloproliferative disorders– Acute myeloid leukemia

• Differences in– Blast percentage– Extent of maturation– Basophilia– Degree of abnormality

CD45/SS

Borowitz et al (1993) AJCP 100:534-40.Steltzer et al (1993) Ann NY Acad Sci 667:265-280

CD45/SS

Wood (2004) Methods Cell Biology 75:559-576

Wood (2007) Clinics in Lab Medicine 27:551-575

Early Progenitors

Blasts• Blasts are morphologically defined

– AML is > 20% blasts in blood or bone marrow

• Morphologic “blasts” include– Stem cells– Committed progenitors (CD34+)– Promyelocytes– Promonocytes– Immature B cell precursors– Immature T cell precursors

Blasts

Blasts• Blast estimates by flow cytometry may not agree with morphology

– Specimen processing

Red blood cell lysing reagents remove variable number of erythroid precursorsVariable degree of peripheral blood dilution

• Blast estimate may be high or low depending on relative contributions– Not a problem for peripheral blood

– Blasts defined differently by morphology and flow cytometry• Include promonocytes, promyelocytes, immature B cells as appropriate

Normal Blast Maturation

Wood (2004) Methods Cell Biology 75:559-576

Normal Granulocyte Maturation

Normal Monocyte Maturation

Acute Myeloid Leukemia

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Abnormal Antigen Intensity

Wood (2007) Clinics in Lab Medicine 27:551-575

Aberrant Lymphoid Antigens

Wood (2007) Clinics in Lab Medicine 27:551-575

Aberrant Maturation

Wood (2007) Clinics in Lab Medicine 27:551-575

Granulocytic Maturation

Normal Granulocytic Maturation

Wood and Borowitz (2006) Henry’s Laboratory Methods

Normal Granulocytic Maturation

Wood (2004) Methods Cell Biology 75:559-576

Wood (2007) Clinics in Lab Medicine 27:551-575

Monocytic Maturation

Normal Monocytic Maturation

Wood and Borowitz (2006) Henry’s Laboratory Methods

Normal Monocytic Maturation

Wood (2004) Methods Cell Biology 75:559-576

Monocytic Aberrancies

Wood (2007) Clinics in Lab Medicine 27:551-575

Acute Myelomonocytic Leukemia

Wood and Borowitz (2006) Henry’s Laboratory Medicine

Acute Monocytic Leukemia

Abnormal Myeloid Maturation

Normal

MDS

Myeloproliferative Disorders

Myeloproliferative disorders• CML

– Decreased CD16 expression on mature neutrophils– Decreased CD32 expression by mature neutrophils– Decreased L-selectin (CD62L) expression on the CD34-positive

cells– Aberrant expression of CD56 on the blasts and myeloid cells– Aberrant expression of lymphoid antigens such as CD2, CD5, and

CD7 on the blasts in CML blast crisis as well as CD7 expression on CD34-positive cells in chronic phase

Chronic Myeloid Leukemia

04-5708

CML in chronic phase - 2.8% blasts

Myeloproliferative disorders

• Non-CML– Increased expression of Bcl-XL in polycythemia vera– Decreased platelet GPIa/IIa, decreased GP1b and

GPIIb/IIIa, and elevated platelet P-selectin, thrombospondin, GPIV, and c-Mpl in essential thrombocytosis

– Aberrant coexpression of CD14 and CD66 on the myeloid cells in a subset of myeloproliferativedisorders

Myeloproliferative Disorders

• 76 cases referred for evaluation of MPD– All t(9;22) negative– Caveat: Diagnosis of all cases studied not known

• Evaluated by 4 color flow cytometry• Analyzed for abnormalities of blast, myeloid

and monocyte maturation• Compared with cytogenetics

Kussick and Wood (2002) Am J Clin Path 20:854-865

Essential Thrombocytosis

Kussick and Wood (2002) Am J Clin Path 20:854-865

Myeloproliferative Disorders

Population n Mean Age M:FAbnormal

Cytogenetics

Positive Flow(cases) 34 69.6 16:18 12 / 29 (41%)

Negative Flow(controls) 40 55.4 18:22 0 / 36

Kussick and Wood (2002) Am J Clin Path 20:854-865

Myeloproliferative disorders

# ofCytog.Abnls. n

Abnl.Blasts

Abnl.Myelos.

Abnl.Monos.

MeanAbnl.Myel.Ags.

MeanNon-Myel.Ags.

MeanTotalAbnl.Ags.

0 17 94% 100% 56% 3.8 0.8 4.61 8 100% 88% 50% 4.4 0.6 5.0

2 ormore 4 100% 75% 75% 5.8 0.5 6.3

TOTAL 29 97% 93% 55% 4.2 0.7 4.9

Kussick and Wood (2002) Am J Clin Path 20:854-865

MPD Conclusions• Many cases of non-CML myeloproliferative

disorders have immunophenotypicabnormalities

• Presence of immunophenotypicabnormalities correlates with cytogenetics– All cases with cytogenetic abnormalities had

flow cytometric abnormalities– Number of immunophenotypic abnormalities

paralleled increase in cytogenetic abnormalities• Abnormalities less frequent in PV and ET

Myelodysplasia

Early Studies• Many isolated abnormalities described

– loss of erythrocyte A, B, and H antigens– decreased expression of c-Mpl, GPIIb/IIIa, and GPIb on platelets in

refractory anemia– dyssynchronous expression of CD11b and CD16 in the developing

neutrophils– aberrant coexpression of CD14 and CD66 on myeloid cells– decreased CD10 on neutrophils– changes in a variety of leukocyte activation antigens, including FcR

I, FcRII, and FcR III– greater variability in the expression of CD38, CD71, CD13, and

CD33 in refractory anemia

– aberrant coexpression of CD56 on myeloid blasts

Elghetany MT. Haematologica. (1998) 83:1104-15.

Clinical utility

• Can distinguish MDS from normal and AA– Large panel of antibodies– Abnormalities in myeloid, erythroid and megs– Compared with morphology and cytogenetics– Abnormalities in subset of indeterminate cases

• Not advocated for screening

Stetler-Stevenson, et al. Blood (2001) 98:979-987.

Clinical utility• Correlation with IPSS and transplant outcome

Wells, et al. Blood (2003) 102: 394-403.

Clinical utility

Wells, et al. Blood (2003) 102: 394-403.

Clinical utility• Correlation with IPSS and transplant outcome

– Scoring system• 0 points = Normal• 1 point = Single abnormality• 2 points = 2 or 3 abnormalities OR CD34/lymphoid on myelomonocytic• 3 points = 4 abnormalities OR 1-3 abnormalities with CD34/lymphoid• 4 points = 2 to 3 abnormalities on myeloid and monocytic• Additional abnormal blasts: <5% (1), 6-10% (2), 11-20% (3), >20% (4)

• 0-1 = Normal/mild• 2-3 = Moderate• 4-9 = Severe

– Diagnosis of MDS• Score of 3 gives specificity of 100% with sensitivity of 55%; 76% correct

– Correlates with IPSS and transplant outcome

Wells, et al. Blood (2003) 102: 394-403.

Clinical utility

Wells, et al. Blood (2003) 102: 394-403.

Clinical utility

Wells, et al. Blood (2003) 102: 394-403.

Blast score in LG-MDS

Ogata, et al (2006) Blood 108:1037-1044

Erythroid Dysplasia

• Patient populations– 104 MDS– 69 Pathologic controls– 19 Normals

• Evaluated MFI– CD71– CD105– Cytosolic H&L-ferritin (HF

or LF)– Mitochondrial ferritin (MtF)

Della Porta, et al. Leukemia (2006) 20:549-555

Erythroid Dysplasia

• Linear Discriminant Analysis– MDS with erythroid dysplasia -

• 52/53 positive = 98.1% sensitivity

– MDS without erythroid dysplasia• 9/15 positive = 60%

– Normal and pathologic controls• 64/65 negative = 98.5% specificity

– MtF positive in all cases with ringed sideroblasts

Della Porta, et al. Leukemia (2006) 20:549-555

Clinical Diagnosis

• 124 cases submitted for myelodysplasia• Assessed by 4 color flow cytometry• Analyzed for maturational aberrancies

– Negative = no abnormalities– Indeterminate = mild abnormalities (< 2)– Positive = more than mild abnormalities (>= 2)

• Compared with cytogenetics and morphology– MDS = either cytogenetics and/or morphology

Kussick, et al. AJCP (2005) 124: 170-181

Distribution of cases by cytogenetic, morphologic, and flow cytometric findings.

CYTOGENETICS MORPHOLOGY  FLOW

CYTOMETRY      Normal Indeterm. Abnormal     Normal 31 12 0

Normal* Indeterminate 12 3 7  Abnormal 3 3 20     Normal 0 0 0

Abnormal* Indeterminate 1 1 1  Abnormal 0 0 30

Myelodysplasia

N = 124

Kussick, et al. AJCP (2005) 124: 170-181

Myelodysplasia

MDS = abnormal morphology and/or cytogeneticsFlow abnormal

89% sensitivity88% specificity

Flow abnormal or indeterminate95% sensitivity67% specificity

Also true for cases with < 5% blasts84% flow abnormal were MDS

Kussick, et al. AJCP (2005) 124: 170-181

Conclusion• Flow cytometry can aid in diagnosis of MDS and MPD

– More confident diagnosis• Identify immunophenotypic abnormalities• Exclude other disorders

– Aid subclassification• More accurate blast identification and quantitation

– Assist in prognostication– Rapid

• Requires– Consistent flow cytometric technique – Knowing normal patterns of antigen expression – Knowing common abnormal patterns