The Diagnosis of Myeloid Neoplasia by Flow Cytometry. Brent L. Wood MD PhD Dept. of Laboratory Medicine University of Washington, Seattle

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1 The Diagnosis of Myeloid Neoplasia by Flow Cytometry Brent L. Wood MD PhD Dept. of Laboratory Medicine University of Washington, Seattle

2 Basic Principles

3 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

Increased or decreased normal antigens Asynchronous

4 Normal B cell Maturation Wood and Borowitz (2006) Henry s Laboratory Medicine

5 Normal B cell Maturation Wood (2004) Methods Cell Biology 75:

6 ALL MRD 0.1% abnormal immature B cells

7 Myeloid Stem Cell Disorders Similar immunophenotypic abnormalities Myelodysplasia Myeloproliferative disorders Acute myeloid leukemia Differences in Blast percentage Extent of maturation Basophilia Degree of abnormality

disorders Acute myeloid leukemia Differences in Blast

8 CD45/SS Borowitz et al (1993) AJCP 100: Steltzer et al (1993) Ann NY Acad Sci 667:

9 CD45/SS Wood (2004) Methods Cell Biology 75:

10 Wood (2007) Clinics in Lab Medicine 27:

11 Early Progenitors

12 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

Stem cells Committed progenitors (CD34+) Promyelocytes

13 Blasts

14 Blasts Blast estimates by flow cytometry may not agree with morphology Specimen processing Red blood cell lysing reagents remove variable number of erythroid precursors Variable 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

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

15 Normal Blast Maturation Wood (2004) Methods Cell Biology 75:

16 Normal Granulocyte Maturation

17 Normal Monocyte Maturation

18 Acute Myeloid Leukemia Wood and Borowitz (2006) Henry s Laboratory Medicine

19 Abnormal Antigen Intensity Wood (2007) Clinics in Lab Medicine 27:

20 Aberrant Lymphoid Antigens Wood (2007) Clinics in Lab Medicine 27:

21 Aberrant Maturation Wood (2007) Clinics in Lab Medicine 27:

22 Granulocytic Maturation

23 Normal Granulocytic Maturation Wood and Borowitz (2006) Henry s Laboratory Methods

24 Normal Granulocytic Maturation Wood (2004) Methods Cell Biology 75:

25 Wood (2007) Clinics in Lab Medicine 27:

26 Monocytic Maturation

27 Normal Monocytic Maturation Wood and Borowitz (2006) Henry s Laboratory Methods

28 Normal Monocytic Maturation Wood (2004) Methods Cell Biology 75:

29 Monocytic Aberrancies Wood (2007) Clinics in Lab Medicine 27:

30 Acute Myelomonocytic Leukemia Wood and Borowitz (2006) Henry s Laboratory Medicine

31 Acute Monocytic Leukemia

32 Abnormal Myeloid Maturation Normal MDS

33 Myeloproliferative Disorders

34 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

35 Chronic Myeloid Leukemia CML in chronic phase - 2.8% blasts

36 Myeloproliferative disorders Non-CML Increased expression of Bcl-X L 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 myeloproliferative disorders

37 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:

38 Essential Thrombocytosis Kussick and Wood (2002) Am J Clin Path 20:

39 Myeloproliferative Disorders Population n Mean Age M:F Abnormal Cytogenetics Positive Flow (cases) :18 12 / 29 (41%) Negative Flow (controls) :22 0 / 36 Kussick and Wood (2002) Am J Clin Path 20:

40 Myeloproliferative disorders # of Cytog. Abnls. n Abnl. Blasts Abnl. Myelos. Abnl. Monos. Mean Abnl. Myel. Ags. Mean Non- Myel. Ags. Mean Total Abnl. Ags % 100% 56% % 88% 50% or more 4 100% 75% 75% TOTAL 29 97% 93% 55% Kussick and Wood (2002) Am J Clin Path 20:

41 MPD Conclusions Many cases of non-cml myeloproliferative disorders have immunophenotypic abnormalities Presence of immunophenotypic abnormalities 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

42 Myelodysplasia

43 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:

44 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:

45 Clinical utility Correlation with IPSS and transplant outcome Wells, et al. Blood (2003) 102:

46 Clinical utility Wells, et al. Blood (2003) 102:

47 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:

48 Clinical utility Wells, et al. Blood (2003) 102:

49 Wells, et al. Blood (2003) 102: Clinical utility

50 Blast score in LG-MDS Ogata, et al (2006) Blood 108:

51 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:

52 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:

53 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:

54 Myelodysplasia Distribution of cases by cytogenetic, morphologic, and flow cytometric findings. CYTOGENETICS MORPHOLOGY FLOW CYTOMETRY Normal Indeterm. Abnormal Normal Normal* Indeterminate Abnormal Normal Abnormal* Indeterminate Abnormal N = 124 Kussick, et al. AJCP (2005) 124:

55 Myelodysplasia MDS = abnormal morphology and/or cytogenetics Flow abnormal 89% sensitivity 88% specificity Flow abnormal or indeterminate 95% sensitivity 67% specificity Also true for cases with < 5% blasts 84% flow abnormal were MDS Kussick, et al. AJCP (2005) 124:

56 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

Subtypes of AML follow branches of myeloid development, making the FAB classificaoon relaovely simple to understand.

Subtypes of AML follow branches of myeloid development, making the FAB classificaoon relaovely simple to understand. 1 2 3 4 The FAB assigns a cut off of 30% blasts to define AML and relies predominantly on morphology and cytochemical stains (MPO, Sudan Black, and NSE which will be discussed later). Subtypes of AML follow