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

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5 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 branches of myeloid development, making the FAB classificaoon relaovely simple to understand. However, with few excepoons (AML M3), subtypes of the FAB do not separate AML into prognosocally significant groups. 5

6 The 2008 WHO classificaoon has 4 primary categories of AML (shown in red). Generally a blast cut off of 20% is used to define AML; however, the cytogeneoc abnormalioes t(15;17), t(8;21), and inv(16) and adequate to diagnose AML even in the presence of fewer than 20% blast equivalents. AML with gene mutaoons (listed in blue) is a provisional enoty AML not falling into categories 1-3 are classified as AML, not otherwise categorized. This group of AML is classified along a system analogous to the FAB classificaoon. 6

7 We have several tools available in the laboratory for diagnosis and classificaoon of acute leukemias. We will touch on other tools but the focus of this presentaoon will be on flow cytometry. 7

8 Morphology is the gold standard for blast enumeraoon with a blast percentage of 20% defining AML in the WHO classificaoon system. Of note, a blast percentage of 30% was used by the FAB classificaoon; however, given the similar prognosis in cases with 20-30% blasts and 30% or more blasts, the WHO classificaoon adopted a blast cutoff of 20% to define AML. 8

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12 Morphology provides a more accurate blast percentage than flow cytometry in the marrow as flow cytometry of the marrow is hindered by both hemodiluoon and variable exclusion of erythroid precursors. HemodiluOon generally lowers the true blast percentage while variable exclusion of the erythroid precursors has the opposite effect. Erythroid elements are variably excluded from the denominator in generaong the blast percentage due to paroal lysis of erythroids during processing, thereby aroficially increasing the blast percentage. Flow cytometry of the peripheral blood generally provides a more accurate assessment of blast percentage; however, again, morphology is considered the gold standard while flow cytometry may be used as a guide for blast enumeraoon. 12

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15 All images in this presentaoon were generated from data collected in the clinical laboratory on an LSR II with 4 lasers (addioonal yellow laser) allowing for the potenoal detecoon of 10 colors in a single tube. The panels we use are shown in the following slide. 15

16 We rouonely collect at least 100,000 events per sample. 16

17 IniOal recognioon of acute leukemia is o`en appreciated on a CD45 versus side scacer (SSC) plot. CD45 versus side scacer gaong allows determinaoon of an approximate 5 part differenoaoon with separaoon of lymphocytes (blue, high CD45 and low SSC), monocytes (magenta, high CD45 and intermediate SSC); myeloid forms (green, high CD45 and high SSC), lymphoid blasts (aqua, low CD45 and low SSC) and myeloid blasts (red, low CD45 and intermediate SSC). The image on the le` shows a normal marrow and is contrasted with the image of the right which shows a marrow from a paoent with AML who has an expanded populaoon occupying the blast gate by CD45 versus side scacer gaong. 17

18 The blast gate by CD45 side scacer gaong contains various cell types in addioon to myeloid blasts including basophils (purple), plasmacytoid dendrioc cells (aqua), myelomonocyoc cells and some lymphoid cells. Blasts (red) should be idenofied specifically with a progenitor cell marker (such as CD34). Basophils and PDCs are disonguished from blasts by a high level of CD123 expression. Basophils are negaove for HLA- DR while PDCs express high HLA- DR. 18

19 Blast equivalents include not only myeloid blasts but also immature monocyoc cells (promonocytes and monoblasts) which may be seen in acute myeloid leukemias with monocyoc differenoaoon and abnormal promyelocytes that are seen in acute promyelocyoc leukemia. Abnormal immature monocyoc cells (shown in aqua in the second panel) may have higher CD45 than typical myeloid blasts and abnormal promyelocytes (shown in orange in the third panel) have higher side scacer than typical myeloid blasts. These alternaove blast equivalents will be discussed further later in the presentaoon while typical myeloid blasts will be the focus of the next few slides. 19

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21 Early progenitor cells (*) express very high levels of CD34 and low to absent CD38. With maturaoon, these early blasts gradually drop CD34 as they acquire CD38. As CD38 reaches maximal blast levels, CD34 drops precipitously and markers associated with maturaoon and differenoaoon (such as CD15 for myeloid differenoaoon and maturaoon), are acquired. Immunophenotypic changes associated with normal blast differenoaoon and maturaoon are discussed in the following references: Kussick SJ, Wood BL. Using 4- color flow cytometry to idenofy abnormal myeloid populaoons. Arch Pathol Lab Med. Sep 2003;127(9): Wood BL. Myeloid malignancies: myelodysplasoc syndromes, myeloproliferaove disorders, and acute myeloid leukemia. Clin Lab Med. Sep 2007;27(3): , vii. 21

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23 Shown on the le` is CD45 versus SSC for all viable cells and on the right is HLA- DR versus CD33 the blast gate. The upper panel depicts an example of normal marrow while the lower panel illustrates an example of AML. Normally CD45 is expressed at an intermediate level while in the paoent with AML, CD45 is expressed at a very variable level with a subset of blasts showing low to absent CD45. Similarly, in the normal blasts, both CD33 and HLA- DR are expressed at varying levels during maturaoon while in the example of AML, HLA- DR is high and uniform while CD33 is abnormally low. 23

24 Shown on the le` is CD34 versus CD15 for the blast gate and on the right is CD38 versus CD15 for the blast gate. The upper panel depicts an example of normal marrow while the lower panel illustrates an example of AML. Normally CD15 is negaove on early progenitors expressing high levels of CD34 and low to absent levels of CD38. CD15 expression is seen on blasts a`er they acquire higher levels of CD38 and is accompanied by a loss of CD34. In the case of AML shown in the lower panel, an aberrantly high level of CD15 (a marker usually indicaong maturaoon) is seen on CD34 high, CD38 low blasts. 24

25 Shown on the le` is CD34 versus CD38 for the blast gate and on the right is HLA- DR versus CD33 for the the blast gate. The upper panel depicts an example of normal marrow while the lower panel illustrates an example of AML. Normal progenitor cells show varying levels of the 4 anogens depicted while the case of AML shown demonstrates uniform high CD34 and HLA- DR with uniform low CD33 and CD38. 25

26 Shown on the le` is CD34 versus CD5 for the blast gate and on the right is CD34 versus CD7 for the blast gate. The upper panel depicts an example of normal marrow while the lower panel illustrates an example of AML. Normal myeloid blasts are predominantly negaove for CD5 and show low level CD7 on a minor subset while the case of AML shown in the lower panel expresses bright CD7 with low level CD5. Of interest this case showed expression of myeloid markers including CD13, CD15, CD33 and CD117 with MPO on a small subset without expression of CD3 (surface or cytoplasmic). 26

27 These will be discussed later in the presentaoon. 27

28 The 2008 WHO classificaoon has 4 primary categories of AML (shown in red). AML with gene mutaoons (listed in blue) is a provisional enoty AML not falling into categories 1-3 are classified as AML, not otherwise categorized. This group of AML is classified along a system analogous to the FAB classificaoon. The next few slides will discuss flow cytometric features of some subtypes of AML including: AML with recurrent cytogene2c abnormali2es AML with t(8;21)(q22;q22), (AML1/ETO) AML with t(15;17)(q22;12), (PML/RARalpha) and variants AML not otherwise categorized Acute myelomonocyoc leukemia Acute monoblasoc and monocyoc leukemia Acute erythroid leukemia Acute megakaryoblasoc leukemia Myeloid sarcoma 28

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30 This slide demonstrates an example of AML with a t(8;21) translocaoon. Concurrent morphology showed just under 20% blasts (17.3%). Flow cytometry demonstrates 20.6% blasts (CD34 and CD117 posiove) that express myeloid markers including heterogeneous CD13 (lower le` panel) and co- express CD19 at a level below that of the hematogones (shown in aqua). The blasts also express CD56 on a subset. 30

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33 This slide demonstrates an example of the typical hypergranular variant of AML with a t(15;17) translocaoon. Note the high SSC of the blasts on the CD45 versus SSC plot. The abnormal promyelocytes/blast equivalents in this case are shown in orange and express CD117, bright CD33, and heterogeneous CD13 with low CD15 and without CD34 or HLA- DR. The low to absent CD15 is helpful in disonguishing abnormal promyelocytes from normal promyelocytes that might be present in a regeneraong marrow as the lacer acquire high levels of CD15. With differenoaoon therapy (such as ATRA), the blast equivalents may show paroal/disordered maturaoon. Although flow cytometry is suggesove of this subtype, correlaoon with geneoc studies to demonstrate the t(15;17) is required for definiove diagnosis. 33

34 This slide shows an example of the microgranular variant of AML with a t(15;17) [M3v]. This case has a SSC is notably lower than seen in the case in the prior slide. The SSC in M3v is variable, ranging from high (at the level of a granulocyte) to intermediate (at the level of a typical myeloid blast). AML M3v is typically posiove for CD117, bright uniform CD33, and heterogeneous CD13 with low to absent HLA- DR similar to typical APL; however, as demonstrated in this case, M3v may have variable CD34 and expression of CD2. Again, although flow cytometry is suggesove, correlaoon with geneoc studies to demonstrate the t(15;17) is required for definiove diagnosis. 34

35 Different clones of CD14 highlight monocyoc cells at different stages of differenoaoon. It is important to be aware of the staining characterisocs of the clone of CD14 being used in your laboratory. 35

36 Abnormal immature monocyoc cells including promonocytes and monoblasts are included as blast equivalents in acute myeloid leukemias with monocyoc differenoaoon (acute monocyoc/monoblasoc leukemia and acute myelomonocyoc leukemia). Immature monocyoc cells express CD4 with bright CD33 and CD64 and typically have bright HLA- DR with variable CD15 and CD13. CD14 is typically low to absent on immature monocyoc populaoons with different clones of CD14 anobody idenofying different stages of monocyoc differenoaoon. CD56 is aberrantly expressed in some cases and monocyoc anogens are described as aberrantly decreased in some cases as well. This case depicted in this slide is of an acute monocyoc leukemia with a mature monocyoc component shown in pink and an immature monocyoc component shows in aqua. Both populaoons show high level expression of CD4, CD33, and CD64. The immature monocyoc populaoon shows low to absent CD13 and CD14 with bright HLA- DR. The mature monocyoc populaoon shows variable CD13 and higher expression of CD14 that the immature populaoon. Both populaoons show expression of uniform CD15 and CD56. 36

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38 This slide shows an example of AML M4. Myeloid blasts expressing CD34 and CD117 are shown in red and comprise 24% of the white blood cells. MyelomonocyOc cells are depicted in the lower right panel and include maturing neutrophilic precursors beyond the blast stage (green) and monocyoc cells. By flow cytometry, both neutrophilic precursors and monocyoc cells each comprise greater than 20% of the white blood cells. MonocyOc cells are shown in pink (mature) and aqua (immature) and are idenofied by CD45 versus SSC characterisocs in conjuncoon with high level expression of CD33 and CD64. The mature monocyoc cells express high levels of CD14 which is decreased on immature monocyoc cells. CD13 is also decreased on the immature monocyoc cells which comprise approximately 6% of the white blood cells bringing the total percentage of blast equivalents (myeloid blasts +immature monocyoc cells) to 30%. In this case an increase in eosinophils is noted as well (shown in orange). Eosinophils have a high level of CD45 and side scacer and lack expression of CD16 and CD64 allowing separaoon from maturing myeloid cells (shown in green) and monocyoc cells. A tandem bone marrow aspirate smear demonstrated the presence of eo- basos and FISH studies shows the presence of an inversion

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41 This slide shows an example of AML M6b or pure erythroid leukemia. The abnormal blast populaoon is shown in light blue and is apparent on the CD45 versus side scacer plot with low to absent CD45 and intermediate side scacer. The abnormal cells lack CD34, HLA- DR and CD117 but express CD36, CD71, and CD235a. The abnormal cells lacked lineage specific markers including CD3 (surface or cytoplasmic), MPO, or B cell markers. In addioon, the blasts lacked megakaryocyoc markers CD41 and CD61. A minor subset of the blasts show aberrant expression of CD7. 41

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43 This slide depicts a case of relapsed AML- M7. The abnormal blasts are easily idenofied by CD45 versus side scacer. The blasts in this case (shown in yellow) express CD117 without CD34 or HLA- DR and express both CD61 and CD41. 43

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45 This slide depicts flow cytometry from the peripheral blood of a newborn (3 days old) with Down Syndrome who has leukocytosis. CD34+ blasts are 16% of the white blood cells and express CD34, CD117, and CD33 (bright) without CD13. CD41 and CD61 are expressed on subsets of the blasts. The blasts express CD7 at a low level with CD56 on a minor subset. This immunophenotype is characterisoc for TAM as is the age of the paoent. 45

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48 This slide shows an example of an MPAL, T/Myeloid. A uniform blast populaoon is noted which expresses the myeloid lineage specific marker MPO at a high level in conjuncoon with cytoplasmic CD3. Of note this neoplasm also expresses CD117, heterogeneous CD13, and bright CD33 with uniform CD2 and variable CD7. However, these lacer markers, although generally associated with myeloid or T lymphoid differenoaoon are not considered lineage specific. Myeloid, T cell and B cell lineage differenoaoon are defined as follows in the 2008 WHO: Myeloid lineage MPO or, MonocyOc differenoaoon Two of the following (NSE, CD11c, CD14, CD64, lysozyme) T cell lineage ccd3 (or surface CD3) B cell lineage Strong CD19 with one of the following or weak CD19 with 2 of the following ccd79a, ccd22, CD10 48

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50 This us a nasopharyngeal biopsy submiced to rule out lymphoma. The inioal lymphoma panel showed no abnormal B cell or T cell populaoons. However, an expanded populaoon of cells was noted in the blast gate. These cells were noted to be posiove for CD34 and CD4 in the T cell tube. A follow- up tube demonstrates expression of myeloid markers including CD117 and CD13 confirming the diagnosis of myeloid sarcoma. A bone marrow biopsy performed 1 week later showed a high grade myelodysplasoc syndrome. 50

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52 Categories of abnormality seen on myeloid progenitors in AML 1. Abnormal intensity of anogen expression (increased, decreased or absent). 2. Asynchronous expression of markers associated with maturity with markers associated with immaturity 3. Homogeneous expression of an anogen usually expressed at varying levels through blast maturaoon 4. Expression of non- lineage anogens 52

53 The first panel demonstrates an abnormal blast populaoon idenofied in a paoent with a history of AML who presented to our insotuoon in relapse. The blasts express bright CD34 with low to absent CD38 with expression of uniform CD33 and increased CD13. CD56 was expressed on a minor subset. A sample was submiced post inducoon chemotherapy and prior to transplant that showed 0.8% CD34+ blasts overall. A small subpopulaoon of the blasts (shown in orange, 0.1% of the total white blood cells) had the same immunophenotype as that seen prior to therapy with bright CD34, low CD38, uniform CD33 with increased CD13 and CD56 expression on a subset indicaong the presence of MRD. 53

54 The upper panel shows abnormal myeloid blasts from a paoent at diagnosis while the lower panel shows the paoent post inducoon therapy. At diagnosis, the abnormal blasts express bright CD34 with variably low CD38, CD15 on a subset and bright uniform CD5. Post inducoon a subset of the blasts appear to be normal and regeneraong (red, 1% of the white blood cells) while a subset are abnormal (aqua. 0.8% of the white blood cells) and express bright CD34, variably decreased CD38, and bright, uniform CD7 similar to the blasts seen at diagnosis. Of interest, some immunophenotypic features of the abnormal blasts have shi`ed. As an example, the abnormal blasts remaining post inducoon chemotherapy have a lower level of CD15 than those seen at diagnosis. 54

55 The upper panel shows the paoent s blasts at diagnosis. The abnormal blasts express CD117 with low to absent CD34, bright CD33, decreased CD13 and variably decreased CD38. CD56 is expressed on a subset of the blasts. The lower panel is from a marrow collected at day 24 post inducoon therapy. Blasts are relaovely increased at 3.5% of the white blood cells but show a normal immunophenotype with expression of CD13, CD34, CD38, CD33, and CD117 at normal levels without CD56, findings suggesove of marrow regeneraoon. No blast populaoon with the immunophenotype of the blasts seen at diagnosis is noted. 55

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