The diagnosis of AML relies on morphologic evaluation (cytochemical stains), immunophenotyping by flow cytometry, and assessment of karyotype and molecular studies (Fig. 1). Blast percentage is best determined by a 500-cell differential of the marrow aspirate. The presence of ≥ 20% myeloid blasts in the peripheral blood or bone marrow is required for the diagnosis of AML, except in AML with t(8;21), inv(16), or t(15;17), in which case, these specific cytogenetic changes/gene fusions are sufficient for the diagnosis of AML regardless of the blast percentage. Three broad types of myeloblasts are described based on the granular content and nuclear features of the blasts (type 1: agranular basophilic cytoplasm, nucleus with fine chromatin, and two to four distinct nucleoli; type 2: basophilic cytoplasm with 20 or fewer azurophilic granules and similar nuclear features as type 1 blasts; type 3: basophilic cytoplasm with more than 20 azurophilic granules), although the morphologic variety of blasts exceeds the defined categories ( Fig. 2 ). Auer rods are linear aggregates of primary granules composed predominantly of myeloperoxidase (MPO) that are found in 30% to 50% of newly diagnosed patients with AML and, if present, are one of the hall mark morphologic features to establish a diagnosis of AML (see Fig. 2C ). Malignant promyelocytes have moderately basophilic cytoplasm with numerous azurophilic granules, frequent Auer rods including cells with multiple Auer rods (“faggot cells”), and bilobed or sliding-plate nuclei. Monoblasts and promonocytes usually exhibit folded or convoluted nuclei and may contain prominent vacuoles. Promyelocytes, promonocytes, and atypical pronormoblasts are all considered blast equivalents. Micromegakaryocytes and pronormoblasts are not considered blasts (see Fig. 2K and L). AML marrows are typically hypercellular with decreased or absent megakaryocytes. Exceptions are marrows of older patients or those with t-AML, which may be hypocellular with dysplastic changes of one or more hematopoietic lineages. Prominent dysplasia may suggest a previous diagnosis of MDS but can also be found in patients with de novo AML, where the prognostic significance of dysplastic changes is less clear. For example, multilineage dysplasia in natural killer (NK) AML with NPM1 or biallelic CEBPA mutations does not appear to affect the otherwise more favorable outcome of AML patients with these mutations. Cases with extensive fibrosis may be due to a preceding myeloproliferative neoplasm or acute megakaryocytic leukemia.
Fig1. WORKUP OF ACUTE MYELOID LEUKEMIA. The diagnostic workup consists of a morphologic assessment, immunophenotyping by flow cytometry, assessment of the karyotype, and a panel of gene mutations. Whereas morphologic assessment by itself is often not sufficient to render a diagnosis, flow cytometry will confirm the lineage assignment (myeloid vs. lymphoid) and stage of differentiation in more than 95% of cases. In the remainder, either no lineage-specific antigens are expressed (acute undifferentiated leukemia) or antigens of more than one lineage are present (mixed-phenotype acute leukemia). In the latter scenario, antigens of several lineages can be found on one (bi-phenotypic) or separate populations of blasts (bilineal). Karyotyping and gene mutation analysis may add diagnostic information in morphologically ambiguous situations but is otherwise of more interest in determining prognosis. Additional information (exposure to previous chemotherapy and/or radiation therapy, history of an antecedent hematologic disorder, dysplasia) forms the basis for the 2016 revision of the WHO classification of AML. WHO, World Health Organization. (Adapted from Dohner H, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424–447.)
Fig2. SPECTRUM OF BLASTS, BLAST EQUIVALENTS, AND OTHER CELLS. Blast cells in acute myeloid leukemia (AML) exhibit a wide spectrum of morphologic features. The French–American–British classification described three types of blasts depending on the granule content (A–C). However, blasts with nuclear invagination (frequently associated with NPM1 and/or FLT3 mutations) (D); blasts with pseudopods (frequently shown to be megakaryoblasts) (E); and monoblasts (F) are also quite distinctive. Blast equivalents include granular or hypogranular promyelocytes (G and H) for acute promyelocytic leukemia, promonocytes (I) for AML with a monocytic component, and atypical pronormoblasts (frequently with cytoplasmic vacuoles) (J) for acute erythroleukemia of the pure erythroid type. Micromegakaryocytes (K) and pronormoblasts (L) are not considered blasts.
Several cytochemical reactions further highlight morphologic characteristics (MPO, periodic acid–Schiff, Sudan black B, naphthol AS-D chloroacetate esterase [specific esterase], α -naphthyl acetate/butyrate esterase [nonspecific esterase], acid phosphatase; Fig. 3 ). MPO is the most specific granulocytic marker, and MPO positivity in at least 3% of the blasts is consistent with a diagnosis of AML. On the other hand, lack of MPO staining does not rule out AML because it is often not present in AML with minimal differentiation, acute monoblastic leukemia, and acute megakaryocytic leukemia. Monoblastic leukemias are stained by nonspecific esterase. The diagnostic utility of cytochemical reactions is limited by the more rapid availability of immunophenotypic data by flow cytometry. Nonetheless, cytochemical stains of a touch imprint of the core biopsy can be very useful in the event of a “dry tap.” Likewise, immunohistochemical staining for CD34 and CD117 can be used to estimate the blast percentage in a bone marrow core biopsy.
Fig3. CYTOCHEMISTRY: MYELOPEROXIDASE, α-NAPHTHYL ACETATE ESTERASE, AND COMBINED ESTERASE REACTIONS. The MPO reaction is easily performed, can be done in less than a few minutes, and provides important initial information about the lineage of the blasts, particularly in cases in which morphologic assessment is difficult. In many laboratories it is routinely performed for all new acute leukemias. The MPO reaction should be interpreted in the blast population and expressed as a percentage of blasts that are positive. (A) The positive MPO reaction is strong. (B) The reaction is weak and seen in only some blasts. A counterstain would have obscured the weak reaction product. A weak MPO reaction is not uncommon in cases of acute myeloid leukemia (AML) associated with myelodysplasia (as in [B]). The neutrophils in such cases are also only weakly positive ( bottom cell [B]). The ANAE reaction is a nonspecific esterase reaction positive in most monocytic cells ([C] orange-brown cell on left compared to negative neutrophil, and erythroid cell, middle and right). The ANAE reaction is interpreted as positive in cells as a percentage of nonerythroid elements. A significant monocytic component is usually defined as 20% or greater of the nonerythroid elements and is usually required for making a diagnosis of acute myelomonocytic leukemia. It is notable that in some cases of AML with inv(16) (i.e., AML with abnormal eosinophils), the monocytes are ANAE negative. A COES reaction uses another nonspecific esterase reaction for monocytes, α -naphthyl butyrate esterase, together with the specific esterase, chloroacetate esterase, for granulocytes. The combination allows simultaneous evaluation of granulocytes ( blue reaction product) and monocytes ( orange-brown reaction product). (D) Acute myelomonocytic leukemia. A monocyte (top), a granulocyte (right), and a myelomonocytic hybrid cell that exhibits both the orange-brown and blue reaction products (bottom). ANAE, α -Naphthyl acetate esterase; COES, combined esterase; MPO, myeloperoxidase.
The first systematic attempt to classify AML was made by the French–American–British (FAB) Cooperative Group in 1976 and updated in 1985, and was based solely on morphology (blast percent age, degree of differentiation, lineage involvement). Because of its limited scope, the FAB system is now considered inadequate. Rapidly growing insights from genetic mutation analyses, their association with prognosis, and, in some cases, prediction of response to therapy triggered a revision of the old system and led to the changes in the 2008 edition of the World Health Organization (WHO) classification of AML and updated in 2016. The focus has shifted to identification of recurrent cytogenetic and molecular abnormalities, information regarding exposure to prior leukemogenic therapy, and morphologic features related to dysplasia-related changes as well as morphologic remnants of the FAB system ( Table 1 ). Several categories have been defined:
1. AML with recurrent genetic abnormalities. This includes AML with relatively common cytogenetic changes: AML with t(8;21) (q22;q22), RUNX1-RUNX1T1 ( Fig. 4 ); AML with inv(16) (p13.1q22) or t(16;16)(p13.1;q22), CBFB-MYH11 ( Fig. 5 ); AML with t(15;17)(q22;q12); PML-RARA ( Fig. 6 ); and AML with t(9;11)(p22;q23), MLLT3-KMT2A ( Fig. 7 ). Less common subtypes are AML with t(6;9)(p23;q34), DEK-NUP214; AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2), GATA1, MECOM; and AML with t(1;22) (p13;q13), RBM15-MKL1 ( Fig. 8 ). Normal karyotype AML with nucleophosmin 1 mutation ( NPM1; see Fig. 60.8G and H ), and biallelic CEBPA mutations (and in the absence of additional mutations) were initially considered as provi sional, but are now incorporated in this subtype, with other molecular subsets expected to be added in subsequent editions.
2. AML with myelodysplasia-related changes (AML-MRC) is defined in one of three ways: (a) AML in patients with a pre ceding diagnosis of MDS or MDS/MPN; (b) AML with multi lineage dysplasia recognized morphologically by the presence of dysplastic features in 50% or more of cells in two separate lineages ( Fig. 9 ); and (c) AML associated with a myelodysplasia-related cytogenetic abnormality. Patients with AML-MRC appear to have a worse outcome compared with AML-not otherwise specified (AML-NOS). However, the OS of patients treated with intensive chemotherapy for AML-MRC as defined solely by multilineage dysplasia is similar to that of patients with AML-NOS.
3. The category of t-AML was retained from the 2001 WHO classification, although the distinctions between AML associated with alkylating agents or radiation and those with topoisomerase II inhibitors were abandoned. Many patients receive complex therapeutic regimens, making this distinction difficult and impractical. Nonetheless, AML following exposure to topoisomerase II inhibitors typically have a shorter latency (less than 2 years) and frequently have monoblastic morphology with fusions of the KMT2A (previously MLL) gene. CBF AML and APL have also been associated with topoisomerase II inhibitors exposure. AML following alkylating agents typically have a longer latency, are pre ceded by MDS, and have complex cytogenetic changes including monosomy and deletions of chromosomes 5 and/or 7 as well as TP53 mutations. Immunosuppressive therapies are now considered potentially leukemogenic.
4. NOS subtypes are AML cases that do not fit the categories already described and for the most part are classified using the FAB scheme (AML with minimal differentiation, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monocytic or monoblastic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute basophilic leukemia). The AML-like disease of acute panmyelosis with myelofibrosis is included here ( Fig10).
5. Lastly, the classification now includes myeloid sarcoma ( Fig. 11 ), myeloid proliferations associated with Down syndrome, and the rather rare entity of blastic plasmacytoid dendritic cell neoplasm (BPDCN) ( Fig. 12 ).
Fig4. ACUTE MYELOID LEUKEMIA WITH T(8;21)(Q22;Q22), (RUNX1-RUNX1T1). (A) Low-power, Wright-stained bone marrow aspirate smear showing increased blasts associated with differentiating myeloid cells. (B) Details illustrating some of the features associated with this leukemia. They include blasts with long thin Auer rods (top left), immature cells with abnormal eosinophilic globules (top and bottom, second from left), abnormal salmon-colored granulation in the maturing cells, sometimes associated with a basophilic periphery (top and bottom, fourth from left), and slightly abnormal features in the mature neutrophils (far right). Pseudo–Chediak-Higashi granules were not seen in this case. (C) Biopsy specimen illustrates the significant degree of maturation that can sometimes be seen. In fact, in some cases the blast count can be less than 20%, but the diagnosis of acute myeloid leukemia is still made with the cytogenetic finding of t(8;21).
Fig5. CUTE MYELOID LEUKEMIA WITH ABNORMAL BONE MARROW EOSINOPHILS AND INV(16)(P13.1;Q22) OR T(16;16) (P13.1;Q22), (CBFB-MYH11). (A) Low-power, Wright-stained bone marrow aspirate showing blasts, monocytic cells, granulocytic cells, and abnormal eosinophils. (B) Features of the abnormal eosinophils in three abnormal eosinophils (left three cells). Note the abnormal basophilic granules in the eosinophilic myelocytes. These granules are large, tend to cluster or coalesce, and are interspersed among the large eosinophilic granules, which are more difficult to see. As the eosinophils mature, the abnormal basophilic granules are less prominent and sometimes disappear (far right). A common misconception is that the basophilic granules are the granules of basophils and that the abnormal eosinophils are “hybrid cells.” (C) A basophil in the same case (top cell) for comparison. (D) Cells from a case of reactive eosinophilia, also for comparison. Immature eosinophils (cell to the right) do have primary blue granules. However, they are usually less prominent and less atypical than the basophilic granules of the abnormal eosinophils. It is notable that the monocytes in cases of AML with inv(16) or t(16;16) are sometimes α -naphthyl acetate esterase (ANAE) reaction negative. (E) Negative ANAE reaction. (F) Abnormal eosinophils cannot be recognized in hematoxylin and eosin-stained sections of the biopsy.
Fig6. ACUTE PROMYELOCYTIC LEUKEMIA, ACUTE MYELOID LEUKEMIA WITH T(15;17)(Q22;Q12), (PML-RARA). (A–C) Acute pro myelocytic leukemia (APL). (D and E) Hypogranular or microgranular subtype. (F) Bone marrow biopsy. (G) APL cells maturing after all-trans retinoic acid (ATRA) therapy. In the typical granular type of APL (A), the abnormal promyelocytes can exhibit variable morphologic features. Even within the same case, the granules can range from coarse, dark, and dense to fine and dust-like (B). The nuclei in the abnormal promyelocytes frequently exhibit a bilobed, dumbbell, or reniform shape. This is a diagnostically important feature, which can sometimes be difficult to recognize beneath the granules (B). Auer rods can be single, multiple, coalesced into Auer bodies, and even present in maturing cells (C). The microgranular type usually presents with an elevated white blood cell count (D). Although granules cannot be readily appreciated at the light microscope (E), granules can be demonstrated by electron microscopy. The abnormal nuclear shapes (bilobed, dumbbell, and reniform) can be easily appreciated (E). Bone core biopsy sample typically shows sheets of cells with abundant granular cytoplasm (F). After ATRA therapy the abnormal promyelocytes mature to abnormal neutrophils ([G] top and bottom right, compared with normal neutrophil, left). These can be seen for weeks after therapy and do not signify a failed response to the differentiating agent.
Fig7. ACUTE MONOBLASTIC LEUKEMIA WITH T(9;11)(P22;Q23), (KMT2A-MLLT3). Acute monoblastic leukemia with t(9;11). (A) Such cases typically present with high counts because of circulating monoblasts. (B and C) Bone marrow aspirate is packed with monoblasts and shows few granulocytic elements. (D) Absence of a granulocytic component can be illustrated with the combined esterase reaction, in which most of the cells show the α -naphthyl butyrate reaction product (orange-brown), with only rare granulocytes with the blue reaction product from the chloroacetate esterase reaction. (E) The biopsy sample is usually packed with sheets of monoblasts with fine nuclear chromatin and abundant pink cytoplasm.
Fig8. ACUTE MYELOID LEUKEMIA WITH LESS COMMON CYTOGENETIC AND GENETIC CHANGES. (A and B) Acute myeloid leukemia (AML) with t(6;9)(p23;q34). These cases are characterized by marrow dysplasia and basophilia. However, the morphologic characteristics can be quite varied among cases. AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) is characterized by normal or high platelet counts (C), and by a bone marrow with numerous micromegakaryocytes (D). AML with t(1;22)(p13;q13) is uncommon and occurs in infants. The blasts are megakaryoblasts (E) but in the bone marrow can have a sarcomatous appearance (F). AML with NPM1 or NPM1 and FLT3 mutations frequently presents with high peripheral blast counts, and the blasts sometimes have nuclear invaginations (G). NPM1 mutation is associated with the abnormal cytoplasmic localization of NPM1 rather than the normal nuclear staining (H).
Fig9. ACUTE MYELOID LEUKEMIA WITH MULTILINEAGE DYSPLASIA. (A) Bone marrow aspirate shows increased blasts and maturing cells with dysplastic features. (B–D) Details of the dysplasia. (B) Maturing erythroid elements exhibit megaloblastoid change and bizarre nuclear abnormalities. (C) Dysplastic granulocytes (bottom three) are compared with a rare normal granulocyte (top). The dysplastic forms have pale cytoplasm and abnormal nuclear shapes with hypolobation, hypersegmentation, and prominent nuclear excrescences. (D) Dysplastic micromegakaryocytes have single or double small nuclei but mature cytoplasm with platelet material within. (E) Increased blasts. (F) Dysplasia is difficult to appreciate on a biopsied section. This is true except for the megakaryocytes. Dysplastic megakaryocytes can be recognized on the biopsy specimen by their abnormally small nuclei, which are sometimes multiple and widely spaced.
Fig10. ACUTE PANMYELOSIS WITH MYELOFIBROSIS. (A) Bone marrow biopsy specimen shows a loosely packed marrow with a swirling appearance to the cellular elements due to underlying fibrosis. (B) The latter is illustrated on the reticulin stain. (C) Proliferations of erythroid cells (top left), megakaryocytes (bottom left), and immature cells within the fibrotic areas (right). (D) Immunohistochemical staining shows increased megakaryocytes ( top, CD61) and increased blasts ( bottom, CD34).
Fig11. MYELOID SARCOMA. Myeloid sarcomas can sometimes present a diagnostic challenge. In this case the touch imprints (A) and frozen section preparation (B) from a mass lesion in the cecal area from a 44-year-old patient were thought to represent a high-grade lymphoma. However, initial immunohistochemical stains did not support the diagnosis, and on closer inspection of the tumor and with subsequent immunomarkers, a diagnosis could be reached. The tumor (C) is composed of sheets of non-cohesive cells. A diagnostic clue to the origin was the presence of eosinophilic myelocytes (D), which indicate that some tumor cells have the capacity to differentiate into eosinophils. The granules of neutrophilic myelocytes cannot be recognized on tissue section. The immunohistochemical stains showed the cell to be CD45 + (not shown), (B) and T marker negative (E and F), but myeloid marker (myeloperoxidase and CD33) positive (G and H). Cytogenetic analysis showed the case was inv(16)(p13.1;q22).
Fig12. BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM. The blastic plasmacytoid dendritic cell tumor is an unusually aggressive malignancy that was previously called hematodermic malignancy. It is now classified in the acute myeloid leukemia category of myeloid neoplasms. It frequently presents in the skin with a blastic proliferation of cells in the dermis (A and B), which inevitably spreads to the blood (C) and bone marrow (D and E).
Table1. WHO 2016 Classification of Acute Myeloid Leukemia (AML) and Related Neoplasms
