Pathology of Acute Myeloid Leukemia Not Otherwise Specified

Updated: Dec 18, 2017
  • Author: Elham Vali (Khojeini) Betts, MD; Chief Editor: Christine G Roth, MD  more...
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In the 2016 update to the World Health Organization (WHO) classification system, [1, 2] the category "acute myeloid leukemia [AML] not otherwise specified" [NOS])" includes those AML subtypes not associated with recurrent genetic abnormalities such as:

  • t(8;21)(q22;q22); (RUNX1-RUNX1)
  • inv(16)(p13q22) or t(16;16)(p13;q22); (CBFβ/MYH11)
  • t(15;17)(q22;q12); (PML/RARα) and variants [ie, t(11;17)(q23;q12), t(5;17)(q35;q12)]
  • 11q23(KMT2A) abnormalities
  • t(6;9)(DEK-NUP214), inv (3) (q21;q26) or t(3;3)(q21;26.2); t(1;22)(p13;q13)
  • BCR-ABL1
  • Mutated NPM1
  • Biallelic mutation of CEBPA
  • Mutated RUNX1

In addition, it is also important to exclude other, more specific AML categories (ie, AML with myelodysplasia-related changes, therapy-related myeloid neoplasm, AML with germline predisposition, myeloid proliferations related to Down syndrome) before classifying as AML NOS. 

AML NOS comprises 30-40% of AML cases. In general, AML NOS is more common in adults, although it may occur in any age group. 

A diagnosis of AML requires the presence of least 20% leukemic cells in the bone marrow (BM) or peripheral blood (PB). It is important to recognize that promonocytes are considered blast equivalents for purposes of defining an AML.

AML NOS may be subclassified further based on morphologic and cytochemical/immunophenotypic features that indicate the major lineages and degree of maturation: AML with minimal differentiation; AML without maturation; AML with maturation; acute myelomonocytic leukemia; acute monoblastic and monocytic leukemia; pure erythroid leukemia; acute megakaryoblastic leukemia; acute basophilic leukemia; and acute panmyelosis with myelofibrosis.

An important change in the 2016 WHO update was the removal the subcategory of "acute erythroid leukemia, erythroid/myeloid type"; in the new classification system, blasts are always enumerated as a percentage of total marrow nucleated cells. Cases with an erythroid predominance but less than 20% blasts among the total number of marrow cells are now classified as myelodysplastic syndrome (MDS). [2]

See also the following:


Clinical Features

Patients with acute myeloid leukemia (AML) with minimal differentiation, AML without differentiation, and AML with differentiation usually present with evidence of bone marrow failure (ie, anemia, neutropenia, and/or thrombocytopenia). Patients with acute myelomonocytic leukemia (AMML) also typically present with anemia and/or thrombocytopenia, but AMML is often associated with a monocytosis that may be composed of promonocytes, monoblasts, and mature monocytic cells; the later may predominant in the peripheral blood. Patients with acute monoblastic and monocytic leukemia may present with bleeding disorders, as well as with extramedullary masses, cutaneous and gingival infiltration, and central nervous system (CNS) involvement. 

Although patients with acute megakaryoblastic leukemia may present with evidence of bone marrow failure associated with pancytopenia, in some cases, thrombocytosis is present. Patients with acute basophilic leukemia often present with evidence of bone marrow failure, as is seen with the other types of AML described above; in addition, they may present with cutaneous involvement, organomegaly, lytic lesions, and symptoms related to hyperhistaminemia. Acute panmyelosis with myelofibrosis is associated with severe constitutional symptoms (ie, weakness, fatigue, fever, and bone pain) and, invariably, pancytopenia; the disease follows a rapidly progressive clinical course.


Morphologic Features

In this section, the morphologic features of the subtypes of acute myeloid leukemia (AML) not otherwise categorized (NOS) are briefly discussed.

AML with minimal differentiation

AML-M0 cannot be diagnosed on morphologic grounds alone, because the blasts are of varying size and are agranular, sometimes resembling lymphoblasts (see the image below). Immunophenotyping is required to determine myeloid lineage. By definition, the blasts are MPO and Sudan Black B negative (<3% positive blasts). 

Blasts of acute myeloid leukemia, type M0. Blasts of acute myeloid leukemia, type M0.

AML without maturation

In AML without differentiation, the predominant blast type, representing more than 90% of the nonerythroid cells, is a poorly differentiated myeloblast with one or more distinctive nucleoli. The blast population, termed blast type I, consists of blasts without any recognizable granules. In blast type II, some blasts have a few fine, azurophilic granules (numbering <20). The presence of occasional Auer rods is consistent with the diagnosis of AML-M1. Because the blast population represents more than 90% of the cells, minimal differentiation is still present, but very few mature cells are found.

AML with maturation

AML with differentiation displays clear evidence of significant maturation, with abnormal differentiating cells ranging from promyelocytes to neutrophils. As defined by the World Health Organization (WHO) criteria, the percentage of blasts is at least 20% and less than 90%. [1]  The percentage of monocytic precursors must be below 20% in the bone marrow. In blast type III, more than 20 granules must be present, with a central nucleus, no Golgi zone, and a fine chromatin with the classic blast characteristics (ie, blast type I without granules, blast type II with <20 granules).

Acute myelomonocytic leukemia

In acute myelomonocytic leukemia (AMML), both granulocytic and monocytic precursors exist in varying proportions. For a diagnosis of AMML, the marrow monocytic component must account for 20% or more of the nonerythroid nucleated cells, but it cannot exceed 80%. The blasts cells show folded nuclei and some blasts may have cytoplasmic granules and Auer rods.

Acute monoblastic and monocytic leukemia

In acute monoblastic leukemia, the marrow is almost exclusively composed of immature monoblasts with less than 20% promonocytes or mature monocytes (see the following image). The blasts are large (≥30 mm), with an abundant rim of cytoplasm; rarely, azurophilic fine granules are present, and vacuolated basophilic cytoplasm is sometimes seen. The nucleus is round to oval, with delicate, lacy chromatin.

Blasts of acute myeloid leukemia, type M5a. Blasts of acute myeloid leukemia, type M5a.

In acute monocytic leukemia, at least 20% of the abnormal cells are recognizable promonocytes or beyond, with twisted or folded nuclei, gray-blue cytoplasm, and scattered azurophilic granules (see the image below). The percentage of mature monocytes is generally much higher in the peripheral blood than in the bone marrow. Rarely, a few blasts contain Auer rods.

Morphology of acute myeloid leukemia, type M5b. Morphology of acute myeloid leukemia, type M5b.

Acute erythroid leukemia (pure erythroid leukemia)

Pure erythroid leukemia without morphologic evidence of erythroid maturation may be difficult to separate from other leukemias (lymphoblastic, megakaryoblastic, minimally differentiated) and immunophenotyping is essential for diagnosis. The differential diagnosis includes megaloblastic leukemia due to folate or vitamin B12 deficiency. 

Acute megakaryoblastic leukemia

Megakaryoblastic leukemia is a fulminant proliferative disease for which bone marrow biopsy is invaluable in establishing the diagnosis. The reason for this is that in only a few cases does the bone marrow aspirate show a significant (>20%) number of blasts. Frequently, the aspirates are very scant, owing to the marked myelofibrosis common in the disease.

For these reasons, bone marrow biopsy specimens are needed. The sections may show many blasts or clusters of micromegakaryoblasts, as well as more mature megakaryoblasts. This is associated with an increase in the reticulin network and a corresponding decrease in the usual myeloid maturation. Megakaryoblastic fragments with changes in red blood cell morphologic structure and circulating small blasts, resembling either type I or type II blasts, may be found in the peripheral blood. The morphologic features of the blasts reveal cells that are pleomorphic; these cells may vary from very small forms with dense nuclear chromatin to large forms with 1-3 prominent nucleoli. Cytoplasmic blebs may be found surrounding some blasts. In more mature cells, such as circulating micromegakaryocytes, these expansions look like platelets.

In pediatric acute megakaryoblastic leukemia, the bone marrow aspiration is much more easily performed; the blasts may resemble lymphoblasts, but cytoplasmic blebs may be identified, as depicted below.

Megakaryoblasts from a pediatric case of acute meg Megakaryoblasts from a pediatric case of acute megakaryoblastic leukemia.

Acute basophilic leukemia

In acute basophilic leukemia, the blasts are characterized by moderately basophilic cytoplasm and a variable number of coarse basophilic granules. Mature basophils are sparse.

Acute panmyelosis with myelofibrosis

Acute panmyelosis with myelofibrosis is characterized by pancytopenia; leukoerythroblastosis may be evident in the peripheral blood. On bone marrow biopsy, hypercellularity with variable hyperplasia of erythroid precursors, granulocytes, and megakaryocytes is evident. There are foci of immature cells, including blasts, clusters of late-stage erythroid precursors, and increased dysplastic megakaryocytes associated with marked reticulin fibrosis. The marked fibrosis usually results in a "dry tap" upon aspiration.


Immunophenotypic Features

Subtyping of acute myeloid leukemia (AML) not otherwise categorized (NOS) depends upon a combination of immunophenotypic features and methods, including enzyme cytochemical staining, flow cytometric immunophenotyping and, in some cases, immunohistochemical (IHC) staining. [3]

Enzyme cytochemical staining

AML with minimal differentiation is characterized by negative myeloperoxidase (MPO) and Sudan black B reaction (results are positive in <3% of blasts). In AML without maturation, more than 3% of blasts are positive on MPO or Sudan black B staining. Occasionally, the specific esterase stain for granulocyte precursors (ie, naphthol ASD chloroacetate esterase [CAE]) is positive when the MPO is negative.

AML with maturation demonstrates blasts with strong MPO positivity. The nonspecific esterase reactions (ie, alpha naphthyl acetate esterase [ANAE] and alpha naphthyl butyrate esterase [ANBE]) cannot identify more than 20% of monocyte precursors of the nonerythroid component of the bone marrow. Acute myelomonocytic leukemia (AMML) demonstrates more than 20% monocytic precursors of the nonerythroid component of the bone marrow by ANAE and/or ANBE staining and over 20% myeloid precursors by differential count and/or by MPO staining of the nonerythroid component of the bone marrow.

In acute monoblastic and acute monocytic leukemias (AMoLs), nonspecific esterase staining (ie, ANAE and/or ANBE), causes a strong positive reaction in more than 80% of the nonerythroid component of the bone marrow in over 90% of cases. Occasionally, the Sudan black B reaction is positive in the absence of an MPO reaction. It should be kept in mind that the blasts of AMoL may show fine, granular staining with CAE. However, AMoLs are characterized by less than 20% myeloid precursors of the nonerythroid component of the bone marrow, primarily as determined on the basis of the differential count, because promonocytes may show some scattered MPO positivity. 

Flow cytometric immunophenotyping

Flow cytometric immunophenotyping (FCI) distinguishes AML from precursor B-cell and precursor T-cell lymphoblastic leukemias. FCI defines AML with minimal differentiation, which requires expression of myelomonocytic markers (ie, CD13, CD33) by flow cytometric analysis. There is no expression of intracytoplasmic MPO, CD3, or lineage-specific B-cell markers. FCI may be useful in distinguishing the hypogranular variant of acute promyelocytic leukemia from acute monocytic leukemia. By FCI, both the classic (hypergranular) and microgranular or hypogranular variants of acute promyelocytic leukemia uniformly lack expression of CD14; they typically lack expression of HLA-Dr, CD34, and CD56; and they may have dim expression of CD64 and CD117.

By contrast, AMoL uniformly expresses HLA-Dr; it often expresses CD56 and CD64; it may express CD34; and it variably expresses CD117. In addition, one should keep in mind that although CD14 is a monocyte-specific marker, CD14 is often absent or is frequently diminished in expression in AMLs with monocytic differentiation (ie, AMML and AMoL). In addition, other markers characteristically expressed by normal monocytic cells (ie, CD11b, CD13, CD15, CD33, and CD64) may be absent or at least partially diminished in AMML and AMoL. [4] CD56 may be aberrantly expressed in up to 50% of cases of AMML and AMoL.

Detection of CD34 and CD117 expression by FCI has also been shown to be indicative of malignancy in monocytic disorders; thus, such expression may be observed in AMML and AMoL. [4] In one study, the combination of any degree of CD64 expression with CD15 expression of 3+ intensity and heterogeneous CD13 expression or CD13 expression of 1+ to 2+ intensity was observed only in the AMML and AMoL subtypes.

Flow cytometric analysis defines acute megakaryoblastic leukemia; the diagnosis requires demonstration of megakaryocytic antigen expression (ie, CD41, CD42b, and/or CD61) by the blasts. In pediatric patients, acute megakaryocytic leukemia may resemble acute lymphoblastic leukemia; thus, in pediatric patients, FCI may be particularly useful. [5] In comparisons of the three megakaryocytic markers, CD42 was the least sensitive for identifying early megakaryoblasts (owing to the lack of CD42 expression by early megakaryoblasts); CD41a was the most sensitive but was least specific; and CD61 was the most specific marker of megakaryoblastic differentiation. [6, 7]

Immunohistochemical staining

Although FCI is the preferred method of immunophenotypically differentiating acute leukemias, paraffin IHC staining may be useful in situations in which it is not possible to perform FCI. For example, when a bone marrow aspirate is markedly hemodiluted or cannot be obtained, bone marrow sections may be the only evaluable means to establish a diagnosis of myelodysplastic syndrome (MDS) or AML, as well as to distinguish these two entities from each other and from other possible hematolymphoid malignancies.

As mentioned earlier, it is important to interpret stains in light of other immunophenotypic markers (ie, an IHC panel) and in combination with the morphologic features of each case. There are IHC markers for some of those antigens typically analyzed by FCI that may be employed on bone marrow sections to detect immature cells (ie, CD34, CD117, and TdT), to detect monocytic cells (ie, CD14), to distinguish erythroid from myeloid precursors (ie, CD33, MPO, and e-cadherin and glycophorin A), and to detect megakaryoblasts (CD41 and CD61).

FCI and IHC are equally sensitive to the detection of CD34. IHC staining appears to be significantly more reliable in detecting CD34 than CD117. Nevertheless, in combination, they are most reliable and are complementary for accurately quantifying blasts. These methods should be performed on both bone clots and cores, owing to their variable reactivity in paraffin-embedded and decalcified tissues. It is also important to remember that not all myeloblasts express CD34 and/or CD117.

TdT may be detected by flow cytometry as well as IHC staining. Flow cytometry has been shown to be very sensitive and can detect as few as 2% blasts; flow cytometry also allows for multicolor analysis to confirm the presence of TdT on T-cells, B-cells, and myeloid cells. [8] In contrast, TdT IHC staining does not necessarily correlate with the percentage of blasts in AML. [9] This lack of significant correlation results from the fact that cells that are positive for TdT on IHC staining of bone marrow tissue specimens most likely represent some myeloblasts that express TdT (given that TdT is a marker of immaturity and is not lineage specific), as well as other immature cells in the bone marrow, such as hematogones.


Molecular/Genetic Features

The molecular or genetic abnormalities detected in patients with acute myeloid leukemia (AML) who have a normal karyotype may be arbitrarily divided into two major groups: (1) abnormalities directly affecting the proliferation and apoptosis of leukemia cells, and (2) abnormalities affecting the proliferation and apoptosis of leukemia cells through interaction with bone marrow stroma. This discussion is restricted to a subset of the abnormalities directly affecting the proliferation and apoptosis of leukemia cells, namely FLT3 gene mutations and NPM1 gene mutations.

FLT3 gene mutations

The FMS-like receptor tyrosine kinase 3 (FLT3), also known as fetal liver kinase 2 (FLK-2) and stem cell tyrosine kinase 1 (STK-1), belongs to a class III receptor tyrosine kinase (TK) family. [10] The human FLT3 gene is located at chromosome 13q12 and contains 24 exons. [11]  FLT3 encodes a protein, which exists in two forms: a 158- to 160-kd membrane-bound protein glycosylated at N-linked glycosylation sites in the extracellular domain, and a 130- to 143-kd non–membrane-bound unglycosylated protein. [12]

FLT3 is preferentially expressed by hematopoietic stem cells and is also expressed in the brain, placenta, and liver. [13] The FLT3 ligand, which is expressed as a membrane-bound or soluble form by bone marrow stromal cells, stimulates stem cells by itself or in cooperation with other cytokines. [12, 14, 15, 16, 17]  FLT3 is also expressed on the surface of a high proportion of AML cells. [18, 19, 20]

Two unique forms of the FLT3 gene mutation have been described: (1) an internal tandem duplication in the juxtamembrane domain-coding sequence (FLT3/ITD), [21] and (2) a missense point mutation at the D835 residue and point mutations, deletions, and insertions in the codons surrounding D835 within the FLT3 TK domain (FLT3/KDM). [22, 23, 24, 25, 26, 27, 28]  FLT3/ITD and FLT3/KDM mutations occur in 15-35% and 5-10%, respectively, of adults with AML; they are associated with a poor prognosis. [29, 30, 31, 32, 33, 34] In addition, extremely high levels of FLT3 transcripts have been demonstrated in a proportion of patients with AML who do not have FLT3 mutations; the presence of such transcripts was also associated with a poor prognosis. [35] Rarely, both types of mutations occur simultaneously in AML; this may be related to clonal progression. [36]

FLT3 mutations are mainly found in de novo cases of AML and are less frequent in cases of AML that are associated with myelodysplastic syndrome (MDS) or that occur as a consequence of therapy. [34] The frequency of FLT3 mutations in patients with AML has been associated with patient age. For example, FLT3/ITD mutations were found in about 25% of all adult patients, but they were more prevalent in patients older than 55 years (31.4%). [24, 37] In contrast, FLT3/ITD mutations have been found in approximately 10% of pediatric patients [38, 39, 40, 41] ; they are rarely seen in infants with AML. [42]

FLT3/ITD mutations are strongly associated with leukocytosis and an increase in the percentage of blast cells in the peripheral blood and bone marrow of patients with AML. [43, 44] However, the relationship between FLT3/KDM mutations and leukocytosis remains controversial. Only one study has demonstrated significant leukocytosis in adult patients with AML with FLT3/KDM mutations. [25]

Several large-scale studies have demonstrated the impact of FLT3 mutations on patients' clinical outcomes. [34, 45]  FLT3/ITD mutations have been found to be a strong adverse predictive factor for overall survival, disease-free survival, and event-free survival within the intermediate-risk cytogenetic category. 

NPM1 gene mutations

The NPM1 gene is located at band 5q35 and contains 12 exons. [46, 47]  NPM1 encodes three alternatively spliced nucleophosmin (NPM) isoforms: B23.1, B23.2, and B23.3. NPM is a highly conserved phosphoprotein that is ubiquitously expressed in tissues. [46, 47] Although the bulk of NPM resides in the granular region of the nucleolus, it shuttles continuously between the nucleus and the cytoplasm. [46, 47, 48] Except for two cases involving splicing the donor site of NPM1 in exon 9 or exon 11, [49] all reported NPM1 mutations occur in exon 12. They typically result in an elongation of the NPM protein remaining in the cytoplasm; this change is relatively specific for AML, whereas almost all other human neoplasms consistently show nucleus-restricted NPM expression. [50]  NPM1 mutations are characteristically heterozygous and retain a wild-type allele.

NPM1 functions both as an oncogene and a tumor-suppressor gene, depending on the gene dosage, expression levels, interacting partners, and compartmentalization. [51] NPM plays a central role in cell growth and proliferation through its involvement in ribosome biogenesis. Its expression increases in response to mitogenic stimuli, and above-normal amounts are detected in highly proliferating and malignant cells. However, NPM1 contributes to growth-suppressing pathways through its interaction with the alternate reading frame (ARF).

Although NPM1 mutations have been described in a few patients with chronic myelomonocytic leukemias and MDS, many patients with NPM1 mutations rapidly progress to overt AML. [52, 53, 54]  NPM1 mutations are common in de novo cases of AML; they are associated with a normal karyotype and do not occur in core binding factor leukemias or acute promyelocytic leukemia [50, 55] ; AML secondary to myeloproliferative disorders/MDS and therapy-related AML rarely show cytoplasmic NPM. [56]

NPM1 mutations occur in 9.0% to 26.9% of all children with AML with a normal karyotype. In adult patients with AML, the incidence of NPM1 mutations ranges from 25.0% to 35.0%, accounting for 45.7% to 63.8% of all adult patients with AML with a normal karyotype. [46, 47, 54, 57, 58, 59, 60, 61]

AML cases associated with NPM1 mutations show a wide morphologic spectrum. NPM1 mutations are more frequent in the acute myelomonocytic and monoblastic/monocytic AML subtypes [54, 58, 62] and in AMLs with prominent nuclear invaginations (ie, "cuplike" nuclei). [63] More than 95% of AML cases associated with NPM1 mutations test negative for CD34. [50]  NPM1 mutations have been demonstrated in several cell lineages (ie, myeloid, monocytic, erythroid, and megakaryocytic but not lymphoid). [64]

As noted above, several studies have shown a strong correlation between NPM1 mutations and FLT3-ITD. [57, 58] Patients with AML that is associated with NPM1 mutations with a normal karyotype often have high blast counts, high FLT3/ITD and lactic dehydrogenase (LDH) serum levels, extramedullary involvement (mainly gingival hyperplasia and lymphadenopathy), and elevated platelet counts. [57, 58]

Bone marrow biopsy specimens from patients with AML that is associated with NPM1 mutations frequently show an increase in the number of megakaryocytes that exhibit dysplastic features. [65] After induction therapy, patients with AML who have a normal karyotype but who carry NPM1 mutations achieve higher rates of complete remission than similar patients who do not harbor an NPM1 mutation.


Prognosis Factors

A summary of the clinical relevance of genetic abnormalities is outlined below.

Abnormalities associated with a favorable clinical course include the following [66, 67] :

  • NPM1 gene mutations (in the absence of FLT3/ITD)

The following are abnormalities associated with a dismal clinical course:

  • MLL (KMT2A) gene rearrangement
  • t(6;9)(p23;q34); DEK-NUP214
  • inv(3)(q21 q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
  • t(1:22)(p13;q13); RBM15-MKL1
  • FLT3 gene mutations
  • RAS gene mutations
  • KIT gene mutations
  • p53 gene mutations
  • ERG gene mutations
  • MN1 gene mutations
  • CXCR4 overexpression
  • BAALC overexpression