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18
CHAPTER
Acute Myeloid Leukemia

Kathryn Foucar, MD [18.1] Definition 377
[18.2] Epidemiology and Pathogenesis 379
Kaaren Reichard, MD
[18.3] Key Steps in the Diagnosis of AML 379
David Czuchlewski, MD [18.4] Blasts and Blast Equivalents 380
[18.5] Classification of AML 385
[18.6] Biologic Subtypes of AML 385
[18.7] AML, Not Otherwise Specified (NOS) 408
[18.8] Prognostic Factors in AML 417
[18.9] Diagnostic Pitfalls in AML Diagnosis 419
[18.10] Minimal Residual Disease, Post-Therapy Changes, and
Relapse 422
[18.11] Differential Diagnosis of AML 422
[18.12] Components of the Diagnostic Interpretation 423
[18.13] Clues and Caveats 424
[18.14] References 424
377
in 2 clinical situations (1.prior cytotoxic chemotherapy

and/or radiation therapy and 2. Down syndrome), cases
T
he successful diagnosis of acute myeloid leukemia with features of myelodysplastic syndrome and those with
(AML) has never been an easy task for the practicing features of AML are lumped together as myeloid neoplasms
pathologist, and the stakes have always been high. without regard to blast count, as there are no significant
Not only are the morphologic features of a case often clinical or biologic differences in behavior tied to exceeding
challenging, but major, urgent, potentially life-threatening the 20% threshold. These basic definitional considerations
treatment decisions hinge on this diagnosis. In acute are summarized in f18.1.
promyelocytic leukemia (APL), the challenge is even
greater, because any delay, even a few hours, will postpone
notification of the clinician that the patient is at high risk
for a potentially catastrophic hemorrhage. Consequently, pathologically increased
the pathologist is faced with the expectation of providing a myeloid blasts present
correct diagnosis as rapidly as possible.
Recent biologic insights in AML have even more dramat-
ically expanded expectations of pathologists, with the advance
from traditional morphologic and immunophenotypic blast percentage Y
evaluations to a biologic assessment of AML. This transition 20%?
requires that the pathologist understand pathogenetic features N
of AML, since these mechanistic parameters are directly
linked to prognosis, curability, many distinctive clinical
and morphologic features, optimal therapy, and appropriate blast equivalents present? Y
minimal residual disease monitoring. The 2008 WHO classi-
fication of AML has set a new diagnostic standard in AML N
diagnosis by utilizing a recurrent genetic abnormality as the
defining feature for many types of AML [Vardiman 2008b]. Thus, blasts +
the state-of-the-art diagnosis of AML requires an integration blast equivalents
N 20%? Y
of clinical, hematologic, morphologic, immunophenotypic,
and genetic features. This process must occur in a systematic
fashion and must be logical and cost effective.
t(8;21), inv(16), Y
t(16;16) or t(15;17)?
[18.1] Definition
N
Acute myeloid leukemia is a clonal hematopoietic
disorder that may be derived from either a hematopoietic
stem cell or a lineage-specific progenitor cell. AML is charac-
terized both by a predominance of immature forms (with E 50% and Y
variable, but incomplete, maturation) and loss of normal 20% non-E blasts?
hematopoiesis. Single or multiple hematopoietic lineages N
may comprise the leukemic clone. The requisite blast/blast
equivalent percentage is 20% in the peripheral blood and
bone marrow; a lower percentage is acceptable in cases with probable MDS, MPN
AML-defining translocations and in acute erythroid leukemia or MDS/MPN* go to f18.2
(see [18.4] Blasts and Blast Equivalents, p 380 for discussion of (p 385)
blasts/blast equivalents) [Vardiman 2008b]. f18.1 Definition of AML algorithm.
The blast percentage is derived from counting all *Note that the MDS-like state in patients with Down syndrome <5 years of age or in
nucleated cells for AML diagnosis with the exception of patients with a prior cytotoxic therapy behaves biologically as AML regardless of blast
acute erythroid leukemias in which the blast percentage count; see Chapters 16 & 17 for further assessment
is based on non-erythroid cells. In extramedullary sites, AML= acute myeloid leukemia; E = erythroid cells; MDS = myelodysplastic syndrome;
a diagnosis of myeloid sarcoma is equivalent to AML MPN = myeloproliferative neoplasm
regardless of blood and bone marrow findings. Finally,
18: A CUTE MYELOID L EUKEMIA
378
t18.1 Selected Risk Factors for AML

Risk Factor Comments
Inherited Genetic Disorders*

Down syndrome Acute leukemia 10 - 20 times that of individuals without trisomy 21
Elevated risk of acute megakaryoblastic leukemia (~600 times normal)
Transient abnormal myelopoiesis is a spontaneously remitting leukemia-equivalent unique to Down syndrome
patients
Severe congenital Cumulative incidence of MDS and AML after 15 years of G-CSF therapy is 34%; risk is 11% at age 20
neutropenia Transformation is particularly associated with the acquisition of secondary mutations to the gene encoding the G-CSF
receptor
Cyclic neutropenia does not appear to share a significant risk of progression to MDS or AML
Shwachman-Diamond Estimated incidence of MDS/AML is 19% at 20 years of age and 36% at 30 years, perhaps due to mitotic spindle
syndrome destabilization in the absence of SBDS protein and consequent chromosomal instability
Dyskeratosis Head and neck squamous cell carcinomas dominate the cancer risk; however, the ratio of observed to expected
congenita cases of AML is 195:1 [Alter 2009]
Fanconi anemia Ratio of observed to expected incidence of AML is 868:1
Risk of early transformation is increased in D1 complementation group and in the Ashkenazi Jewish population
Environmental Exposures
Ionizing radiation Linked to the risk of secondary AML
Benzene exposure Strong dose-response relationship with the development of AML
Benzene-related cases may be reminiscent of therapy-related AML, a finding that recalls the use of benzene as an
early form of chemotherapy
Cigarette smoking Slightly increased risk of AML (average relative risk = 1.6); positive dose-response relationship with the number of
cigarettes smoked; cigarette smoke contains benzene, which may be responsible for the increased risk
Pesticides/herbicides Slightly increased risk of AML (meta-relative risk: 1.55)
Chemotherapy
Topoisomerase II Agents include etoposide and teniposide
inhibitors Developing t-AML after such epipodophyllotoxins ~2% - 12%, depending on regimen-related factors (see t18.6)
Alkylating Agents include: melphalan, cyclophosphamide, nitrogen mustard, chlorambucil, busulfan, carboplatin, cisplatin,
chemotherapy dacarbazine, procarbazine, carmustine, mitomycin C, thiotepa, and lomustine
Early regimens: relative risks for secondary AML exceed 300
Less toxic regimens (eg, ABVD): somewhat decreased the risk (see t18.6)
Anthracyclines and Agents include daunorubicin, doxorubicin, and mitoxantrone (intercalating agents, but also act as topoisomerase II
anthracenediones inhibitors)
Increased risk of AML may be attributable to the latter mechanism of action
Risk of secondary AML with these agents is less than with strong topoisomerase II inhibitors such as the
epipodophyllotoxins, but incidence still substantial (1 study found relative risk with anthracycline therapy to be on
the order of 2.7)
Anti-tubulin agents Agents include taxanes, vincristine, and vinblastine (frequently administered with other classes of chemotherapeutic
agents)
Some anecdotal reports suggest an increased risk with taxanes, but other studies show no increase in incidence
when taxanes are added to an anthracycline-based regimen
WHO 2008 classification endorses these drugs as cytotoxic agents implicated in therapy-related hematologic
neoplasms
*Many other inherited disorders are more weakly associated with increased risk for AML, including Li-Fraumeni syndrome, neurofibromatosis, Noonan
syndrome and Klinefelter syndrome; patients with congenital amegakaryocytic thrombocytopenia are also at some increased risk of developing AML
~1/2 reported cancers associated with Diamond-Blackfan anemia are AML; the rarity of these bone marrow failure syndromes somewhat hinders precise
risk quantification
ABVD = adriamycin (doxorubicin), bleomycin, vinblastine, and dacarbazine; AML = acute myeloid leukemia; G-CSF = granulocyte colony-stimulating factor;
MDS = myelodysplastic syndrome
References: [Alter 2009, Belson 2007, Bhatia 1996, Burroughs 2009, Cimino 1991, Dale 2003, Donadieu 2005, Felix 1998, Freedman 2002, Geddis 2009, Green 2009, Hijiya 2009, Le Deley 2007, Leone 2007, Lipton 2006, Natelson 2007, Patt 2007,
Rosenberg 2008a, 2008b, Van Maele-Fabry 2007, Vineis 2004, Xavier 2009, Yeasmin 2008]

379
[18.2] Epidemiology and Pathogenesis t18.2 Class I and Class II Mutations in AML
The age-adjusted incidence of AML in the United
Class I Mutations Class II Mutations
States is 3.4 cases per 100,000 persons [Deschler 2006]. AML can (Proliferation) (Impaired Differentiation)
occur in patients of any age, but in general, both the overall FLT3 PML-RARA
incidence and the proportion of total acute leukemias that
KIT RUNX1-RUNX1T1
are myeloid increase with age. Thus, acute lymphoblastic
RAS CBFB-MYH11
leukemia (ALL) predominates in children, with only one
PTPN11 MLL fusions
case of AML diagnosed for every 5 cases of ALL [Belson 2007].
For childhood AML, peak incidence occurs in the first year JAK2 CEBPA
of life, then decreases until age 4, and thereafter remains *The mechanism of leukemogenesis for NPM1 mutations remains
somewhat unclear
relatively constant until adulthood [Gurney 1995]. The incidence of
AML then increases through adulthood, during which period Reference: [Vardiman 2008b]
70% - 80% of acute leukemias are AML, with a marked spike
in incidence in the elderly. Much of this increased incidence
myeloproliferative process rather than outright leukemia.
is attributable to AML with myelodysplasia-related changes,
However, leukemia supervenes when both abnormalities
which becomes more common with age, while the incidence
are present in the mice simultaneously [Grisolano 2003]. This and
of de novo AML remains approximately constant across all
similar observations have given rise to the designation of
adult age groups [Deschler 2006].
AML-associated molecular genetic changes as either Class I
One of the central themes in this chapter will be that
or Class II, with the former conferring increased proliferation
AML may best be considered an umbrella term for a hetero-
and the latter contributing to arrested hematopoietic differen-
geneous group of myeloid leukemias that differ substantially
tiation t18.2. This distinction is largely irrelevant for diagnostic
in cause, age of onset, clinical features, and prognosis. For
purposes, but it does underscore the important idea that
example, AML with t(1;22)(p13;q13); RBM15-MKL1
cooperating alterations in multiple pathways are usually
is a de novo megakaryoblastic AML arising in infants and
present in AML.
very young children, while megakaryoblastic leukemia
without the t(1;22) often arises in children in the setting of
Down syndrome. Epidemiologic and pathogenetic hetero-
[18.3] Key Steps in the Diagnosis of AML
geneity is the necessary consequence of the increasingly fine
An attempt to outline key steps in the diagnosis of AML
distinctions among subtypes of AML. Despite this hetero-
is made in t18.3 (general approach strategies to all myeloid
geneity, we can make some general observations about the
neoplasms are presented in Chapter 13). Although some of
disease states and environmental exposures that appear to
these steps are performed simultaneously or are overlapping,
increase an individuals risk for developing AML of various
it is important for the diagnostician to understand the
subtypes. These are listed and discussed in t18.1. The risk
importance of basic CBC information in determining the
of AML is also substantially increased in patients with
likelihood that a patient has AML. This is especially important
other hematopoietic disorders, including myelodysplastic
for rapid diagnosis of AML and appropriate utilization of
syndrome (MDS), some myeloproliferative neoplasms
specialized tests. Although the WBC is highly variable in
(MPNs), MDS/MPN overlap syndromes, aplastic anemia,
AML, it is essential for the diagnostician to assess for hemato-
and paroxysmal nocturnal hemoglobinuria, in which case
poietic failure (ie, neutropenia, anemia, and thrombocy-
the development of AML may be due to progression of the
topenia). Although preservation of a single hematopoietic
underlying disease.
lineage may be observed rarely, hematopoietic failure is an
In general, AML arises following the accumulation
expected finding in AML.
(through inherited genetic mechanisms, environmental
If hematopoiesis is preserved (normal absolute neutrophil
influences, sheer random chance, or some combination
count, RBC parameters, and platelet count), a diagnosis
of these) of specific translocations, mutations, and other
of AML is very unlikely, and other diagnostic possibilities
genetic alterations. Crucially, leukemogenesis often
should be considered. In our experience, the rare examples of
requires the acquisition of several cooperating genetic
exceptions to this general rule have occurred in circumstances
lesions. For example, the introduction of the fusion protein
in which the blood and bone marrow examinations were
RUNX1-RUNX1T1, formed by the t(8;21)(q22;q22), into
performed for reasons unrelated to hematologic dysfunction,
mice is insufficient to cause leukemia [Mrozek 2008b]. Similarly,
and early/partial involvement by acute leukemia was
introduction of an abnormally activated tyrosine kinase,
detected.
which provides a pro-proliferative signal, results in a
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t18.3 Acute Myeloid Leukemia: Diagnostic Steps*

Simultaneously with CBC assessment, the blood smear
should be reviewed systematically for blasts, blast equivalents,
1. Evaluation of an abnormal CBC for possible AML (usually step 1
for the pathologist) other abnormal cells, and dysplasia (all hematopoietic lineages).
Confirm HP failures, assess for blasts/blast equivalents (see step The identification, lineage determination, and enumeration
2), and dysplasia of blasts/blast equivalents is also an integrative process that
WBC: non-specific; in AML can be low, normal, or high encompasses routine morphology, cytochemical staining,
ANC: severe neutropenia characteristic of HP failure; typical in
AML, but exceptions occur and flow cytometric immunophenotyping on blood or bone
Circulating blasts: variable number and percent in AML, but key marrow (see [18.4] Blasts and Blast Equivalents). Throughout
feature to assess in blood this diagnostic process, the pathologist must remain aware of
RBC features: severe anemia characteristic of HP failure, an
expected feature of AML both the urgency in rapid diagnosis of APL and the numerous
Polychromasia: reduced, since anemia is result of bone diagnostic pitfalls/key problem areas in AML diagnosis (see
marrow production failure [18.9] Diagnostic Pitfalls in AML Diagnosis, p 419).
Other RBC pathology: non-specific
Platelets: severe thrombocytopenia characteristic of HP failure The molecular genetic data essential for the diagnosis of cases
2. Identify morphologic blasts and blast equivalents in blood (and of AML with recurrent genetic abnormalities is often delayed
subsequent bone marrow, if performed) compared to rapid morphology, cytochemical, and immuno-
Morphologic assessment of nuclear features key for blast phenotypic assessment. Consequently, a final integrated report
designation may not be generated for several days to several weeks. However,
Distinguish from blast look-alikes and other immature
hematopoietic cells fluorescence in situ hybridization (FISH) testing offers a more
Consider APL specifically; alert clinician if suspected based on rapid turnaround for assessment of specific genetic abnormalities
morphology of immature cells and CBC data
Cytochemistry, flow cytometric immunophenotyping, genetics can depending upon probe availability. FISH testing is ideal for rapid
all be performed on blood as needed assessment of AML-defining translocations, including t(15;17)
3. Bone marrow examination often performed to address differential (q22;q21)* in APL, which results in the fusion of PML-RARA.
diagnoses from blood assessment or for protocol requirements A lineage-based classification can be used for an initial diagnosis
4. Determine lineage of blasts/blast equivalents (can be performed if data supporting a specific biologic subtype is not available.
on blood or bone marrow)
However for the most part, with the exception of APL, lineage-
Morphology (nucleus and cytoplasm)
Cytochemistry based classification alone is not predictive of genotype. In addition,
Immunophenotype in the absence of a diagnostic biologic parameter, the diagnostician
Consider APL specifically and alert clinician if suspected must exclude many differential diagnostic considerations. This can
5. Enumeration of blasts/blast equivalents by morphology and be especially problematic when specimens are suboptimal, fibrotic,
differential cell count
necrotic, or show a blast percentage approximating 20%.
Blood (percentage; threshold 20%)
Bone marrow (percentage; threshold 20%): blast count <20%, but Frequent discussions with the clinical team are essential
Auer rods or other distinctive morphology suggests AML throughout the diagnostic process. Particular attention must
Unique situations compromising blast count: be paid to rapid communication about possible APL, including
Fibrosis and/or necrosis
Predominance (50%) of erythroid lineage recommendations for coagulation assessment. This dialogue
Marked hypocellularity with the clinical team is also essential to determining factors that
Technically poor specimen can potentially influence the pathologists interpretation, such as
6. Assess all lineages for dysplasia (core biopsy better for antecedent hematologic disorders, prior cytotoxic therapy, and
megakaryocyte assessment)
recent granulocyte colony-stimulating factor (G-CSF) therapy.
7. Identify biologic subtypes of AML based on:
Distinctive morphologic features (eg, multilineage dysplasia)
Recurrent cytogenetic subtypes
Molecular assessment for NPM1, CEBPA, and FLT3 [18.4] Blasts and Blast Equivalents
Prior chemotherapy/radiation Blasts, blast equivalents, and other immature hematopoietic
Antecedent hematologic disorder
Constitutional disorder (eg, Down syndrome or other
cells must be identified and distinguished from various blast
constitutional disorders) look-alikes t18.4. For AML diagnosis, blasts include myeloblasts,
8. For cases lacking diagnostic biologic features, exclude other monoblasts, and megakaryoblasts, while promonocytes are
differential diagnostic possibilities blast equivalents in all types of AML and promyelocytes are
9. Lineage-based classification for AML, NOS cases lacking blast equivalents exclusively in acute promyelocytic leukemia.
identifiable biologic features
*Some diagnostic steps overlapping and/or simultaneous *The t(15;17) PML-RARA translocation has been historically denoted as both
ANC = absolute neutrophil count; APL = acute promyelocytic leukemia; t(15;17)(q22;q21) and t(15;17)(q22;q12). Currently the most accurate
CBC = complete blood count; HP = hematopoietic; NOS = not otherwise description is actually thought to be t(15;17)(q24;q21). For the sake of simplicity
specified; RBC = red blood cell; WBC = white blood cell and consistency, we have chosen to use the t(15;17)(q22;q21) designation.

381
t18.4 Blasts, Blast Equivalents, Other Immature Cells in Blood and Bone Marrow
Cell Type Key Morphologic Features Cytochemistry Immunophenotype/Comments*
Myeloblast Large nucleus with finely dispersed chromatin and variably MPO+ CD34+, CD13+, CD33+, MPO+, HLA-DR+,
prominent nucleoli vCD11c+, wCD45+, CD117 usually +
Relatively high nuclear/cytoplasmic ratio
Variable number of cytoplasmic granules, may be
concentrated in limited portion of cytoplasm
Promyelocyte Nuclear chromatin slightly condensed; nucleoli variably Strong, uniform CD13+, CD33+, MPO+, wCD45+, CD34,
prominent; nucleus often eccentric, and Golgi zone may MPO+ HLA-DR
be apparent
Loss of HLA-DR and acquisition of strong
Numerous cytoplasmic granules that may be more CD15 and CD11c associated with maturation
dispersed throughout cytoplasm
Gradual loss of CD33 also characterizes
Blast equivalent in APL only successive maturation stages
In APL, intense cytoplasmic granularity usually present CD34 usually negative in hypergranular
Nuclear configuration variable, but nuclear folding and variant; often positive in microgranular
lobulation characteristic of microgranular variant of APL variant
Monoblast Moderate to low nuclear to cytoplasmic ratio, nuclear NSE+ CD34, HLA-DR+, CD13+, CD33 bright +,
chromatin finely dispersed with variably prominent CD36/CD64 coexpression, vCD4+, CD11c+,
nucleoli; nuclei round to folded wCD45+
Abundant, slightly basophilic cytoplasm containing fine Usually CD34
granulation and occasional vacuoles
Occasional cases moderate CD45+
Promonocyte Slightly condensed nuclear chromatin; variably prominent NSE+ CD36/CD64 coexpression, HLA-DR+, CD13+,
nucleoli CD33 bright +, vCD14+, CD4+, CD11c+,
Abundant finely granular blue/gray cytoplasm that may be CD45+
vacuolated
Very monocytic appearance with nuclear immaturity
Consistent blast equivalent in AML
Erythroblast Relatively high nuclear/cytoplasmic ratio PAS+ Glycophorin A+, hemoglobin A+, CD71+,
Nucleus round with slightly condensed chromatin; nucleoli CD34, CD45, MPO, myeloid antigens
variably prominent negative
Moderate amounts of deeply basophilic cytoplasm that may CD117 often positive
be vacuolated
Included in blast percentage only in acute erythroid
leukemia
Megakaryoblast Highly variable morphologic features; often not NA CD34, CD41+, CD61+, CD33 bright +, CD13,
recognizable without special studies HLA-DR (or dim)
May be lymphoid-appearing with high nuclear to Progressive maturation characterized by loss
cytoplasmic ratio of CD34 and acquisition of CD42 and von
Nuclear chromatin fine to variably condensed Willebrand factor
Cytoplasm may be scant to moderate, is usually agranular
or contains a few granules; blebbing or budding of
cytoplasm may be evident
Blasts may form cohesive clumps
Blastic plasmacy- Variable morphology; not identified in either normal bone MPO CD4+, CD56+, CD123+, CD43+, CD45+,
toid dendritic cell marrow or AML HLA-DR+, vTdT+
Often not recognizable without immunophenotyping NSE CD123 not specific; seen in subset of AMLs
May be lymphoid-appearing (Discussed in detail in Chapter 19)
May show cytoplasmic tadpoles
*Key flow cytometric clues to lineage assignment in boldface
Rare AML lacks surface CD13 and CD33
False positive CD41 expression by flow cytometry may be secondary to platelet adherence to blasts
See Chapter 19
AML = acute myeloid leukemia; APL = acute promyelocytic leukemia; MPO = myeloperoxidase; NSE = non-specific esterase; PAS = periodic acid-Schiff;
v = variable antigen expression; w = weak antigen expression
References: [Dohner 2010, Vardiman 2008b]

382
a b c
i18.1 This bone marrow aspirate composite illustrates the spectrum of i18.2 Bone marrow aspirate smear from a patient with acute myeloid leukemia
cytoplasmic granulation ranging from agranular a to granular (b and c) includes both granular and agranular blasts. Note Auer rod (upper left cell).
blast. Myeloblasts with granules lack other features of promyelocytes such as a (Wright)
prominent paranuclear hof. (Wright)
i18.3 Megakaryoblasts show prominent cytoplasmic blebbing in a peripheral i18.4 Several erythroblasts with deeply basophilic cytoplasm are present on this
blood smear. (Wright) bone marrow aspirate smear. (Wright)
Erythroblasts are included in the blast percentage only in acute

pure erythroid leukemia. Nuclear morphologic features are
most critical in the decision that a cell is a blast exhibiting finely
dispersed rather than condensed nuclear chromatin. Other
useful nuclear features of blasts to assess include overall size,
nucleoli, and nuclear configuration. Cytoplasmic features are
very helpful in lineage determination, ie, sparse fine granules
and Auer rods in myeloblasts, cytoplasmic blebbing in
megakaryoblasts, and deeply basophilic, vacuolated cytoplasm
in erythroblasts t18.4, i18.1, i18.2, i18.3, i18.4, i18.5. As their
name implies, blastic plasmacytoid dendritic cell neoplasms
can be derived from cells with morphologic features of blasts.
These cells are not evident in normal bone marrow and are
i18.5 The erythroblasts in this case of acute pure erythroid leukemia account
not generally included in the discussion of immature cell
for 80% of cells. These erythroblasts show deeply basophilic cytoplasm with populations that need to be identified in cases of possible AML.
prominent vacuolization. (Wright) However, for completeness, blastic plasmacytoid dendritic cells

383
i18.6 A promyelocyte with an eccentric nucleus, a paranuclear hof, and abundant i18.7 A spectrum of myeloblasts is present in this bone marrow aspirate smear
cytoplasmic granules is present in the center of this bone marrow aspirate smear including 2 blasts with Auer rods. (Wright)
from a patient with acute myeloid leukemia with maturation. Promyelocytes are
not included within the blast percentage for this type of AML. (Wright)
a b
i18.8 This bone marrow aspirate smear illustrates 4 myeloblasts, one of i18.9 This composite shows a spectrum of unique features of myeloblasts,
which contains pseudo Chdiak-Higashi granules. This granular blast is not a including a myeloblast with deeply basophilic and sharply vacuolated cytoplasm
promyelocyte, because it lacks a prominent paranuclear hof. (Wright) resembling Burkitt lymphoma a, while distinct clumping of myeloblasts mimics a
metastatic tumor b. (Wright)
are highlighted in t18.4, but the reader is referred to Chapter 19
for comprehensive discussion and illustrations.
Myeloblasts can be morphologically diverse, and the
distinction between a granular blast and a promyelocyte with
a distinct paranuclear hof (Golgi zone) is key i18.1, i18.2, i18.6.
Auer rods are a key cytologic feature of myeloblasts, although
Auer rods can be seen in promyelocytes in APL and even rarely
in mature cells i18.7. Other unique features of myeloblasts
include pseudo Chdiak-Higashi granules, nuclear indentation,
deeply basophilic cytoplasm with vacuoles, and, rarely,
clumping on aspirate smears mimicking a metastatic process
i18.8, i18.9. Associations of some of these unusual morphologic
features with immunophenotypic and molecular genetic
properties have been reported [Chang 2006, Kussick 2004]. Cytochemical i18.10 Cytochemical staining for myeloperoxidase illustrates a spectrum of scant
staining for myeloperoxidase is important in establishing to moderate positivity, which is characteristic of early granulocytic maturation in
the lineage of myeloblasts i18.10. Features of AML on bone cases of acute myeloid leukemia. (myeloperoxidase cytochemical stain)

384
i18.11 This bone marrow core biopsy section is effaced by a homogeneous i18.12 Monoblasts (right lower) and promonocytes (upper right and upper
infiltrate of acute myeloid leukemia cells. Note the large nuclear size, variably left) are evident in this peripheral blood smear from a patient with acute
prominent nucleoli, and moderately abundant amounts of cytoplasm. (H&E) monocytic leukemia. Promonocytes are blast equivalents. (Wright)
i18.13 This bone marrow aspirate smear highlights 2 promonocytes (upper i18.14 Intense reddish brown cytoplasmic nonspecific esterase positivity is
cells) and an immature, atypical monocyte (lower cell) in a case of acute evident in this acute monocytic leukemia. (cytochemical stain for nonspecific
monocytic leukemia. Promonocytes are blast equivalents. (Wright) esterase)
marrow core biopsy sections are also heterogeneous, but may show falsely low blast percentages due to hemodi-
prototypic findings include a predominance of immature lution or falsely elevated blast percentages due to lysis of
cells with dispersed chromatin, variably prominent nucleoli, erythroid cells. Furthermore, many types of blasts (erythro-
and moderate amounts of eosinophilic cytoplasm i18.11. blasts, monoblasts, megakaryoblasts) and blast equivalents
Monoblasts and promonocytes (blast equivalents) must (promyelocytes, promonocytes) typically lack CD34
also be successfully identified based on morphologic and expression.
cytochemical features i18.12, i18.13, i18.14. Based on morphologic differential cell counts, the blast
Flow cytometric immunophenotyping (and to a lesser threshold in blood and bone marrow for AML diagnosis
extent immunohistochemical staining) is a mainstay in is 20% [Vardiman 2008b]. However, as noted earlier, there are
assessing immaturity (weak CD45, CD34, CD117) and exceptions to this general rule f18.1, and the blast/blast
lineage in AML t18.4. Integration of morphology, limited equivalent percentage must always be assessed in the overall
cytochemistry, and immunophenotype is optimal in AML context of a given case. Determining an accurate blast/
diagnosis t18.4, t18.5. However, the actual blast percentage blast equivalent percentage can be especially challenging in
is best assessed by morphology, and the percentage of technically suboptimal, necrotic, or fibrotic specimens (see
CD34+ cells should not substitute for the morphologic [18.9] Diagnostic Pitfalls in AML Diagnosis, p 419). In these
percentage. Keep in mind that flow cytometry specimens situations, immunohistochemical techniques may be helpful

385
in determining lineage and immature cell populations on
tissue sections. Similarly, cases with a marked expansion of
the erythroid lineage can be difficult to diagnose (see [18.9] continued from
Diagnostic Pitfalls in AML Diagnosis, p 419). Key features f18.1 (p 377)
to assess include the overall percentage of erythroid lineage
cells, the degree of maturation/differentiation of the
erythroid lineage, the percentage of myeloid blasts among Down syndrome Y probable transient
non-erythroid cells, and the extent of dysplasia. in neonate/infant? abnormal myelopoiesis
N
[18.5] Classification of AML Down syndrome Y myeloid leukemia associated

In most cases, the integration of traditional clinical, in child <5 years of age? with Down syndrome
hematologic, morphologic, cytochemical, and immuno- N
phenotypic data results in a confident diagnosis of AML.
If a patient has Down syndrome, has a known history of prior cytotoxic therapy? Y therapy-related
antecedent myelodysplasia, or has had prior chemotherapy/ (see t18.1) myeloid neoplasm
radiation therapy, additional biologic information can be N
provided at the time of traditional assessment. However,
the biologic classification of AML will often hinge upon AML with recurrent genetic
recurrent cytogenetic Y
molecular genetic studies. If a positive molecular genetic abnormality (subclassify
abnormality? (see t18.1)
result is obtained, the AML will be subcategorized among the for the specific abnormality)
N
9 molecular genetic subtypes in the 2008 WHO classification
t18.5. If non-contributory molecular genetic and clinical
results are obtained, the case will be subclassified based MDS related Y AML with myelodysplasia-
on traditional lineage-based criteria (see [18.7] AML, Not changes? related changes
Otherwise Specified (NOS), p 408). N
NPM1 or CEBPA AML with recurrent genetic

Y
[18.6] Biologic Subtypes of AML mutation? abnormality (subclassify
by the specific mutation)
As illustrated in f18.2 and t18.5, there are essentially 2 N
ways to categorize specific biologic subtypes of AML: genetic
findings and clinical setting. Many details pertaining to these continue
subtypes are listed in t18.5, which serves as a quick reference to t18.5
guide for this section. In this section we will describe in some
detail those cases of AML categorizable by clinical setting, as
well as the genetically defined subtypes of AML. f18.2 Flow chart for classification of AML by biologic subtypes.
AML= acute myeloid leukemia; MDS = myelodysplastic syndrome
[18.6.1] Molecular Genetic Biologic Subtypes of AML
Core Binding Factor Acute Myeloid Leukemias contribute to leukemogenesis by dysregulating the normal
Core binding factor (CBF) AML includes AML with CBF transcriptional activity [Downing 2003, Helbling 2005].
t(8;21) and AML with inv(16)/t(16;16). These 2 AML
cytogenetic groups are often grouped together based on AML with t(8;21)(q22;q22); RUNX1-RUNX1T1
involvement of related CBF machinery and relatively overall AML with t(8;21)(q22;q22) is defined by the presence
favorable prognosis [Marcucci 2005]. The CBF genes are RUNX1 of this translocation, a variant translocation, or molecular
(21q22, aka AML1, CBFA2) and CBFB (16q22). As part of RUNX1-RUNX1T1 fusion, regardless of blast count f18.3
the CBF heterodimer transcription factor complex, RUNX1 [Arber 2008a]. Clues and confirmatory tests for this distinct type of
binds to DNA promoter sequences of genes needed for AML are listed in t18.5.
hematopoiesis, while CBFB protects the complex from The peripheral blood findings are variable, but circulating
proteolysis [Okumura 2008, Paschka 2008a, Speck 2002]. The chimeric proteins, blasts accompanied by cytopenias are common i18.15.
produced as a result of the chromosomal rearrangements, Circulating mature granulocytes including neutrophils are a

386
t18.5 Clues and Confirmatory Tests for Biologic Types of AML (continued on next spread)
AML Subtype Clinical Features CBC/Blood Morphology BM Aspirate
Recurrent
Abnormalities
AML t(8;21) Young adults, myeloid sarcomas Cytopenias, circulating blasts Blasts may be <20%
(q22;q22); 10% - 15% of pediatric AML with evidence of neutrophilic Long tapered Auer rods
(RUNX1- 7% of adult AML maturation Salmon-colored cytoplasm, rim of basophilia
RUNX1T1)
Manifests as AML with maturation
AML inv(16) Young adults Although variable, higher WBC Blasts may be <20%
(p13.1;q22) Extramedullary disease and blast counts vs t(8;21) Abnormal eosinophils with admixed
t(16;16) 6% - 12% pediatric AML Monocytic cells, myeloid blasts eosinophil/ basophil granules
(p13.1;q22) 8% adult AML predominate Variable monocytic component
CBFB-MYH11 Cytopenias
AML t(15;17) Young adults Leukocytosismicrogranular Abnormal promyelocytes predominate (blasts

(q22;q21); DIC major complication variant may be <20%)
PML-RARA* 8% - 15% pediatric AML Leukopeniahypergranular Hypergranular variant (majority of cases):
5% - 12% adult AML variant packed with granules, numerous Auer rods
Profound thrombocytopenia in single cell
Rare hyperbasophilic variant Microgranular variant (20% of cases):
reniform, bilobed nuclei with scant to
inconspicuous granules
Strong uniform cytochemical MPO positivity
AML t(9;11) More common in children Anemia, thrombocytopenia Monocytic blasts/blast equivalents
(p22;q23); Extramedullary disease (gingival, skin), Variable WBC count and blasts
MLLT3-MLL DIC
5% - 20% pediatric AML
4% - 5% adult AML
AML t(6;9) Young adults (median age 30 years) Basophilia (2%) in 50% of Background myelodysplasia
(p23;q34); 1% of pediatric and adult AMLs cases May see monocytic features, occasional ring
DEK-NUP214 WBC may be lower than typical sideroblasts
AML
AML inv(3) Possible hepatosplenomegaly Normal or increased platelets Many small, hypolobated megakaryocytes
(q21;q26.2) Giant, hypogranular Multilineage dysplasia
or t(3;3) platelets; circulating, bare
(q21;q26.2) megakaryocytic nuclei
RPN1-EVI1
AML t(1;22) Restricted to infants or young children Megakaryoblasts with Megakaryoblasts with basophilic cytoplasm
(p13;q13); (<3 years of age) without Down basophilic cytoplasm and and cytoplasmic blebbing; smaller blasts
RBM15-MKL1 syndrome cytoplasmic blebbing; smaller may show a more lymphoblastic appearance
Marked organomegaly blasts may show a more Micromegakaryocytes are present
lymphoblastic appearance Granulocytic and erythroid dysplasia not
prominent
AML with Typically, de novo acute leukemia in WBC may be relatively high Many, but not all cases show monocytic
mutated older adult differentiation
NPM1 80% - 90% of acute monocytic leukemias have
mutated NPM1
High blast percentage
AML with Occurs in 6% - 15% of de novo AML; no Relatively high hemoglobin and Most cases show features of AML with or
mutated age or sex predilection blood blast count without maturation; no specific morphologic
CEBPA Relatively low platelet count features
*The t(15;17) PML-RARA translocation has been historically denoted as both t(15;17)(q22;q21) and t(15;17)(q22;q12); currently the most accurate description
is actually thought to be t(15;17)(q24;q21); for the sake of simplicity and consistency, we have chosen to use the t(15;17)(q22;q21) designation
AML = acute myeloid leukemia; APL = acute promyelocytic leukemia; ATRA = all trans retinoic acid; BM = bone marrow; CBC = complete blood count;
DIC = disseminated intravascular coagulation; HP = hematopoiesis; IHC = immunohistochemistry; MDS = myelodysplastic syndrome;
MPN = myeloproliferative neoplasm; MPO = myeloperoxidase; MRD = minimal residual disease; PML = promyelocytic leukemia; WBC = while blood cell count
387
BM Biopsy Flow Cytometry Confirmatory Tests/Other
Hypercellular Blasts: CD34+, CD117+, CD13+, Favorable subtype

Increased blasts with evidence of CD33+, CD19+, CD79a+, TdT+ or Cytogenetics
granulocytic maturation CD56+ KIT activating mutations (12% - 47%) may confer worse overall
Megakaryopoiesis and Frequent subpopulations reflecting survival
erythropoiesis decreased granulocytic maturation in Molecular monitoring of RUNX1/RUNX1T1 for MRD
IHC: PAX5+, CD10+ background
Hypercellular; admixture of immature 2 distinct populations: Favorable subtype
and mature monocytic and blasts (CD34+, CD117+, weak CD45+, Cytogenetics
granulocytic components myeloid antigens +) Molecular or FISH for CBFB-MYH11 if suspect cryptic fusion
monocytic cells (CD45+, bright Trisomy 22 often also seen
CD36/CD64 coexpression, CD33 If +22 identified, may need FISH/molecular if karyotype
bright+, CD34, CD117, HLA-DR+) otherwise normal
KIT mutations (25%); significance unclear
Molecular negativity may be associated with long-term
remission
Hypercellular, dominated by Classically CD34, HLA-DR, CD33 Favorable subtype
cells with obvious granules in bright+ Cytogenetics
hypergranular variant CD2 and CD34 expression common in Molecular for PML-RARA needed in rare cases with negative
Antibodies against PML show a microgranular variant cytogenetics and FISH
nuclear granular pattern vs a CD56 in 20% of cases; associated MRD monitoring (see Chapter 36)
speckled pattern in non-APL with worse outcome
promyelocytes ATRA overcomes maturation block; used in combination with
chemotherapy
Arsenic trioxide also used in therapy of APL
FLT3 mutations (~30% - 40%); likely adverse effect
Hypercellular, monoblasts and Coexpression of CD36/CD64, Intermediate risk
promonocytes HLA-DR+, CD33 bright+, CD4 Cytogenetics
IHC: CD68+, lysozyme+, CD163+ weak+, CD56+, CD45+ FISH for MLL
CD34, CD117
CD14 variable, often negative/dim
Background myelodysplasia (>60% Typical myeloid phenotype: Poor prognosis
of cases) CD45 weak+, CD34+, CD117+, CD13+, Cytogenetics ( abnormality may be overlooked)
CD33+ FLT3-ITD+ in ~70% - 80%
Small, hypolobated megakaryocytes Blasts typically express CD34, CD13, Poor risk
Cellularity and fibrosis variable CD33; may express CD7, CD41 and Cytogenetics
CD61
Micromegakaryocytes are present Blasts typically express CD41 and Cytogenetics

There is usually collagen and/or CD61; CD42b more variable. CD36 Patients may respond to intensive chemotherapy
reticular fibrosis may be positive. CD34, MPO, CD45
Overall appearance may suggest frequently negative, somewhat
metastatic tumor complicating lineage identification.
Usually markedly hypercellular Blasts typically CD34 negative, with Molecular (typically sizing via capillary electrophoresis for exon
expression of CD14, CD11b and 12 mutations; type A mutation, a 4 base pair TCTG insertion
CD68 most common); favorable outcome
Monocytic antigens typically IHC for abnormal cytoplasmic NPM1 protein endorsed by WHO;
expressed (CD36/CD64) some studies suggest molecular analysis may be preferred
Most cases show features of AML Blasts express typical myeloid Molecular (mutations described throughout gene, with 2
with or without maturation; no antigens: usually CD34+, express hot-spots at N-terminal and C-terminal)
specific morphologic features assist CD7 in the majority of cases Favorable outcome
in identifying CEBPA mutated cases
HP = hematopoiesis; IHC = immunohistochemistry; DIC = disseminated intravascular coagulation; MDS = myelodysplastic syndrome;
388
t18.5 Clues and Confirmatory Tests for Biologic Types of AML (continued from previous spread)
AML Subtype Clinical Features CBC/Blood Morphology BM Aspirate
Other Biologic Subtypes
AML with Elderly; patient may have antecedent Cytopenias Dysplasia of 50% of erythroid and/or
MDS-related MDS, MDS/MPN (#1 criterion) Single or multilineage dysplasia granulocytic cells (#2 criterion)
changes (3
criteria; only 1
required)
Therapy- History of chemotherapy and/or Cytopenias Cases may meet criteria for AML, MDS, or
related radiation therapy Single or multilineage dysplasia MDS/MPN (see t18.8)
myeloid common
neoplasm
Down syndrome (DS)-related myeloid proliferation:
Transient Diagnosis of DS (including mosaic Blasts are morphologically Blasts are morphologically indistinguishable
abnormal cases) indistinguishable from those in from those in myeloid leukemia associated
myelopoiesis Neonate/fetus myeloid leukemia associated with DS
(TAM) Possible co-morbidities include with DS Erythroid and megakaryocytic dysplasia
cardiopulmonary failure, Basophilia may be present typically present
hyperviscosity, and hepatic fibrosis Possible thrombocytopenia with
Spontaneous remission leukocytosis
Myeloid Diagnosis of DS (including mosaic Blasts have megakaryoblastic Myeloid neoplasms falling into this category
leukemia cases) features, with basophilic may show <20% blasts, as MDS and AML
associated Infant/young child (usually <4 - 5 yrs of cytoplasm, coarse granules, in this clinical setting are not clinically or
with DS age) and cytoplasmic blebbing biologically distinct
Erythroid precursors, marked Dyserythropoiesis and dysgranulopoiesis
anisopoikilocytosis, and giant
platelets may be seen
Often cytopenias
DIC = disseminated intravascular coagulation; HP = hematopoiesis; IHC = immunohistochemistry; MDS = myelodysplastic syndrome;
References by subtype
8;21 [Boissel 2006, Cairoli 2006, Care 2003, Chen 2008, De 2007, Gamerdinger 2003, Huang 2006, Khoury 2003, Kozlov 2005, Leroy 2005, Marcucci 2003, 2005, Miyoshi 1993, Mrozek 2001, Paschka 2006, Rowe 2000,
Schlenk 2004, Schnittger 2006, Tiacci 2004, Valbuena 2006, Weisser 2007]
inv(16) [Boissel 2006, Cairoli 2006, Care 2003, Hung 2007, Marcucci 2003, 2005, Mrozek 2001, Paschka 2006, Rowe 2000, Schlenk 2004, Spencer 2007, Xu 2008]
15;17 [Ades 2005, Arber 2008a, Arbuthnot 2006, Bennett 2000, de Botton 2004, Doyle 2009, Foley 2001, Gomis 2004, Ito 2004, Kaito 2005, Kelaidi 2009, Lee 2003, Leu 2009, Mohamedbhai 2008, Mueller 2006,
Ravandi 2009, Sanz 2009b, Stankova 2006, Tallman 2009, Wang 2008]
9;11 [Arber 2008a, Balgobind 2009, Dunphy 2007, Krasinskas 1998, Mrozek 1997, Xu 2006b]
6;9 [Alsabeh 1997, Chi 2008, Oyarzo 2004, Parcells 2006, Slovak 2006, Thiede 2002]
8 21
f18.3 Partial karyotype showing chromosomes 8 and 21 illustrates t(8;21) i18.15 Peripheral blood smear from a patient with acute myeloid leukemia and
(q22;q22). The abnormal chromosome of each pair is each on the right. t(8;21) shows prominent myeloid and monocytic maturation. Blasts are present, but
(Giemsa trypsin Wright) maturation is prominent. Note profound anemia and thrombocytopenia. (Wright)

389
BM Biopsy Flow Cytometry Confirmatory Tests/Other
Abnormal megakaryocytes Aberrant antigen maturation profile MDS-associated cytogenetic abnormalities (#3 criteria), not all
variable; myeloid blasts (CD34+) cases (see t18.7)
Cases may meet criteria for AML, Variable; often aberrant Two common subtypes: chromosomal losses/gains or 11q23
MDS, or MDS/MPN (see t18.8) (MLL) translocation
Blast % may be lower than blood Positive for CD34, CD56, CD13, CD33, GATA1 mutation
blast count CD7, CD4, CD41, CD61, CD42b.
Cellularity and blast % variable (in
some cases explained by presence
of blasts at sites of fetal HP)
Very dense fibrosis may be present In contrast to TAM, blasts are CD34- GATA1 mutation; cases of MDS or AML occurring in patients
Highly atypical megakaryocytes in ~50% of cases. CD56 and CD41 with DS who are older than age 5 and lack GATA1 mutation
also more likely to be negative are considered conventional MDS or AML
HP = hematopoiesis; IHC = immunohistochemistry; DIC = disseminated intravascular coagulation; MDS = myelodysplastic syndrome;
inv(3) [Jotterand Bellomo 1992, Martinelli 2003, Nucifora 1997, Raza 2004, Testoni 1999]
t(1;22) [Duchayne 2003, Mercher 2001, Niu 2009, Paredes-Aguilera 2003, Ribeiro 1993]
NPM1 [Becker 2010, Falini 2005, 2007, Konoplev 2009, Schnittger 2005, Tsou 2006, Yu 2006, Yun 2003]
CEBPA [Baldus 2007, Bienz 2005, Dufour 2010, Frohling 2004, Koschmieder 2009, Pabst 2009, Schlenk 2008, Wouters 2009]
MDS-related changes [Arber 2008b, Wandt 2008]
Therapy-related changes [Czader 2009, Knight 2009, Pedersen-Bjergaard 2008, Rund 2005, Seedhouse 2007, Vardiman 2008a]
Down syndrome [Massey 2006, Xavier 2009, Zipursky 2003]
usual finding; monocytes may also be present i18.15. In the predominant after successful therapeutic reduction of the
bone marrow, the blast count is variable and in some cases is acute leukemia i18.22, i18.23.
less than the otherwise required 20% [Chan 1997, Wong 2009b, Xue 1994]. Immunophenotypically, blasts express typical immature
Blasts with distinctive, long, thin Auer rods with tapered ends (CD34, CD117) and myeloid markers [CD13, CD33,
are characteristic and may even be seen in mature neutrophils myeloperoxidase (MPO)]. Expression of particular aberrant
and eosinophils i18.16, i18.17. Additional distinctive findings markers (CD19, CD56, TdT) may serve as key flow
include salmon-colored cytoplasm with a rim of basophilia cytometric clues to this diagnosis [Chen 2008, Hurwitz 1992, Khalidi 1998,
in maturing granulocytes, and dysplastic features of the Khoury 2003, Kozlov 2005, Tiacci 2004, Valbuena 2006]. Diminished CD19 and
mature granulocytes, including pseudo Pelger-Hut nuclei positive CD56 expression on the leukemic blasts may suggest
and nuclear/cytoplasmic dyssynchrony i18.18 [Nakamura 1997]. The an accompanying underlying KIT activating mutation in
bone marrow core biopsy is hypercellular with evidence of AML with t(8;21) [De 2007]. By immunohistochemistry, OCT2
granulocytic maturation i18.19 [Nakamura 1997]. and BOB1 are not reported to associate with the known
AML with t(8;21) may be associated with systemic PAX5 expression [Gibson 2006].
mastocytosis i18.20, i18.21. In some cases the mastocytosis Patients with de novo AML with t(8;21) have a
may be occult at diagnosis, becoming more apparent and relatively favorable prognosis [Mrozek 2008a]. t(8;21) AML may
390
i18.16 Long, tapered Auer rods and abnormal cytoplasmic granulation are i18.17 This neutrophil from a case of acute myeloid leukemia with t(8;21)
evident in this bone marrow aspirate smear from a patient with acute myeloid shows Auer rods within the cytoplasm. (Wright)
leukemia and t(8;21). (Wright)
i18.18 This bone marrow aspirate smear from a patient with acute myeloid i18.19 Substantial maturation is evident in this bone marrow core biopsy
leukemia and t(8;21) shows abnormal salmon-colored cytoplasmic granulation with section from a patient with acute myeloid leukemia and t(8;21). (H&E)
a rim of basophilia, a morphologic feature of this genetic type of AML. (Wright)
i18.20 Systemic mastocytosis was evident in conjunction with acute myeloid i18.21 This bone marrow core biopsy section from a patient with concurrent
leukemia associated with t(8;21) in this bone marrow aspirate smear. Note features acute myeloid leukemia with t(8;21) and systemic mastocytosis shows numerous
of acute myeloid leukemia with t(8;21) as well as numerous mast cells. (Wright) admixed mast cells. (immunoperoxidase for tryptase)

391
i18.22 This bone marrow aspirate smear is taken following chemotherapy for i18.23 A striking persistence of tryptase-positive mast cells is evident in this
acute myeloid leukemia with t(8;21) and associated systemic mastocytosis. The core biopsy from a case of acute myeloid leukemia with t(8;21) and associated
AML is in remission, but mast cells persist and are markedly increased. (Wright) systemic mastocytosis. Chemotherapy effectively eradicated the AML, while
systemic mastocytosis persists. (immunoperoxidase for tryptase)
i18.24 Monoblasts and promonocytes predominate in this peripheral blood i18.25 Circulating leukemic cells with a myelomonocytic appearance charac-
smear from a patient with marked leukocytosis and inv(16). (Wright) terize this case of AML with inv(16). (Wright)
also occur after previous chemotherapy (therapy-related)

or in blast phase of chronic myelogenous leukemia (CML)
[Arber 2002, Gustafson 2009, Yin 2004]. Frequent secondary cytogenetic
aberrations include loss of a sex chromosome (Y in men
and X in women) and deletion 9q [Kuchenbauer 2006, Mrozek 2008a]. 16
KIT activating mutations have been identified in t(8;21) and f18.4 Normal (left) and abnormal (right) chromosome 16 illustrate inv(16)
confer an inferior overall survival [Boissel 2006, Cairoli 2006, Paschka 2006, (p13.1;q22).
Schnittger 2006]. FLT3 mutations are seen in 4% - 12% of AML with
t(8;21), although only a minority are of the internal tandem
duplication type tied most definitively to prognosis in cytoge- AML with inv(16)/t(16;16)(p13.1;q22); CBFB-MYH11
netically normal cases (see[18.8]Prognostic Factors in AML, AML with inv(16)/t(16;16) is defined by the translo-
p 417) [Boissel 2006, Care 2003, Shimada 2006, Sritana 2008]. Nevertheless, some cation and/or molecular fusion of CBFB and MYH11
reports suggest that FLT3 mutation is an adverse prognostic regardless of blast count f18.4. Clues and confirmatory tests
finding in AML with t(8;21) [Boissel 2006]. are listed in t18.5. Morphologically, this type of AML most
often appears as a subtype of acute myelomonocytic leukemia
with abnormal eosinophils.
392
i18.26 Numerous eosinophils with admixed aberrant basophilic granules are i18.27 Numerous eosinophils with aberrant cytoplasmic granulation are
evident in this bone marrow aspirate smear from a patient with acute myeloid evident in this bone marrow aspirate smear from a patient with acute myeloid
leukemia and inv(16). (Wright) leukemia and inv(16). (Wright)
a b
i18.28 Significant maturation with relatively low numbers of myeloblasts, i18.29 This composite of low and high magnification of a bone marrow
monoblasts, and promonocytes is evident on this bone marrow aspirate smear core biopsy section from a patient with acute myeloid leukemia and inv(16)
in acute myeloid leukemia with inv(16). Note predominance of eosinophils and shows numerous eosinophils in conjunction with myelomonocytic cells showing
maturing myeloid cells. (Wright) abundant cytoplasm. (H&E)
The peripheral blood abnormalities are variable, but myeloperoxidase and toluidine blue reactivity, distinguishing
leukocytosis, anemia, and thrombocytopenia are common. them from basophil granules. Significant maturation may
Monocytic cells are abundant with variable numbers of be evident with relatively low numbers of blasts i18.28. Both
monoblasts, myeloblasts, and promonocytes i18.24, i18.25. maturation and significant eosinophilia are evident on bone
The abnormal eosinophils (described below) are generally marrow biopsy sections i18.29.
inconspicuous or absent in the blood. Immunophenotypically, AML with inv(16)/t(16;16)
The bone marrow is hypercellular with a variable reveals distinct populations corresponding to the admixture of
blast count. The morphologic features show a spectrum of blasts, granulocytes, and monocytic cells. An immunohisto-
mature and immature monocytic cells and myeloblasts. The chemical antibody stain (AH107) against the CBFB-MYH11
monocytic and granulocytic components are confirmed fusion protein may aid in the diagnosis [McKenna 2009, Zhao 2006].
by positive nonspecific esterase and myeloperoxidase Patients with AML with inv(16)/t(16;16) have a
cytochemical stains, respectively. In addition, the bone favorable prognosis similar to t(8;21). The inv(16) may
marrow contains abnormal eosinophils with mixed eosino- occur rarely after previous chemotherapy or in blast phase of
philic and basophilic granules i18.26, i18.27. The abnormal CML [Merzianu 2005, Wu 2006]. KIT mutations are present in ~30%
basophil granules within these eosinophils lack both of patients, the significance of which is unclear [Paschka 2006].

393
15 17
f18.5 Partial karyotype showing chromosomes 15 and 17 illustrates t(15;17)

(q22;q21). The abnormal chromosome of each pair is on the right. (Giemsa
trypsin Wright)
Activating mutations in NRAS, KRAS, KIT, or FLT3 are seen

in ~70% of cases [Boissel 2006, Valk 2004].
APL with t(15;17)(q22;q21); PML-RARA f18.6 Dual color dual fusion probe set for PML and RARA shows the classic
AML with t(15;17)(q22;q21)/PML-RARA (aka APL) is abnormal fusion pattern. Note 2 fused signals, one red signal and one green
a distinct clinicopathologic entity defined by the presence of signal, indicating PML-RARA fusion characteristic of acute promyelocytic
the PML-RARA fusion, regardless of blast count f18.5, f18.6, leukemia.
t18.5 [Arber 2008a]. Because of the propensity for life-threatening
coagulopathy, it is imperative that cases of APL be rapidly
identified [Lock 2004, Stein 2009]. This can be accomplished with a bone marrow core biopsy sections, APL cells show moderate
myeloperoxidase cytochemical stain, antibody against the amounts of eosinophilic cytoplasm i18.35. Because of the
PML protein, or FISH f18.6. degree of maturation, CD34 is characteristically negative
Two typical morphologic variants of APL are recognized i18.36.
(hypergranular and microgranular), plus a third variant Microgranular APL is morphologically distinct from
that is quite rare (hyperbasophilic). Hypergranular APL the hypergranular variant in both nuclear and cytoplasmic
often presents with peripheral leukopenia with rare, if any, features. Microgranular promyelocytes exhibit marked
circulating promyelocytes, while the bone marrow is typically nuclear irregularities including reniform, lobulated, and
packed with abnormal, hypergranular promyelocytes i18.30, monocyte-like nuclei, while the cytoplasm shows subtle
i18.31, i18.32. Occasional/rare cells packed with Auer rods are often inconspicuous granulation i18.37, i18.38, i18.39. The
noted i18.33 [Bennett 2000]. There is little to no maturation beyond rare hyperbasophilic variant is characterized by nuclear
the promyelocyte stage. These hypergranular promyelocytes irregularity and basophilic cytoplasm; features overlap
are intensely myeloperoxidase or Sudan blackB positive, a with microgranular APL i18.40. It is essential to distinguish
very useful feature in the rapid diagnosis of APL i18.34. On microgranular APL from a true monocytic leukemia i18.41.
i18.30 This bone marrow aspirate smear from a patient with acute i18.31 This bone marrow aspirate smear from a patient with acute
promyelocytic leukemia shows many hypergranular promyelocytes, which are promyelocytic leukemia shows variable nuclear irregularity in conjunction with
blast equivalents in only this AML subtype. (Wright) moderate amounts of hypergranular cytoplasm. (Wright)

394
i18.32 Intense cytoplasmic granulation, especially in the upper left cell, i18.33 Numerous Auer rods are present within the cytoplasm of acute
is evident on this bone marrow aspirate smear from a patient with acute promyelocytic leukemia cells on this cytospin preparation. (Wright)
promyelocytic leukemia. (Wright)
i18.34 Cytochemical staining for myeloperoxidase shows intense uniform i18.35 Little evidence of maturation beyond the promyelocyte state is evident
positivity in acute promyelocytic leukemia. (myeloperoxidase cytochemical stain) in this bone marrow core biopsy section from a patient with acute promyelocytic
leukemia. Note relative uniformity of the leukemic population and moderate to
abundant amounts of eosinophilic cytoplasm. (H&E)
a b
Cytochemically, both the hypergranular and
microgranular APL cases show intense myeloperoxidase and
Sudan black B positivity in the leukemic cells i18.42. This
marked degree of staining is quite compelling for a diagnosis
of APL and is very useful in distinguishing APL from
morphologically similar disorders.
Immunophenotypically, hypergranular and microgranular
APL show some similarities but also key differences. The
hypergranular variant is typically CD34 negative, HLA-DR
negative, with bright CD33, whereas the microgranular
variant is often CD34 positive with aberrant CD2 expression
(90%) [Edwards 1999, Kaleem 2003, Lin 2004, Paietta 2003]. The lack of CD34 and
i18.36 This composite highlights the morphologic features a and usual absence
HLA-DR is not specific for APL, thus complete reliance on
of CD34 staining b in this case of acute promyelocytic leukemia. Note CD34 flow cytometric immunophenotyping to diagnose APL is to
positivity of only blood vessels. (H&E and immunoperoxidase for CD34) be avoided [Moon 2007, Oelschlaegel 2009].

395
i18.37 The distinctly folded, sliding plate appearance of nuclei in microgranular i18.38 The distinctive nuclear features of microgranular acute promyelocytic
acute promyelocytic leukemia is evident in this peripheral blood smear. Note leukemia are evident on high magnification of this peripheral blood smear. Note
marked leukocytosis. (Wright) marked leukocytosis. (Wright)
a b
i18.39 Rare hypergranular promyelocytes are present in microgranular acute i18.40 This composite compares microgranular acute promyelocytic leukemia
promyelocytic leukemia in blood. (Wright) a with the basophilic variant of acute promyelocytic leukemia b. (Wright)
a b a b
i18.41 This composite compares microgranular acute promyelocytic leukemia i18.42 Intense myeloperoxidase positivity, characteristic of all types of acute
a with acute monocytic leukemia b. Note the differences in nuclear configuration promyelocytic leukemia, can be identified by either cytochemical stain a or
and the greater amounts of cytoplasm in acute monocytic leukemia. (Wright) immunoperoxidase stain b. (myeloperoxidase cytochemical and immunoper-
oxidase stains)
396
t18.6 Characteristics of AML with Variant RARA Translocations

Variant Partners Comments
ZBTB16 (11q23) Morphologic differences from classic APL: blasts with regular nuclei, increased Pelger-Hut-like neutrophils;
ATRA resistant
NUMA1 (11q13) ATRA-sensitive
NPM1 (5q35) Morphologic differences: no Auer rods
Predominantly hypergranular
ATRA-sensitive
STAT5B (17q11.2) ATRA-resistant
PRKAR1A (17q23 - 24) Single case report
Cryptic fusion
Typical APL: hypergranular, morphology, and immunophenotype
AML = acute myeloid leukemia; APL = acute promyelocytic leukemia;
ATRA = all trans retinoic acid
References: [Arnould 1999, Bennett 2000, Catalano 2007, Chen 1993, Redner 1996, Sainty 2000, Wells 1997]
The differential diagnostic considerations vary between

hypergranular and microgranular APL largely due to their
different cytologic appearances. A wave of granulocytic
recovery after transient agranulocytosis, G-CSF therapy, or
any other prominent promyelocyte population can mimic 9 11
hypergranular APL. Key cytologic factors in granulocytic
recovery include prominent paranuclear hofs (Golgi regions), f18.7 Partial karyotype showing chromosomes 9 and 11 illustrates t(9;11)
round nuclei, and absence of Auer rods [Harris 1994, Innes 1987]. In (p22;q23). The abnormal chromosome of each pair is on the right. (Giemsa
addition, CD117 and CD11b expression may aid in distin- trypsin Wright)
guishing APL from a benign process [Rizzatti 2002]. As noted
above, the microgranular variant of APL may be mistaken
for acute monocytic leukemia (AMoL); however, the from classic APL include less prominent nuclear bilobation,
typical bilobed (sliding plate) appearance of microgranular absence of Auer rods, and pelgeroid neutrophils. The respon-
APL is not seen in AMoL (see i18.41). Striking uniform siveness of these variants with ATRA varies t18.6.
myeloperoxidase cytochemical activity in the leukemic cells
also indicates APL, while greater amounts of cytoplasm AML with t(9;11)(p22;q23); MLLT3-MLL
and nonspecific esterase positivity characterize monocytic In the WHO 2008 classification, AML with t(9;11) is
leukemias (see i18.42). the only translocation involving the MLL gene included as
Patients with de novo t(15;17) have a favorable a distinct biologic subtype f18.7 [Arber 2008a]. Clues and confir-
prognosis. t(15;17) AML may also arise after previous matory tests in AML with t(9;11) are detailed in t18.5.
chemotherapy or in blast phase of CML [Beaumont 2003, Chung 2008, In the peripheral blood and bone marrow, AML with
Hasan 2008, Pulsoni 2002]. Occasionally the molecular fusion is t(9;11) manifests as a proliferation of monoblasts and/
present but is cytogenetically and/or FISH cryptic requiring or promonocytes (blasts/blast equivalents 20%) i18.43,
PCR-based confirmation [Wang 2009, Wong 2009a]. FLT3 mutations i18.44. Full monocytic differentiation is rarely evident. t18.4
are detected in 30% - 40% of APL cases and may be associated delineates the key morphologic, cytochemical, and immuno-
with shorter survival times [Callens 2005]. phenotypic features of monoblasts and promonocytes
i18.45. Prominent nonspecific esterase (NSE) cytochemical
Acute Myeloid Leukemias with Variant RARA positivity and negative/weak MPO in the blasts is a key clue
Translocations to the diagnosis (see i18.14).
Occasionally, a case will exhibit many features suggestive Immunophenotyping reveals monocytic differentiation
of APL but lack the t(15;17) translocation; alternative typified by CD36/CD64 coexpression, weak CD4, bright
translocation partners for RARA on chromosome 17 have CD33, strong HLA-DR, variable (often negative) CD14,
been noted in these cases t18.6. Morphologic differences
397
i18.43 This peripheral blood smear shows profound pancytopenia and a rare i18.44 This bone marrow aspirate smear illustrates acute monocytic leukemia
circulating monoblast. (Wright) and t(9;11). Note predominance of monoblasts and promonocytes (blast
equivalents). (Wright)
while both CD34 and CD117 are typically negative [Dunphy 2007,
a b
Krasinskas 1998, Xu 2006b].
AML with t(9;11) is associated with an intermediate
prognosis and reportedly fares better than AMLs with other
MLL translocation partners [Balgobind 2009, Mrozek 1997]. AMLs with
MLL rearrangements other than t(9;11) are diagnosed
as AML, not otherwise specified, or as therapy-related
leukemia with t(v;11q23) in the setting of prior cytotoxic
treatment. If the cytogenetics reveal t(2;11)(p21;q23) or
t(11;16)(q23;p13), these should be diagnosed as AML with
myelodysplasia-related changes [Arber 2008a].
AML with Variant MLL Translocations

Although >60 fusion partner genes are known for
MLL, the more common translocations in AML include i18.45 This composite highlights the morphologic features of acute monocytic
6q27 (MLLT4), 10p12 (MLLT10), 19p13.1 (ELL), and leukemia in bone marrow core biopsy sections a, while strongly positive CD68 is
19p13.3 (MLLT1), all resulting in fusion genes [Martineau 1998, noted, compatible with monocytic lineage differentiation b. (H&E and immuno-
peroxidase for CD68)
Meyer 2009, Moorman 1998]. AML with MLL abnormalities accounts
for ~3% of all AML [Schoch 2003]. In neonates, acute monoblastic
leukemias frequently harbor MLL translocations, and high-level expression of Homeobox genes such as HOXA9
overall survival may depend on the translocation partner [Faber 2009, Roth 2009].
[Balgobind 2009]. Given that the MLL gene is not invariably involved
when abnormalities of 11q23 are detected by conventional AML with t(6;9)(p23;q34); DEK-NUP214
cytogenetics and that MLL rearrangements (including AML with t(6;9)(p23;q34) is rare and estimated
those resulting from MLL partial tandem duplication) may to comprise approximately 1% of all adult and pediatric
be missed by standard cytogenetics, molecular or FISH AML cases f18.8 [Byrd 2002, Slovak 2000]. Detailed clues and confir-
techniques may be necessary to identify and verify an MLL matory tests in this type of AML are shown in t18.5 [Alsabeh 1997,
abnormality [Abdou 2002, Caligiuri 1998, Mathew 1999, Watanabe 2003]. Since 11q23 Slovak 2006]. The CBC exhibits findings of bone marrow failure
is a topoisomerase II cleavage site, it has been experimentally (ie, thrombocytopenia, neutropenia, anemia), circulating
demonstrated that topoisomerase II inhibitors (eg, etoposide) blasts, and often basophilia. The blasts may contain Auer
increase chromosomal recombination events involving this rods and be cytochemically positive for myeloperoxidase
locus (see Therapy-Related AML, p 403) [Bueno 2009, Chatterjee 1990, and/or nonspecific esterase, thus exhibiting hybrid myeloid
Le 2009, Libura 2005]. MLL amplification in AML is associated with and monocytic features [Arber 2008a]. The bone marrow is
poor outcome. MLL-rearranged leukemias often cause hypercellular with 20% blasts. Background trilineage

398
der(6)
6 9
der(9)
f18.8 G-banded Wright-stained chromosomes 6 and 9 illustrate t(6;9) i18.46 This cytospin smear in acute myeloid leukemia and t(6;9) shows a
(p23;q34). The abnormal chromosome of each pair is on the right. myeloblast with fine granules in association with dysplastic neutrophils. (Wright)
dysplasia is common as is basophilia i18.46, i18.47 [Oyarzo 2004,

Pearson 1985, Slovak 2006]. Increased ring sideroblasts may be seen
[Alsabeh 1997, Slovak 2006]. CD34 and CD117 are generally positive on
the blasts with a subset expressing TdT.
The t(6;9) tends to be the sole cytogenetically evident,
although easily overlooked, abnormality, which results in
the formation of a chimeric fusion gene DEK-NUP214
(NUP214 once known as CAN) [Soekarman 1992, von Lindern 1992]. This
resultant oncoprotein is likely to work together with other
cryptic genetic aberrations to ultimately contribute to the
development of AML [Deguchi 2002].
Up to 80% of t(6;9) AML cases may have an internal
tandem duplication mutation of the tyrosine kinase FLT3,
which may play a key role in leukoemogenesis by impeding
normal cellular proliferation [Oyarzo 2004, Parcells 2006, Thiede 2002]. i18.47 This bone marrow aspirate smear in acute myeloid leukemia and
Increased global protein synthesis specifically in t(6;9) t(6;9) shows myeloblasts with fine cytoplasmic granules. (Wright) (courtesy J
Mooney, MD)
myeloid cells as a result of increased translation may be a
mechanism underlying leukemogenesis [Ageberg 2008].
Patients with t(6;9) AML have an overall poor prognosis topographic relationship with MDS1 [Martinelli 2003, Nucifora 1997].
(5-year survival estimate in adults is 9%) [Slovak 2006]. Higher The morphologic hallmark of these cases is the presence of
WBC counts predict shorter overall survival, while higher conspicuously atypical megakaryocytes (usually small in
bone marrow blast counts predict shorter disease-free survival size with monolobated or bilobated nuclei) in a background
[Slovak 2006]. Allogeneic stem cell transplant may improve overall of multilineage dysplasia. A similar picture is present in
survival [Slovak 2006]. the peripheral blood, with notably prominent platelet-
associated abnormalities (including giant, hypogranular
AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); forms and circulating megakaryocytic nuclei) in a
RPN1-EVI1 background of granulocytic dysplasia [Jotterand Bellomo 1992]. EVI1
Rarely, a case of AML will bear either the inv(3) also serves as a translocation partner with genes other than
(q21;q26.2) or the t(3;3)(q21;q26.2), both of which RPN1, in which case the process is not considered to fall
generate fusion of RPN1 and EVI1 t18.5. The latter gene within this specific biologic subtype. AML with RPN1-EVI1
codes for a transcription factor and is itself transcriptionally is a highly aggressive disease with a median survival of
complex, with alternate forms that include exons spliced only 5.5 months, though response to alternate treatment
in from an upstream gene MDS1. The fusion with RPN1 regimens may be better [Raza 2004, Testoni 1999]. It is to be hoped
causes aberrant expression of EVI1 and disturbs the normal that wider recognition of the entity, facilitated by its specific

399
i18.48 This bone marrow aspirate smear composite is from 2 patients with i18.49 This bone marrow core biopsy section from a patient with acute
acute megakaryoblastic leukemia and t(1;22). Note prominent clumping of megakaryoblastic leukemia and t(1;22) shows numerous blasts as well as differen-
blasts (right). (Wright) (courtesy A Vendrell, MD) tiating megakaryocytic cells as evidenced by enlarging overall cells size and moderate
to abundant amounts of eosinophilic cytoplasm. (H&E) (courtesy S Geaghan, MD)
inclusion in the WHO 2008 system, may foster more
effective therapies.
AML with t(1;22)(p13;q13); RBM15-MKL1

AML with t(1;22)(p13;q13) is an exceedingly rare
diagnosis (<1% of cases) that should be considered only in
children <3 years of age; most commonly, presentation is
in infancy t18.5. The t(1;22) involves RBM15 and MKL1.
The latter gene is named for its association with megakaryo-
blastic leukemia and encodes a protein involved in chromatin
organization, while RBM15 regulates c-myc; the fusion
event may disturb both of these activities [Mercher 2001, Niu 2009]. As
befits an AML involving MKL1, this is a megakaryoblastic
leukemia, and these blasts may show distinct clumping
on aspirate smears and are often mixed with micromega- i18.50 On high magnification, megakaryoblasts and minimally maturing
karyocytes i18.48, i18.49, i18.50 [Duchayne 2003]. Other clues to megakaryocytes are evident on this bone marrow core biopsy section from a
patient with acute megakaryoblastic leukemia and t(1;22). (H&E) (courtesy S
the possible presence of this subtype are organomegaly, the
Geaghan, MD)
lack of significant erythroid or granulocytic dysplasia, and
the presence of bone marrow fibrosis [Paredes-Aguilera 2003, Ribeiro 1993]. prognostically significant. However, the inclusion of these
The fibrosis is often so extensive as to suggest a differential gene mutations has occasioned a good deal of strenuous
diagnosis of metastatic disease, an impression heightened by debate, centering on a fundamental (almost philosophical)
the relatively cohesive nature of the megakaryoblasts. Given a question: at what point does a genetic aberration tied to
megakaryoblastic proliferation in an infant, it is also prudent a given biologic behavior cease to be simply a prognostic
to completely exclude a diagnosis of Down syndrome and its factor and instead become definitional of a specific subtype
associated myeloid proliferations [Hama 2008]. The importance of AML? FLT3, for example, is associated with a relatively
of recognizing the t(1;22) subtype as a distinct diagnostic poor prognosis but is found in a wide array of otherwise
entity is underscored by studies showing a significant survival well-defined AML subtypes, such as APL. For this reason,
advantage with intensive chemotherapy [Duchayne 2003]. the WHO relegates FLT3 mutation status to a prognostic
factor rather than a diagnostic subgroup. In contrast, NPM1
AML with Mutated NPM1 and CEBPA and CEBPA mutations define specific subtypes of AML,
Several recurrent gene mutations now share the stage though these entities are only provisional categories. We will
with the well-established translocations described above. adhere to this scheme and discuss NPM1 and CEBPA here as
The WHO 2008 scheme endorses mutations in 3 genes biologic subtypes of AML, while reserving consideration of
(NPM1, CEBPA, and FLT3) as especially diagnostically or FLT3 for the subsequent section on prognostic factors.

400
AML with Mutations of NPM1 Diagnostic Strategies for Molecular Assessment in AML
NPM1 mutations are frequent in karyotypically normal The inclusion of NPM1 and CEBPA mutation status
AML, occurring in between 50% and 60% of such cases in the AML classification scheme presents a challenge
[Falini 2005]. NPM1 encodes the protein nucleophosmin, which to the diagnostician. In contrast to conventional cytoge-
appears to function primarily as a shuttle for other proteins netics and flow cytometric immunophenotyping, which
between the nucleus and the cytoplasm. It may also play a are routinely performed in newly diagnosed cases of AML,
role in cell cycle control and in the regulation of centromere molecular analysis for gene mutations represents a new
duplication to facilitate orderly mitosis [Falini 2007, Tsou 2006, Yu 2006, frontier. When should this specialized testing be ordered?
Yun 2003]. NPM1 mutations are typically insertions of variable The best answer, as of this writing, is, in cytogenetically
length that produce a frameshift sufficient to alter the nuclear normal cases and/or when, after consultation with the
localization sequence, leading to aberrant accumulation of clinical team, it is determined that gene mutation status
the protein in the cytoplasm t18.5. Though immunohisto- will provide information useful in patient management.
chemical techniques may be applied to detect this abnormal The prognostic significance of NPM1, CEBPA (and
cytoplasmic nucleophosmin, direct PCR amplification of FLT3) mutations is clearly established in cytogenetically
the affected region with sizing by capillary electrophoresis normal cases [Mrozek 2007]. In other situations, including AML
is widely available in molecular diagnostic laboratories with myelodysplasia-related changes and cytogenetically
[Konoplev 2009, Luo 2010]. NPM1 mutations have been especially abnormal cases that do not fulfill criteria for AML with
associated with AML showing monocytic differentiation myelodysplasia-related changes, the significance of these gene
and absence of CD34 expression. Although neither feature mutations remains less certain. However, our understanding
is completely sensitive or specific for predicating an NPM1 of gene mutations is constantly evolving, with recent data
mutation, the fact that up to 90% of acute monocytic demonstrating that NPM1 mutation status may carry similar
leukemias have mutated NPM1 provides a powerful prognostic connotations even in karyotypically abnormal
diagnostic clue [Schnittger 2005]. Mutated NPM1 is associated with AML [Haferlach 2009a]. While, in general, strict adherence to
a good prognosis with one exceptionally important caveat: the WHO classification scheme is desirable, communi-
NPM1 mutations are rather frequently (ie, 40%) present cation with clinicians on a case-by-case basis is essential to
in tandem with FLT3 mutations, in which case the adverse its rational application in this area. Thus, at large research
prognostic effect of the FLT3 mutation essentially negates institutions with multiple treatment protocols and studies,
the otherwise beneficial behavior associated with the NPM1 maximum effort in all cases (even those carrying cytogenetic
mutation [Baldus 2007, Schlenk 2008]. Thus, testing for NPM1 should abnormalities or myelodysplasia-related changes) to identify
always be performed together with FLT3 analysis. these and other mutations might indeed be indicated. In
contrast, such efforts might be excessive in an elderly or infirm
AML with CEPBA Mutations patient ineligible for aggressive treatment options. It is likely
Mutations of the CCAAT/enhancer binding protein that diagnostic strategies employing these gene mutations will
(CEBPA) gene define the second provisional subtype of AML continue to evolve as further studies more clearly define their
with gene mutations. CEBPA encodes a transcription factor impact in the full spectrum of morphologic subtypes of AML.
essential for granulocytic maturation [Koschmieder 2009]. Mutations
occurring in AML cluster in the N-terminal and C-terminal [18.6.2] Clinical Biologic Subtypes of AML
regions, inducing frameshift and missense mutations, respec-
tively, that presumably contribute to leukemogenesis by AML with Myelodysplasia-Related Changes
interfering with the proteins contribution to terminal differ- AML with myelodysplasia-related changes is a broad
entiation. CEBPA mutations are somewhat less common category that may be invoked by clinical setting, genetic
than NPM1 mutations, and are present in between 15% and abnormality, or morphologic findings [Arber 2008b]. Specifically,
20% of cytogenetically normal casesmost commonly, these cases of AML satisfy one or more of the following
but not exclusively, in cases with the morphologic features criteria:
of AML with or without maturation [see [18.7] AML, Not 1. the patient has a history of previously diagnosed MDS or
Otherwise Specified (NOS), p 408] [Baldus 2007]. Mutated CEBPA MDS/MPN overlap syndrome
in isolation confers a relatively good prognosis, though recent 2. cytogenetic abnormalities associated with MDS are
data suggest that this effect is limited to only those cases that present t18.7, f18.9
carry biallelic CEBPA mutations [Bienz 2005, Frohling 2004, Ho 2009, Pabst 2009, 3. there is multilineage dysplasia, defined for this purpose
Schlenk 2008, Wouters 2009]. as dysplasia involving at least half of the cells in 2
separate bone marrow lineages i18.51, i18.52, i18.53, i18.54

401
t18.7 Cytogenetic Abnormalities Sufficient to

Diagnose AML with Myelodysplasia-Related
Changes*
Complex karyotype (>3 unrelated abnormalities, none of which is included
in the AML with recurrent cytogenetic abnormalities subgroup); such
cases should be classified in the appropriate cytogenetic group
7/del(7q)
5/del(5q)
i(17q)/t(17p)
13/del(13q)
del(11q)
del(12p)/t(12p)
del(9q)
idic(X)(q13) i18.51 This peripheral blood smear shows profound pancytopenia, a
t(11;16)(q23;p13.3)* circulating blast, and dysplastic neutrophils. (Wright)
t(3;21)(q26.2;q22.1)*
t(1;3)(p36.3;q21.1)
t(2;11)(p21;q23)
t(5;12)(q33;p12)
t(5;7)(q33;q11.2)
t(5;17)(q33;p13)
t(5;10)(q33;q21)
t(3;5)(q25;q34)
*Patients with a diagnosis of AML with myelodysplasia-related changes
should not have a history of prior cytotoxic therapy; in such a case, the
therapy-related myeloid neoplasm category takes precedence over any
MDS-related changes, and the disease should be classified as such;
t(11;16)(q23;p13.3) and t(3;21)(q26.2;q22.1) occur in therapy-related cases
with particular frequency
Reference: [Arber 2008a]
i18.52 This bone marrow aspirate smear in acute myeloid leukemia with
myelodysplasia-related changes shows highly abnormal multinucleated erythroid
precursors, increased myeloblasts, and dysplastic maturing granulocytic cells.
(Wright)
f18.9 This karyogram shows a complex karyotype including monosomy of i18.53 This bone marrow core biopsy in acute myeloid leukemia with
many chromosomes as well as multiple marker chromosomes. The abnormal myelodysplasia-related changes shows highly atypical multinucleated erythroid
chromosomes are on the right. (Giemsa trypsin Wright) cells in the approximate size range of megakaryocytes. Note increased blasts and
dysplastic megakaryocytes. (H&E)
402
i18.54 In this case of acute myeloid leukemia, the mature neutrophils exhibit i18.55 This bone marrow core biopsy section is from an elderly female with new
features of myelokathexis with exceedingly long, thin interlobar strands. (Wright) onset of pancytopenia. Note hypercellularity, lymphoid aggregate, and increased
megakaryocytes. (H&E)
i18.56 On higher magnification, tremendously increased numbers of small i18.57 This bone marrow core biopsy section shows abnormal localization of
dysplastic megakaryocytes are evident on this bone marrow core biopsy section myeloperoxidase-positive immature granulocytic cells in acute myeloid leukemia
in acute myeloid leukemia with myelodysplasia-related changes (see i18.55). with myelodysplasia-related changes. (immunoperoxidase for myeloperoxidase)
(H&E)
Morphologic and immunohistochemical assessment
of bone marrow core biopsies can be used to enumerate
immature cells and assess for multilineage dysplasia i18.55,
i18.56, i18.57, i18.58 [Ngo 2008].
In addition, these cases by definition lack the
clinical history (eg, prior cytotoxic therapy) and genetic
abnormalities [eg, t(15;17)] that would identify them
as belonging to other specific biologic subtypes of AML.
However, there is a minor exception to this rule: some
cases of AML with myelodysplasia-related changes will
also carry mutated NPM1 or CEBPA, which, as described
above, could theoretically place them into the provisional
categories reserved for cases with such mutations. Because
i18.58 Small dysplastic megakaryocytes are highlighted by immunoperoxidase
the prognostic significance of NPM1 and CEBPA mutations
staining for CD31 in acute myeloid leukemia with myelodysplasia-related is unclear in the setting of myelodysplasia-related changes,
changes. (immunoperoxidase for CD31) the WHO 2008 classification recommends that these rare

403
t18.8 Comparison of Clinicopathologic Features of 2 Types of Therapy-Related Myeloid Neoplasms*

Feature Secondary to Alkylating Agent Therapy Secondary to Topoisomerase II Inhibitor Therapy
Therapy Alkylating agents or radiotherapy induce permanent Epipodophyllotoxins and related agents that inhibit
genetic abnormalities DNA-topoisomerase II
Benzene exposure also linked to AML
Susceptibility Linked to polymorphisms in genes involved in 11q23 is cleavage site for topoisomerase II; double-stranded
detoxification or major DNA repair pathways DNA cleavage; repair blocked by inhibitor
Repair defects linked to polymorphisms in genes that detoxify
drugs
Latent period 2 - 11 years; shorter for patients with higher cumulative Brief, <1 - 3 years
alkylating agent dose
Blood Myelodysplastic phase characterized by severe Abrupt onset of cytopenias in most cases; some cases have
cytopenias, trilineage dysplasia, and basophilia short cytopenic prodrome phase
Leukocytosis common
Bone marrow Usually hypercellular, but may be hypocellular Hypercellular

Fibrosis common Most cases acute monocytic leukemia
Multilineage dysplasia common; variable blast Occasional cases of AML with t(8;21), acute promyelocytic
percentage leukemia, and AML with inv(16)
Cases may fulfill criteria for MDS, MDS/MPN, or AML
Cytogenetics/ 5/del(5q), 7/del(7q) (multistep leukemogenesis) Balanced 11q23 translocations in most cases
molecular Complex karyotypic abnormalities frequent Balanced 21q22 translocation, t(15;17), and inv(16) in occasional
Rare translocations (of type found in de novo AML) cases
Cooperating Class I and Class II mutations
Response to Poor response to conventional antileukemic therapy Good achievement of complete remission; relapse frequent
therapy Expression of multidrug resistance-associated proteins especially in 11q23-associated cases
and drug efflux
Survival Poor Variable; poor in 11q23 group
Worse than de novo AML with comparable features Somewhat better survival for t-AML with translocations
compared to chromosomal loss, but worse than de novo
cases with same translocations
*t-AMLs, t-MDS, and t-MDS/MPN merged in WHO classification; alkylating agent type t-AML merged with topoisomerase II inhibitor group, since most
patients have received both types of therapeutic agents [Vardiman 2008a]
Radiation/radiotherapy also leukemogenic; may also occur after either autologous stem cell or bone marrow transplantation [Beauchamp-Nicoud 2003, Smith 2003];
rarely reported after G-CSF for stem cell donor [Hsia 2008]
AML = acute myeloid leukemia; MDS= myelodysplastic syndrome; MPN = myeloproliferative neoplasm; t- = therapy-related
References: [Czader 2009, Knight 2009, Pedersen-Bjergaard 2008, Rund 2005, Seedhouse 2007, Vardiman 2008a]
cases be assigned to the MDS-related category, with a note chemotherapy and/or radiation treatment [Vardiman 2008a]. Agents
appended to the diagnostic line identifying the coexisting that have been implicated are listed in t18.1. There are 2 broad
mutation. Cases of AML with myelodysplasia-related changes categories of therapy-related myeloid neoplasms: those
have a generally poor prognosis, though this may be primarily that follow treatment with alkylating agents and those that
attributable to the frequent presence of high-risk cytogenetic follow agents directed against the enzyme topoisomerase II.
abnormalities rather than history or morphology per se, as Although in both cases, AML arises following direct DNA
suggested by recent authors [Wandt 2008]. However, in comparison damage induced by the therapeutic agent, the genetic and
to AML, NOS, cases of AML with MDS-related changes have clinical features of the resulting processes are quite different,
a significantly worse overall survival [Weinberg 2009]. as summarized in t18.8 [Czader 2009, Knight 2009, Pedersen-Bjergaard 2008, Rund 2005,
Seedhouse 2007, Vardiman 2008a]. Because chemotherapeutic regimens
Therapy-Related AML often include both alkylating agents and topoisomerase II
One clinical setting that automatically triggers a specific inhibitors, the WHO 2008 recommendation is to group all
subclassification of AML is a history of prior cytotoxic therapy-related myeloid neoplasms (t-AMLs) together rather
404
i18.59 This peripheral blood smear is from a patient who developed therapy- i18.60 This peripheral blood smear shows a therapy-related myeloid neoplasm.
related acute myeloid leukemia. Note profound cytopenias and striking red blood Note profound cytopenias, circulating myeloblasts, and markedly atypical
cell pathology. (Wright) eosinophil. (Wright)
i18.61 This bone marrow aspirate smear shows abundant myeloid blasts and i18.62 Both dysplastic erythroid and granulocytic lineage cells are evident
abnormal erythroid cells in a therapy-related acute myeloid leukemia. (Wright) on this bone marrow aspirate smear from a patient with therapy-related acute
myeloid leukemia. (Wright)
than specifying subtypes [Vardiman 2008a]. Furthermore, therapy-

related cases with the morphologic and clinical features of
myelodysplastic syndrome or MDS/MPN overlap syndromes
share the poor prognosis of therapy-related cases with features
of acute leukemia. For this reason, all such processes are
grouped together as therapy-related myeloid neoplasms,
regardless of blast count [Czader 2009, Vardiman 2008a]. Multilineage
dysplasia is frequently noted in cases of t-myeloid neoplasms,
and persistence of the primary neoplasm may be noted in
conjunction with t-AML i18.59, i18.60, i18.61, i18.62, i18.63,
i18.64, i18.65, i18.66, i18.67, i18.68, i18.69, i18.70. Fibrosis
is common in t-myeloid neoplasms i18.71, i18.72 [Czader 2009,
Vardiman 2008a].
i18.63 This bone marrow aspirate smear shows an admixture of residual chronic
lymphocytic leukemia and concurrent therapy-related acute myeloid leukemia.
Note abundant small lymphocytes and associated increased blasts. (Wright)

405
i18.64 This bone marrow aspirate smear composite shows prominent i18.65 This bone marrow core biopsy section shows side-by-side therapy-related
monoblastic features in this case of therapy-related acute myeloid leukemia linked acute myeloid leukemia a with residual chronic lymphocytic leukemia b. (H&E)
to topoisomerase II inhibitor therapy. (Wright)
i18.66 Immunoperoxidase staining for CD34 shows markedly increased i18.67 This bone marrow core biopsy section from a case of therapy-related
myeloblasts in the area of therapy-related acute myeloid leukemia, while the portion acute myeloid leukemia shows markedly increased blasts in conjunction with
of the bone marrow effaced by persistent chronic lymphocytic leukemia shows CD34 increased and dysplastic megakaryocytes. (H&E)
positivity only in blood vessels (see i18.65). (immunoperoxidase for CD34)
a b
c d
i18.68 Composite of therapy related-acute myeloid leukemia shows increased i18.69 Residual myeloma is readily apparent by immunohistochemical staining
myeloblasts a, disrupted erythroid coloniesb, abnormal localization of for CD138, with persistence of highly atypical plasma cells on bone marrow aspirate
immature granulocytic cells c, and increased and dysplastic megakaryocytes d. smear (inset). Areas not effaced by residual myeloma were effaced by therapy-related
(immunoperoxidase for CD34, hemoglobin A, myeloperoxidase, and CD42b) acute myeloid leukemia. (immunoperoxidase for CD138 and Wright)
406
a b Down Syndrome-Associated AML and Transient Abnormal

Myelopoiesis
Perhaps the paradigmatic example of AML that is
defined by the clinical setting in which it arises is myeloid
leukemia associated with Down syndrome, which is very
often the sequela to an unusual phenomenon, specific to
neonates with Down syndrome, called transient abnormal
myelopoiesis (TAM, aka transient myeloproliferative
disorder) [Baumann 2008]. Immunophenotypically, morpho-
logically, and clinically, TAM is essentially indistinguishable
from acute myeloid leukemia, but it shows the unique and
mysterious property of spontaneous regression i18.73, i18.74,
i18.75, i18.76. The incidence of TAM is quite high in Down
i18.70 This composite of therapy-related acute myeloid leukemia following syndrome, occurring in at least 1 in 10 neonates. The true
therapy for Ewing sarcoma shows myeloid leukemia a, and admixed clusters of incidence could be even higher, since differential counts
metastatic Ewing sarcoma on the bone marrow aspirate smear b. (Wright) on the CBCs of apparently healthy newborns with Down
syndrome may not be routinely obtained. In addition,
TAM can occur in utero, in which case it may cause fetal
hydrops and spontaneous abortion. If series of both in
utero and neonatal cases are combined, it is estimated that
perhaps 20% of conceptions with trisomy 21 that reach
at least late-term gestation experience TAM [Zipursky 2003].
The natural history of TAM is spontaneous hematologic
remission, though approximately 20% of patients succumb
before this occurs to associated morbidities, including
cardiopulmonary failure in the setting of ascites, and fatal
progressive hepatic fibrosis secondary to the leukemic
infiltration and cytokine secretion [Massey 2006, Zipursky 2003]. Some
20% of patients with TAM later develop a non-remitting
myeloid leukemia associated with Down syndrome
(MLADS), which is usually megakaryoblastic in nature
i18.71 This low magnification of bone marrow core biopsy section illustrates
a therapy-related myeloid neoplasm with irregular areas of hypercellularity and and occurs before the age of 4 i18.77, i18.78, i18.79,
fibrosis. (H&E)
a b c
i18.72 This composite of fibrotic therapy-related acute myeloid leukemia i18.73 This peripheral blood smear is from a premature infant with Down
shows the morphologic features on high magnification a, the prominent reticulin syndrome, illustrating a marked leukocytosis with a predominance of blasts charac-
fibrosis b, and the collagen fibrosis c (see i18.71). Collagen fibrosis is unusual in teristic of transient abnormal myelopoiesis. (Wright) (courtesy R McKenna, MD)
therapy-related myeloid neoplasms. (H&E, reticulin, and trichrome)
407
i18.74 Both megakaryoblasts and differentiating megakaryocytes are evident i18.75 This bone marrow core biopsy is from a 4-week-old Down syndrome
in this peripheral blood smear from a Down syndrome neonate with transient baby with transient abnormal myelopoiesis. Note increased immature cells and
abnormal myelopoiesis. Note large megakaryocyte fragments. (Wright) markedly increased small abnormal megakaryocytes. (H&E)
i18.76 This bone marrow core biopsy section is from a 4-week-old Down i18.77 This peripheral blood smear is from a 22-month-old Down syndrome
syndrome infant with transient abnormal myelopoiesis. At high magnification, baby with acute megakaryoblastic leukemia. Note profound pancytopenia,
a marked increase in immature blastic cells is evident in conjunction with circulating megakaryoblasts, and dysplastic platelet. (Wright)
abnormal megakaryocytes. (H&E)
a b
i18.78 This bone marrow aspirate smear shows Down syndrome-associated i18.79 This composite of Down syndrome-associated acute megakaryoblastic
acute megakaryoblastic leukemia. (Wright) leukemia shows a comparison of the morphologic features a in conjunction
with the markedly increased megakaryocytic component highlighted by CD31.
(H&E and immunoperoxidase for CD31)
408
[sidebar 18.1] GATA1, Down Syndrome, and

Myeloid Leukemia
M
yeloid leukemia associated with Down syndrome (MLADS) in
the pediatric age group is overwhelmingly megakaryoblastic in
nature, as is transient abnormal myelopoiesis (TAM) described
further in Down Syndrome-Associated AML and Transient Abnormal
Myelopoiesis, p 406. Interestingly, both processes harbor acquired mutations
in GATA1, a gene on the short arm of the X chromosome that encodes a
transcription factor. GATA1 mutations seem to be surprisingly specific for
these settings. The GATA family of proteins contain zinc finger domains
that bind specific DNA sequences. In fact, the name GATA is derived from
the key portion of the sequence recognized: G-A-T-A [Bates 2008]. GATA1 also
binds a series of partner proteins, including the endearingly named friend of
GATA1 (FOG1) [Hamlett 2008, Johnson 2007]. Depending on the partner protein
in the complex, GATA1 can act as an activator or repressor of transcription
at its target sites, many of which bear upon erythroid and megakaryocytic
i18.80 Both blasts and small dysplastic megakaryocytes characterize this differentiation. DS-associated GATA1 mutations introduce early stop
case of Down syndrome-associated acute megakaryoblastic leukemia. (H&E) codons that result in the utilization of an alternative downstream start site
and, consequently, the production of a shorter GATA1 protein lacking the
(courtesy S Peiper, MD)
N-terminal domain necessary for proper regulation of its transcriptional
targets [Xavier 2009].
i18.80 [Hitzler 2007]. The megakaryoblasts seen in MLADS GATA1 mutations alone do not produce acute leukemia, a point
underscored by the existence of a family cohort with an inherited mutation
are largely indistinguishable from TAM; however, they similar to those seen in Down syndrome. These individuals do not develop
have been shown to harbor more complex cytogenetic TAM or leukemia [Hollanda 2006]. Thus, there is not only a special propensity to
abnormalities compared to blasts from TAM [Massey 2006]. develop acquired GATA1 mutations in the setting of trisomy 21, but also a
specific leukemogenic effect directly dependent on the presence of the extra
MLADS is extremely chemosensitive with an overall survival chromosome 21. Some investigators have implicated RUNX1 in this regard
in excess of 80% [Creutzig 2005, Webb 2005, Xavier 2009]. The diagnostic [Hitzler 2007]. The gene encoding the RUNX1 transcription factor is present
distinction of TAM from MLADS depends largely on the in the Down syndrome critical region on chromosome 21, and RUNX1
clinical setting, with the former occurring exclusively in physically associates with GATA1 [Elagib 2007]. RUNX1 is also a key player
in translocations that defi ne certain types of AML and ALL, though its
neonates and, by definition, resolving by 3 months of age. connection with DS-associated processes remains at present circumstantial
MLADS includes a phase of disease with the features of and controversial [Xu 2006a].
myelodysplastic syndrome, which usually precedes the Another unexplained mystery in these cases is why some, but only some,
onset of more pronounced blastemia. Because of this close TAM progresses to MLADS. Researchers have analyzed, via microarray,
blasts from TAM and from the subsequent MLADS, and found that, despite
temporal and prognostic relationship, both of these disease being largely immunophenotypically and morphologically similar, they
states (MDS-like and AML-like) fall under the umbrella form 2 distinct groups at the level of gene expression [Lightfoot 2004]. In other
term MLADS without regard to blast percentage. TAM and words, there appears to be a progression, with many gene pathways affected,
that underlies the reappearance and the aggressive behavior of the GATA1+
MLADS are further compared in t18.5. See sidebar18.1 for clone [Roy 2009]. Th is contention is also supported by the observation that
further discussion of the significance of GATA1 mutations in blasts in MLADS have often acquired cytogenetic abnormalities in addition
these processes. to the constitutional trisomy 21 [Forestier 2008, Massey 2006]. Similarly, while 12%
of TAM had detectable telomerase activity, this number increases to 52%
when considering cases of MLADS [Holt 2002]. Further investigation of TAM
and MLADS will likely yield additional insights into both the similarities
[18.7] AML, Not Otherwise Specified (NOS) and the differences between these unique disorders.
Despite attempts to utilize a biologic-based classification
of AML to the greatest extent possible, the 2008 WHO classi-
fication has included 11 subtypes of AML, not otherwise [Arber 2008c]. Although up to 1/3 of AML cases currently fall
specified (NOS) [Arber 2008c, Vardiman 2009]. A lineage-based system is into AML, NOS, it is likely that this proportion will steadily
used to subclassify those cases of AML that lack any specific decline as more biologic entities are defined by molecular
AML-defining biologic characteristic. Consequently, the genetic studies.
AML, NOS category is reserved for cases that fulfill general The fairly complex lineage and degree of maturation-
criteria for AML but lack: a) an AML recurrent cytogenetic based classification criteria for AML, NOS cases and their
or molecular abnormality, b) a link to prior chemotherapy, key features and associations are delineated in t18.9 [Arber 2008c,
c) multilineage dysplasia involving the majority of cells, d) Barbaric 2007, Bene 2001, Dunphy 2004, Haferlach 2009b, Hama 2008, Kaleem 2001, Malinge 2008,
MDS-type cytogenetic abnormalities, e)an association with Oki 2006]. Each subtype will be briefly reviewed and illustrated to
Down syndrome, or f)a history of MDS or MDS/MPN complement the information in t18.9.

409
t18.9 Acute Myeloid Leukemia, Not Otherwise Specified (AML, NOS)

Type Typical Features Other/Comments
AML with Predominance of agranular blasts
Myeloid lineage confirmed by imunophenotyping; aberrant features common
minimal Lineage of blasts not apparent by
Cytogenetic abnormalities common; if MDS-related cytogenetic abnormalitiy,
differentiation morphology or cytochemistry classify case as AML with MDS-related change
No recurrent cytogenetic abnormalities; poor prognosis
AML without Blasts 90%; blasts exhibit myeloid features Myeloid lineage confirmed by flow cytometric immunophenotyping, aberrant
maturation by morphology (granules, Auer rods) and/ features common
or cytochemistry (MPO+) No recurrent cytogenetic abnormalities
AML with 20% blasts in blood or bone marrow Myeloid antigens expressed; occasional aberrant features
maturation 10% maturing granulocytic lineage cells Key differential diagnostic considerations are RAEB-2 and AMML
<20% monocytic cells No recurrent cytogenetic abnormalities
AMML Blasts and blast equivalents Myelomonocytic lineage confirmed by flow cytometry; aberrant features common
(promonocytes) 20% in blood or bone No recurrent cytogenetic abnormalities
marrow Monocytosis common
20% maturing granulocytic lineage cells Blood picture may closely resemble CMML
20% monocytic lineage cells Key differential diagnoses include CMML, AMoL and AML with maturation,
Cytochemically stains MPO+ and NSE+ and microgranular APL
AMML t(8;16) Fulfills AMML criteria Disruption and fusion of MYST3 (8p11) and CREBBP (16p13)
(p11;p13) Ingestion of erythrocytes by Rare AML subtype, <0.5% de novo AML (see text) and <2% t-AML
myelomonocytic blasts DIC, extramedullary disease including CNS infiltration common
Acute 80% monocytic lineage cells Monocytic lineage confirmed by immunophenotyping; aberrant features
monocytic 20% neutrophilic lineage cells common
leukemia, 2 If monoblasts predominate, termed acute Extramedullary lesions common (CNS, skin, gingiva)
types monoblastic leukemia; if promonocytes Key differential diagnosis includes CMML, microgranular APL, AMML
predominate, termed acute monocytic
leukemia
NSE+ in majority of cells
Acute erythroid Erythroleukemia consists of 50 erythroid Dysplasia often prominent; if 50% in 2 lineages, designation of AML with
leukemia, 2 cells and 20% myeloblasts (percent based MDS-related changes should be used; if MDS-related cytogenetic features
types on non-erythroid cells) present, should be called AML with MDS-related changes
Must also distinguish from RAEB and non-neoplastic disorders
Pure erythroid leukemia 80% erythroblasts Erythroid lineage must be confirmed by immunophenotyping; CD45; may be CD36+
If MDS-related cytogenetic abnormalities detected, should be classified as AML
for MDS-related changes
Differential diagnosis includes Burkitt leukemia, megaloblastic anemia, and
other acute leukemias
Acute 20% blasts in blood or bone marrow Maturation of megakaryocytic lineage may be present
megakaryo- >50% of blasts are megakaryoblasts Megakaryocytic lineage confirmed by immunophenotyping (CD61+, CD41+,
blastic Clumps (pseudometastasis) CD42b+); may also be CD36+
leukemia Variable cytological features Down syndrome associated cases excluded
Pure megakaryoblastic vs multilineage Differential diagnoses include APMF, metastatic lesions, MPN
Fibrosis common
Micromegakaryocytes not included in blast
percentage
Acute basophilic AML with primary basophilic differentiation Immunophenotyping to confirm basophilic differentiation (CD123+, CD203c+, CD9+)
leukemia Coarse basophilic granules (toluidine blue, Must exclude BCR-ABL1 cases and t(6;9) cases
Alcian blue positive) Key differential diagnosis is mast cell leukemia
Acute 20% myeloid blasts Immunohistochemical staining to confirm increased myeloblasts and other
panmyelosis Proliferation of all hematopoietic lineages lineage involvement
with Fibrosis If MDS-related cytogenetic features detected, should be classified as AML with
myelofibrosis MDS-related changes
Absent or minimal splenomegaly If dysplasia 50% of 2 lineages classify as AML with MDS-related changes
Differential diagnoses include acute megakaryoblastic leukemia, primary
myelofibrosis, post polycythemic PV, metastatic lesions
*These AML cases lack any distinctive clinical (ie, not therapy-related), morphologic (ie, multilineage dysplasia), or molecular/genetic features to allow
inclusion in biologic categories
AEL = acute erythroblastic leukemia; AML = acute myeloid leukemia; AMML = acute myelomonocytic leukemia; AMoL = acute monocytic leukemia;
APL = acute promyelocytic leukemia; APMF = acute panmyelosis with myelofibrosis; CMML = chronic myelomonocytic leukemia; CNS = central nervous system;
DIC = disseminated intravascular coagulation; MDS = myelodysplastic syndrome; MPN = myeloproliferative neoplasm; MPO = myeloperoxidase;
NSE = nonspecific esterase; RAEB = refractory anemia with excess blasts
References: [Arber 2008c, Barbaric 2007, Bene 2001, Dunphy 2004, Haferlach 2009b, Hama 2008, Kaleem 2001, Malinge 2008, Oki 2006]

410
a b
i18.81 Acute myeloid leukemia with minimal differentiation is illustrated in this i18.82 This bone marrow aspirate smear is from a patient with acute myeloid
composite, showing the appearance on bone marrow aspirate smear a and bone leukemia without maturation. Note predominance of blasts with little evidence of
marrow core biopsy sections b. There were no convincing morphologic or cytochemical differentiation. (Wright)
features to document myeloid lineage differentiation. (Wright and H&E)
i18.83 Confirmation of myeloid lineage differentiation is evidenced by scant i18.84 This bone marrow core biopsy section from a patient with acute myeloid
myeloperoxidase positivity in this case of myeloid leukemia without maturation. leukemia without maturation shows a striking predominance of blasts with
(myeloperoxidase cytochemical stain) moderate amounts of cytoplasm. (H&E)
[18.7.1] AML with Minimal Differentiation [18.7.3] AML with Maturation

Agranular, lineage-indeterminant blasts predominate The designation with maturation is applied to AML
in this AML, NOS subtype, and flow cytometric immuno- cases (20% blast threshold met) in which at least 10% of
phenotyping is required for delineation of myeloid lineage cells are promyelocytes, myelocytes, metamyelocytes, or
i18.81 [Arber 2008c, Barbaric 2007, Bene 2001, Kaleem 2001]. There are no recurrent neutrophils i18.85, i18.86. However, a significant monocytic
cytogenetic abnormalities, and prognosis is poor. component is not present. Although dysplasia is common in
AML with maturation, both the extent of dysplasia and the
[18.7.2]AML without Maturation cytogenetic findings cannot fulfill AML with MDS-related
Myeloid lineage blasts, based on morphologic and changes criteria (see AML with Myelodysplasia-Related
cytochemical properties, predominate in this AML, NOS Changes, p 400).
subtype i18.82, i18.83, i18.84. The blast percentage exceeds
90%, and there is no significant maturation of these leukemia [18.7.4]Acute Myelomonocytic Leukemia (AMML)
cells. Blasts and promonocytes must be at least 20%,
and substantial granulocytic and monocytic lineage
involvement must be present to justify a diagnosis of acute
myelomonocytic leukemia (AMML) t18.9, i18.87, i18.88. Both

411
i18.85 This peripheral blood smear of acute myeloid leukemia with maturation i18.86 A wide spectrum of intensity of myeloperoxidase staining characterizes
shows circulating dysplastic neutrophils. (Wright) acute myeloid leukemia with maturation. (myeloperoxidase cytochemical stain)
i18.87 Both myeloid and monocytic differentiation are evident in this i18.88 Moderate amounts of myeloperoxidase cytochemical staining are
peripheral blood smear of acute myelomonocytic leukemia characterized by evident within a subset of the blasts in this case of acute myelomonocytic
marked leukocytosis, anemia, and thrombocytopenia. Note dysplastic neutrophil leukemia. Note the admixed monoblasts are myeloperoxidase negative.
and promonocytes. (Wright) (myeloperoxidase cytochemical stain)
cytochemical staining and flow cytometric immunopheno-
typing are useful in confirming the dual lineage involvement
in AMML i18.88. Careful attention to promonocyte
enumeration is key to distinguishing AMML from CMML
t18.4, i18.13. Similarly, both strong uniform myeloperoxidase
positivity and confirmation of PML-RARA fusion are critical
in distinguishing AMML from microgranular APL.
AMML with t(8;16)(p11;p13); MYST3-CREBBP

A very rare subtype of AMML has distinctive genetic and
morphologic features. Cases of AML with t(8;16)(p11;p13)
show prominent erythrophagocytosis by blasts, prominent
cytoplasmic granulation, and frequent extramedullary disease
t18.5, i18.89, i18.90 [Haferlach 2009b]. Cytochemical nonspecific
i18.89 This cytospin smear is from a patient with acute myeloid leukemia and
esterase and myeloperoxidase positivity are present, t(8;16). Note erythrophagocytosis by blasts. (Wright)
supporting a granulocytic and monocytic lineage despite the

412
i18.90 Ingestion of multiple red blood cells by a leukemic blast is evident on the i18.91 This peripheral blood smear illustrates the typical cytologic features of
cytospin smear from a patient with acute myelomonocytic leukemia and t(8;16). acute monoblastic leukemia. Note that virtually all nuclei are oval to round in
shape and there is little evidence of monocytic maturation. (Wright)
a b
i18.92 This composite illustrates the cytologic and cytochemical features i18.93 Acute monocytic leukemia with maturation including atypical
of acute monoblastic leukemia. Note strong nonspecific esterase positivity b. monocytes is evident in this peripheral blood smear from a patient with marked
(Wright and nonspecific esterase cytochemical stain) leukocytosis and profound anemia and thrombocytopenia. (Wright) (courtesy
T Keith, MD)
a b more monocytoid appearance of the blasts. The incidence
of t(8;16) is low (0.2%-0.4% of de novo AML) [Haferlach 2009b,
Mitelman 1992]. These patients have a poor prognosis [Becher 1988,
Bernasconi 2000, Haferlach 2009b, Heim 1987, Velloso 1996].
[18.7.5] Acute Monocytic Leukemias

Immature monocytic cells predominate in this
AML, NOS type; 2 subtypes are designated based on
whether monoblasts predominate (ie, acute monoblastic
leukemia) or minimally more differentiated blast equivalent
promonocytes predominate (ie, acute monocytic leukemia)
t18.9, i18.91, i18.92, i18.93, i18.94. Very rare acute monocytic
leukemias can exhibit histiocytic differentiation [Boeckx 2007,
Esteve 1995]. Both cytochemical nonspecific esterase staining
i18.94 This composite illustrates the features of acute monocytic leukemia on
bone marrow core biopsy sections. Note widely spaced nuclei due to abundant and flow cytometric immunophenotyping can be used
amounts of cytoplasm, homogeneous uniform cell infiltrates, and increased to document monocytic differentiation, but nonspecific
tingible body macrophages. (H&E)
413
i18.95 This peripheral blood smear is from an elderly patient with i18.96 This bone marrow aspirate smear shows acute erythroid leukemia with
pancytopenia. Note circulating erythroblasts. (Wright) increased myeloblasts and erythroblasts. (Wright)
a b
i18.97 This composite illustrates abnormal PAS positivity in dysplastic i18.98 This bone marrow core biopsy section from a patient with acute
erythroid cells a and ring sideroblasts b in acute erythroid leukemia. (Periodic erythroid leukemia shows hypercellularity. Areas of dark blue are erythroid,
acid-Schiff and Prussian blue) while areas that are slightly more eosinophilic are myeloid infiltrates. (H&E)
esterase stains may be negative or weak in up to 20% of cases

[Arber 2008c, Dunphy 2004]. Extramedullary disease is common in
acute monoblastic/monocytic leukemias. Key differential
diagnostic considerations include CMML, microgranular
APL, and AMML.
[18.7.6] Acute Erythroid Leukemia

The acute erythroid leukemias are further divided into
2 subtypes. In cases designated as erythroleukemia, both a
myeloblast component (20% of non-erythroid cells) and an
erythroid component (50% of all cells) are present, while
the term pure erythroid leukemia is reserved for cases with
80% erythroblasts with little, if any, granulocytic lineage
involvement i18.95, i18.96, i18.97, i18.98, i18.99, i18.100, i18.101, i18.99 High magnification of this bone marrow core biopsy section from a
i18.102, i18.103 [Arber 2008c]. Dysplasia is common in erythro- patient with acute erythroid leukemia shows abundant immature erythroid and
leukemia, but it must be 50% of the lineage cells. If 50% of myeloid lineage cells. (H&E)

414
a b
i18.100 This composite of immunohistochemical stains from a case of acute i18.101 This bone marrow aspirate smear in acute pure erythroid leukemia
erythroid leukemia shows increased myeloblasts by CD34 staining a and shows an overwhelming predominance of erythroblasts. (Wright)
numerous erythroblasts by CD117 staining b. (immunoperoxidase for CD34
and CD117)
i18.102 Both abnormal erythroblasts, marked anisopoikilocytosis, and i18.103 Markedly increased erythroblasts with dispersed chromatin and
dysplastic neutrophils are evident in the peripheral blood in a patient with acute oblong nucleoli efface this bone marrow core biopsy section in acute pure
erythroid leukemia. (Wright) erythroid leukemia. (H&E)
cells in at least 2 lineages are dysplastic, a diagnosis of AML

with MDS-related changes is warranted. The diagnosis of pure
erythroid leukemia is challenging because erythroblasts can
closely resemble Burkitt leukemia, acute megakaryoblastic
leukemia, and other neoplasms. Immunophenotyping is
essential in lineage delineation i18.101.
[18.7.7] Acute Megakaryoblastic Leukemia

The diagnosis of acute megakaryoblastic leukemia can be
uniquely challenging because of the frequent assocation with
fibrosis, which can preclude aspiration for morphology and flow
cytometric immunophenotyping t18.9. Consequently, immuno-
histochemical stains are often required to help confirm both the
i18.104 A marked predominance of uniform megakaryoblasts with striking
minimal 20% blast percentage necessary for a diagnosis of AML
cytoplasmic blebbing is evident on this bone marrow aspirate smear from a and to confirm that at least 50% of the blasts are megakaryo-cytic
15-month-old male with acute megakaryoblastic leukemia not related to Down i18.104, i18.105, i18.106, i18.107, i18.108, i18.109, i18.110, i18.111
syndrome. (Wright)
415
i18.105 This bone marrow clot section is effaced by megakaryoblasts in this i18.106 This circulating megakaryoblast is nondescript and megakaryoblastic
15-month-old male with acute megakaryoblastic leukemia not related to Down lineage is not apparent. (Wright)
syndrome. (H&E)
i18.107 Markedly increased immature megakaryocytic lineage cells are evident i18.108 Striking bone marrow fibrosis is evident in this bone marrow core
in this bone marrow core biopsy section from an adult with acute megakaryo- biopsy section of acute megakaryoblastic leukemia. (reticulin)
blastic leukemia. (H&E)
i18.109 Immunoperoxidase staining for CD61 highlights tremendous i18.110 Admixed immature granulocytic cells are highlighted by myeloper-
numbers of megakaryocytic cells on this bone marrow core biopsy section of acute oxidase staining in this bone marrow core biopsy section in acute megakaryo-
megakaryoblastic leukemia. (immunoperoxidase for CD61) blastic leukemia (see i18.109). (myeloperoxidase cytochemical stain)

416
i18.111 Both circulating megakaryoblasts and more differentiated i18.112 This bone marrow aspirate smear shows acute basophilic leukemia.
megakaryocytic lineage cells are evident in this peripheral blood smear from (Wright) (courtesy R McKenna, MD)
a patient with a high-grade megakaryocytic neoplasm. Note tremendously
enlarged and agranular circulating megakaryocyte fragments. (Wright)
[Arber 2008c]. As emphasized, early differentiating megakaryocytic

lineage cells and dysplastic megakaryocytes such as
micromegakaryocytes are not included in the blast percentage
i18.107, i18.111. Similarly, cases of acute megakaryoblastic
leukemia linked to biologic genetic features such as Down
syndrome or t(1;22) are not included in this AML, NOS
category.
Acute megakaryoblastic leukemia is more common in
children than adults. Pediatric cases of this leukemia show a
similar favorable outcome regardless of whether or not they
are linked to Down syndrome [Hama 2008]. Outcome in adults is
unfavorable [Oki 2006].
The differential diagnosis of acute megakaryoblastic i18.113 Basophilic blast phase of chronic myelogenous leukemia is evident in
leukemia includes other acute leukemias as well as diverse this bone marrow aspirate smear. (Wright)
disorders associated with bone marrow fibrosis, including
chronic myeloproliferative neoplasms and metastatic lesions.
[18.7.9] Acute Panmyelosis with Myelofibrosis
[18.7.8] Acute Basophilic Leukemia The diagnosis of this rare subtype of AML, NOS, is
De novo acute leukemias that exhibit basophilic differen- problematic both because of the obligatory myelofibrosis and
tiation are very uncommon and must be distinguished from because diagnostic requirements include confirmation that
BCR-ABL1-related disorders such as basophilic blast phase of blasts exceed 20% and documentation of expanded myeloid,
chronic myelogenous leukemia t18.9, i18.112, i18.113. Genetic erythroid, and megakaryocytic populations in a bone marrow
testing is essential in making this distinction as well as in that is typically inaspirable t18.9, i18.114, i18.115 [Arber 2008c,
identifying cases of AML with t(6;9), which are excluded Orazi 2005, Thiele 2004]. There is a heavy reliance on immunohisto-
from this AML, NOS group. Basophilic differentiation is best chemical staining to establish this diagnosis i18.115.
determined by either metachromatic staining (toluidine blue The potential overlap with myeloproliferative neoplasms
and alcian blue positivity) or by flow cytometric immuno- is substantial. Therefore, cases of acute panmyelosis with
phenotyping to exclude mast cell leukemia and other myeloid myelofibrosis (APMF) must exhibit an acute onset, lack
lineage leukemias, as well as to provide support for basophilic significant splenomegaly, and lack teardrop erythrocytes.
lineage differentiation (CD123+, CD203c+, CD9+) [Arber 2008c]. Confirmation of an AML-defining blast threshold by
Electron microscopy can also be used to identify theta immunohistochemical staining for CD34 is critical in distin-
granules [Peterson 1991, Shvidel 2003]. guishing APMF from myelodysplasia with fibrosis i18.115

417
a b
t18.10 Prognostic Risk of Cytogenetic Abnormalities
in Patients <60 Years with De Novo AML
Prognostic Risk Group Cytogenetic Finding
Favorable t(15;17)(q22;q21)
t(8;21)(q22;q22)
inv(16)(p13;q22)/t(16;16)(p13;q22)
Intermediate Normal karyotype
t(9;11)(p22;q23)*
del(7q), del(9q)*
Y, +11, +13, +21
Unfavorable Complex karyotype
inv(3)(q21;q26)/t(3;3)
i18.114 This peripheral blood smear composite is from a 56-year-old male t(6;9)(p23;q34)
with acute panmyelosis with myelofibrosis. The patient did not have spleno- 5, 7
megaly. This peripheral blood smear composite shows a circulating myeloblast, a *Some may classify as unfavorable
dysplastic neutrophil, and profound cytopenias. (Wright)
3 chromosomal abnormalities

In intermediate risk group in [Byrd 2002]
a b c AML = acute myeloid leukemia
References: [Breems 2008, Byrd 2002, Cheng 2009, Dohner 2010, Farag 2006, Grimwade 1999, Grimwade 2001,
Kelly 2009, Kolitz 2004, Mrozek 2004, 2008a, Schlenk 2004, Slovak 2000, Weinberg2010]
(submicroscopic) characterization of the karyotypically

normal AMLs is an area of active investigation (see [18.8.2]
Molecular Genetics).
Based on several large studies, a cytogenetic risk stratifi-
cation system has been proposed for AML that categorizes
specific karyotypic abnormalities as favorable, intermediate,
or unfavorable t18.10 [Breems 2008, Byrd 2002, Cheng 2009, Dohner2010, Farag 2006,
i18.115 This bone marrow core biopsy composite from a patient with acute Grimwade 1999, 2001, Kelly 2009, Kolitz 2004, Mrozek 2004, 2008a, Schlenk 2004, Slovak 2000].
panmyelosis with myelofibrosis shows increased immature myeloid lineage cells and These data may be used to stratify therapy [Kolitz 2004]. The
abnormal megakaryocytes a, while increased fibrosis b and markedly increased majority of large studies agree that the AML patients with
blasts are evident c. (H&E, reticulin, and immunoperoxidase for CD34). t(15;17), inv(16)/t(16;16), or t(8;21) have a favorable
(see t16.12). Estimation of the extent of lineage dysplasia is prognosis compared with those that have a complex karyotype
important to distinguish APMF from AML with MDS-related and monosomy 7 [Mrozek 2008a]. Of note, while additional
changes. Finally, a metastatic lesion must be excluded by chromosomal changes with t(8;21), inv(16)/t(16;16), or
morphologic and immunohistochemical assessment. t(15;17) may be seen, these have not been generally shown
to affect prognosis [Byrd 2002, Kelly 2009, Schlenk 2004]. FLT3 mutations
may occur in t(8;21) and inv(16), and although not well
[18.8] Prognostic Factors in AML established, may suggest an adverse prognosis [Boissel 2006]. A
monosomal karyotype in AML may portend a particularly
[18.8.1] Cytogenetics unfavorable prognosis [Breems 2008 , Weinberg2010].
Pretreatment conventional cytogenetic studies identify In patients 60 years with de novo AML, the signif-
an acquired clonal abnormality in approximately 50% - 60% of icance of some of cytogenetic risk groups may differ from
patients with de novo AML, of which 10% - 20% are complex younger patients. A very complex karyotype, defined as 5
(3 chromosomal aberrations). In approximately 40% - 50% chromosomal abnormalities, is associated with an unfavorable
of cases, no karyotypic abnormality is detected using typical risk in the older age group, are non-complex karyotypes
banding techniques, yielding an AML with normal karyotype showing a rare aberration such as trisomy 4, abnormalities of
[Byrd 2002, Cheng 2009, Grimwade 1999, 2001, Mrozek 2004, Slovak 2000]. Molecular 3q, t(6;9)(p23;q34), and double minutes [Farag 2006, Grimwade 2001].

418
[18.8.2] Molecular Genetics t18.11 Potential Immunophenotypic Markers with
In addition to the gene mutations (NPM1 and CEBPA) Prognostic Significance in AML at Diagnosis
that currently define provisional biologic subtypes of AML
Marker Prognostic Significance
(see [18.6] Biologic Subtypes of AML, p 385), there is an
ever-expanding catalogue of additional genetic alterations CD25 Adverse overall and relapse-free
survival
that occur in significant numbers in AML of various subtypes.
These include mutations (eg, TET2, MLL, KRAS, NRAS, CD15 Favorable prognosis
WT1) and alterations in gene expression levels (eg, BAALC, CD7 Controversial; lower complete
remission rate; adverse prognosis
ERG, MN1, EVI1, MN1, PRAME, MLL, WT1, RHAMM) in several studies
[Abdel-Wahab 2009, Baldus 2007, Gaidzik 2008, Paschka 2008b, Santamaria 2009, Schlenk 2008].
CD11b Adverse prognosis
As mentioned in [18.6] Biologic Subtypes of AML, KIT
CD56 Lower complete remission rate
mutations are particularly associated with a relatively
adverse prognosis in the core binding factor AMLs [t(8;21) Expression of lymphoid Lower complete remission rate
antigens (adults)
and inv(16)/t(16;16)] [Cairoli 2006, Care 2003, Paschka 2006]. In general,
No prognostic significance (majority
these alterations contribute to leukemogenesis and carry of pediatric patients)
prognostic significance, but, in contrast to NPM1 and CEBPA Leukemia-associated Lower complete remission rate;
mutations, they do not presently define distinct biologic or phenotype (LAP) (aberrant/ adverse prognosis
clinical entities. Scoring systems incorporating many of these asynchronous antigen profile
alterations have been developed and appear to be useful in patterns)
predicting prognosis [Santamaria 2009]. A major challenge in the Early clearance of peripheral Better disease-free survival
coming years will be the rational and cost-effective implemen- blasts
tation of such multivariate molecular assays. References: [Al-Mawali 2009b, Casasnovas 1998, Del Poeta 1995, Lacombe 2009, Mason 2006, Smith 1992,
Standing out from this alphabet soup is FLT3, which Terwijn 2009]
encodes a membrane-bound receptor tyrosine kinase.
Activating FLT3 mutations occur in 2 forms, only 1 of which
is at present incontrovertibly significant for AML prognosis.
So-called internal tandem duplications (ITD) affecting the exact prognostic significance of FLT3 mutations may be
the juxtamembrane portion of the protein correlate with variable or undocumented in the context of these distinct
poor prognosis, an association that has been particularly AML subtypes.
documented in karyotypically normal cases [Kottaridis 2001, Schlenk 2008,
Stirewalt 2003, Zwaan 2003]. In contrast, the significance of mutations
[18.8.3] Flow Cytometry
affecting the tyrosine kinase domain (TKD) of the protein Aside from the fact that certain immunophenotypic
is controversial, with recent data suggesting that FLT3-TKD profiles tend to track with specific AML categories, certain
mutations may impact prognosis, though perhaps differently, aberrant immunophenotypic findings have prognostic
in distinct subtypes of AML [Bacher 2008, Frohling 2002, Mead 2007, Whitman 2008, significance. These distinctive immunophenotypic features
Yanada 2005]. Clinical trials are currently evaluating the pharma-
are also useful in the assessment for minimal residual disease.
cologic effectiveness of specific FLT3 inhibitors [Sanz 2009a]. Potential predictive markers in AML diagnosis are listed in
As of this writing, a few general observations may be made t18.11 [Al-Mawali 2009b, Casasnovas 1998, Del Poeta 1995, Lacombe 2009, Mason 2006, Smith 1992,
Terwijn 2009].
about the clinical situations in which FLT3 analysis is
indicated. First, FLT3 must be examined in all cases submitted The prognostic value of immunophenotyping in AML
for NPM1 mutation analysis, since an FLT3 mutation in is controversial [Bradstock 1993, Ball 1991]. Many of the studies have
an NPM1-mutated case drastically reduces the beneficial been small, single-institution studies, with few results being
prognosis otherwise associated with the latter finding. confirmed in large clinical trials. The different results likely
Second, because FLT3-ITD status is among the strongest stem from a variety of technical, analytic, and population-
independent prognostic factors in cytogenetically normal based factors. In particular, how the blast population is gated
AML, molecular analysis for alterations at this location is and the cut-off point (or percentage) for selection of antigen
eminently defensible. Third, given the burgeoning interest positivity is problematic.
in targeted anti-FLT3 therapy, the practicing pathologist
may find him- or herself fielding requests for FLT3 mutation [18.8.4]Other Prognostic Factors
analysis for a wide array of AML subtypes. In such a situation, Additional prognostic factors delineated in various
the diagnostician should remain cognizant of the fact that AML outcome studies in adults include age, WBC, de novo
vs secondary AML, performance status, and rapidity in the
419
a b a b c
i18.116 Hypocellular acute myeloid leukemia is evident in this composite i18.117 This composite highlights the morphologic and immunohistochemical
illustrating morphologic features a and markedly increased immunophenotypic features of hypocellular acute myeloid leukemia a, characterized by increased
blasts b. (H&E and immunoperoxidase for CD34) CD34+ blasts b that are myeloperoxidase positive c. (H&E and immunoper-
oxidase for CD34 and myeloperoxidase)
clearance of blasts from either blood or bone marrow [Derolf 2009, aplastic anemia can usually be achieved by the integration of
Dohner2010, Hussein 2008, Lacombe 2009, Wheatley 2009]. Although overall survival morphology and immunohistochemical stains i18.117.
in adults with AML has improved over time, the survival time
for advanced elderly patients (80 years) with AML has not [18.9.3] AML with Necrosis
improved [Derolf 2009]. Improved survival times for children with The delineation of lineage and stage of maturation is
AML have also been noted, although factors predictive of uniquely challenging in extensively necrotic specimens. Bone
inferior survival include age >16 years, non-white ethnicity, marrow aspirate smears may contain insufficient intact cells
absence of a related donor, WBC 100 109/L, and adverse for adequate assessment. Similarly, both flow cytometric
karyotype [Lange 2008]. immunophenotyping and conventional karyotyping are
frequently unsuccessful due to low viability and paucicel-
lularity. The successful diagnosis of extensively necrotic
[18.9] Diagnostic Pitfalls in AML Diagnosis AML is most often achieved by serial sectioning of generous
core biopsy sections and extensive immunohistochemical
[18.9.1] Low Blast Count AML assessment i18.118, i18.119, i18.120. Distinction from other
AMLs with t(8;21), inv(16)/t(16;16), or t(15;17) as necrotic bone marrow infiltrates rests largely with compre-
described earlier and in t18.5 may be diagnosed when the blast hensive immunohistochemical staining. However, blood
count is <20% i18.15, i18.28. Clues to identifying these low smears should be reviewed for evidenced of circulating viable
blast count AML cases include severe peripheral cytopenias neoplastic cells that could be assessed for lineage and stage of
with variable numbers of blasts, Auer rods, abnormal salmon- maturation by flow cytometric techniques.
colored granules and/or abnormal eosinophil precursors,
or distinctive flow cytometric studies. All of these features [18.9.4] AML with Fibrosis
are associated with the more overt AMLs with these same Fibrosis is a defining feature of APMF and is highly
cytogenetic abnormalities t18.5. Cytogenetic studies are characteristic of acute megakaryoblastic leukemia and
essential in confirming the AML-defining translocations and therapy-related myeloid neoplasms. Beyond these specific
excluding other differential diagnostic considerations. AML subtypes, mild to moderate reticulin fibrosis can be
encountered in almost any other AML subtype. In light of
[18.9.2] Hypocellular AML the inaspirability of these fibrotic bone marrows, definitive
Rare cases of AML present in a markedly hypocellular diagnosis of AML often relies upon immunohistochemical
bone marrow i18.116, i18.117. Causes of this uniquely reduced assessment of good quality bone marrow core biopsy sections
cellularity are unknown. The key challenge in these cases is to confirm by estimate that CD34+ blasts are 20% and to
to document that blasts exceed the 20% threshold. Further confirm myeloid lineage by CD33, MPO, and other myeloid
subclassification may or may not be feasible. Distinction from antigen expression i18.121, i18.122. CD117 staining is also
hypocellular MDS, hypocellular hairy cell leukemia, and useful in delineating immature myeloid and some monocytic
cells. Distinction from other fibrotic neoplastic and
420
i18.118 This low magnification of a bone marrow core biopsy section is i18.119 Necrotic acute myeloid leukemia is highlighted on higher magnifi-
extensively necrotic in this patient with massive relapse of acute myeloid leukemia cation of this bone marrow core biopsy section. The necrotic areas are eosinophilic
following bone marrow bone transplantation. (H&E) (right), while scattered collections of intact leukemia cells are evident (left).
(H&E)
i18.120 Necrotic acute myeloid leukemia is highlighted by immunoperoxidase i18.121 This bone marrow core biopsy section shows prominent fibrosis in
staining for CD43 in the focally viable areas. (immunoperoxidase for CD43) association with acute myeloid leukemia. Note spindling of cells reflecting fibrosis.
(H&E)
a b non-neoplastic disorders is achieved largely through immuno-

histochemical studies (see t16.12).
[18.9.5] Myeloid Proliferations with Abundant Erythroid

Cells
Myeloid proliferations with abundant erythroid cells
can generate a challenging set of differential diagnostic
possibilities, including both benign and malignant processes
t18.12 [Arber 2008c, Morice 2005, Tso 2009, Vardiman 2008b]. Arriving at the correct
diagnosis most often requires the assimilation of a substantial
amount of data, including hematologic indices, morphology,
additional laboratory studies (eg, vitamin B12, folate, and
zinc levels), and genetics. In cases that are clearly malignant,
adherence to the diagnostic criteria for each myeloid
i18.122 A predominance of blasts is evident on H&E stain a, while strikingly
increased reticulin fibers are highlighted b in acute myeloid leukemia with neoplasm category will lead to the correct WHO 2008
fibrosis. (H&E and reticulin) subclassification [Vardiman 2008b]. By convention, erythroblasts

421
t18.12 Differential Diagnostic Possibilities for Myeloid Proliferations with Abundant Erythroid Cells
Diagnostic Possibility Comments/Bone Marrow Features
Acute erythroid leukemia: 50% erythroid cells
pure erythroid type 80% erythroblasts
erythroleukemia (erythroid/myeloid) Myeloblasts 20% of non-erythroid cells
Myelodysplastic syndrome <20% myeloblasts; variable erythroid percentage (see Chapter 16)
AML with myelodysplasia-related changes 20% blasts; 50% of dysplastic cells in at least 2 lineages
AML, NOS, with increased erythroid precursors 50% erythroid lineage cells
Polycythemia vera Erythroid predominance with complete maturation; minimal/no dysplasia
Blasts not increased
Peripheral erythrocytosis
Low erythropoietin level
Increased megakaryocytes, usually hyperlobated
Bone marrow hypercellularity with megakaryocyte abnormalities
JAK2V617F mutation positive in 95% of cases
Non-neoplastic erythroid proliferations:
megaloblastic anemia Erythroid hyperplasia with left shift, sieve-like chromatin, giant metamyelocytes (see
Chapter 6)
florid hemolytic anemia Erythroid hyperplasia with intact maturation; nuclear budding, multinucleation (see
Chapter 6)
florid erythroid regeneration post chemotherapy Intact maturation, minimal dysplastic change (mainly seen in most mature cells)
recombinant erythropoietin administration Erythroid hyperplasia with left shift (see Chapter 35)
congenital dyserythropoietic anemia Ineffective erythropoiesis, marked dyserythopoiesis; multinucleation (see Chapter 6)
Tumors mimicking erythroblasts: Immunophenotypic studies reveal lineage

Burkitt/ other lymphoma types
metastatic tumors
other acute leukemias
myeloma
AML = acute myeloid leukemia; NOS = not otherwise specified
References: [Arber 2008c, Morice 2005, Tso 2009, Vardiman 2008b]
should predominate in acute erythroid leukemia, while rods, megaloblastic anemia must remain in the differential
myelodysplasia tends to show an admixture of all stages diagnosis. Genetic studies are often helpful in addition to
of erythroid maturation i18.96, i18.101 (see Chapter 16). serum vitamin B12, folate, and methylmalonic acid levels (see
Distinguishing high-grade myelodysplasia with abundant Chapter 6).
erythroid cells (50%) from AML with MDS-related
changes or acute erythroid leukemia hinges on the percent [18.9.6] G-CSF Therapy
of non-erythroid cells that are myeloblasts. Clinical hetero- Therapeutic doses of recombinant G-CSF or granulocyte
geneity is well-described in acute erythroleukemia; many macrophage colony-stimulating factor (GM-CSF) act
patients may not require the urgent therapy needed in other similarly and may induce a variety of cellular changes
settings of acute leukemia (ie, clinically more akin to MDS). (eg, transient increases in peripheral blood or bone marrow
In such scenarios it is prudent to communicate with the blasts and/or neutrophil dysplasia) that may mimic a myeloid
submitting physician regarding the diagnostic criteria utilized neoplasm (see t10.6) [Meyerson 1998]. Consequently, it is essential
and the clinical heterogeneity of cases with 50% erythroid that the diagnostician have information about cytokine
lineage cells that fulfill AML criteria. therapy as part of routine bone marrow examination. In de
Megaloblastoid change is a frequent dysplastic novo presentations of bone marrow left-shifted myeloid
morphologic feature in myeloid malignancies. Without hyperplasia and increased blasts, but lacking definitive
other types of overt dysplasia, an increase in blasts, or Auer dysplasia, Auer rods, or abnormal karyotype, one should
422
a b exercise extreme caution prior to diagnosing a myeloid

malignancy. A reactive process such as CSF effect, infection,
or paraneoplastic phenomenon secondary to an underlying
solid tumor must be ruled out.
[18.10] Minimal Residual Disease, Post-Therapy

Changes, and Relapse
Minimal residual disease monitoring in AML by
either flow cytometric immunophenotyping or molecular
techniques can potentially improve assessment of ongoing
treatment response and ultimately improve outcomes
[Al-Mawali 2009a]. Established minimum residual disease (MRD)
assays include PCR amplification of known genetic
i18.123 This composite illustrates the appearance of markedly increased
benign megakaryocytes on a bone marrow aspirate smear a and bone marrow aberrations or flow cytometric detection of aberrant immuno-
clot section b in this abnormal megakaryocytic proliferation following induction phenotypes [Kern 2008, Shook 2009]. Quantitative PCR assessment of
chemotherapy for acute myeloid leukemia and t(11q23). (Wright and H&E) APL is addressed in Chapter 36.
Flow cytometric immunophenotyping may attain a
sensitivity of aberrant cell detection of 10-4 compared with the
traditional techniques of morphology (1% - 5%) and cytoge-
netics (1% - 5%) (see Chapter 36). The sensitivity of detection
of MRD by molecular techniques is in the range of 10-5.
Several published reports indicate that the detection
of MRD is a valuable tool in predicting relapse [Feller 2004,
San Miguel 1997, 2001, Venditti 2002]. The lack of flow cytometric detection
of leukemic cells after induction chemotherapy may argue
against early AML relapse, and a threshold of 0.15% residual
leukemic cells post induction and post consolidation therapy
predicts for better overall survival (see Chapter 36 for more
details) [Al-Mawali 2009a, Drach 1992].
The morphologic spectrum of bone marrow features
i18.124 This bone marrow clot section is from a patient with early relapse of during induction chemotherapy is detailed in Chapter 35.
acute promyelocytic leukemia in which only half (right side) of the particle is Distinctive post-therapy findings in AML include striking
effaced by recurrent leukemia. (H&E) non-neoplastic dysplastic megakaryocytic proliferations
and ATRA-induced morphologically abnormal maturing
promyelocytes in APL i18.123 [Rosenthal 1991, Tohyama 2003]. Relapse
of AML is generally characterized by a marked decline in cell
counts in blood, variable numbers of circulating blasts, and
variable extent of bone marrow effacement i18.124, i18.125.
These relapsed AMLs generally exhibit similar morphologic,
immunophenotypic, and genetic features to the original
leukemia, although clonal evolution may occur. Rare AML
patients develop therapy-related secondary AML, creating
a unique diagnostic challenge. In general, the characteristic
morphologic and cytogenetic features of t-AML will be
distinct from those of the original leukemia.
i18.125 Markedly increased CD117 positive promyelocytes are evident in a [18.11]Differential Diagnosis of AML
distinct zoning pattern in this bone marrow clot section from a patient with early Depending upon the lineages involved and the extent
relapse of acute promyelocytic leukemia(see i18.124). (immunoperoxidase for of maturation, the differential diagnosis of AML is diverse.
CD117)
423
The primary differential diagnostic considerations for Morphologic look-alikes of myeloid blasts include
specific AML subtypes have been included earlier in the granular ALL, blastic plasmacytoid dendritic cell neoplasm,
discussion of specific AML subtypes. Similarly, the differ- aggressive NK cell leukemia, myeloma, and lymphomas
ential diagnosis of hypocellular, fibrotic, and necrotic AML [Groom 1991, Pitman 2007]. Although bone marrow core biopsy
is included in [18.9] Diagnostic Pitfalls in AML Diagnosis, section morphology generally allows distinction, the bone
p 419. Because myeloid neoplasms with abundant erythroid marrow aspirate smear appearance of rhabdomyosarcoma,
lineage cells are particularly problematic, an earlier section neuroblastoma, medulloblastoma, and other metastatic
focuses on the differential diagnosis of these erythroid lesions can closely mimic acute leukemia (see Chapter 29)
predominant lesions. For AML, definitive diagnosis hinges [Chen 2004, Etzell 2006]. Even immunophenotypic overlap between
on accurate blast enumeration to allow distinction from metastatic lesions and AML has been noted [Etzell 2006].
high-grade MDS and MDS/MPN. The diagnostician must A systematic evaluation including cytochemical stains
be aware that a diagnosis of AML is warranted in cases with and immunophenotyping can aid in these distinctions.
20% blasts if an AML-defining translocation is identified Benign disorders that can mimic selected AML subtypes
(see Low Blast Count AML, p 419). Transformations include megaloblastic anemia, G-CSF therapy, arsenic
into AML by other myeloid neoplasms must also be toxicity, and other toxic bone marrow insults.
recognized. This is best achieved when comprehensive
clinical information is readily available. Previous bone
marrow specimens should be compared systematically to [18.12] Components of the Diagnostic
current specimens to clarify issues of transformation of an Interpretation
underlying hematologic disorder such as MDS, MPN, and The components of the diagnostic interpretation of AML
MDS/MPN. Auer rods are for the most part indicative of must include information regarding blast/blast equivalent
AML, but they may be present in rare patients with MDS enumeration, lineages involved, extent of maturation, and
(see Chapter 16). However, Auer rod-like inclusions have extent of dysplastic features of all lineages. The integration of
also been noted in lymphomas and myeloma (see i23.20) molecular genetic information is also essential. t18.13 provides
[Groom 1991, Hutter 2009]. tips and strategies for this process.
t18.13 Diagnosis of AML: Key Tips/Strategies

Parameter WHO Criteria Comments/Tips/Caveats
Percent 20% for AML (blast threshold based on 20% threshold may be met in blood or BM
blasts/blast percentage of non-erythroid cells for AML Difficult to count blasts on inaspirable specimens
equivalents subtypes with 50% erythroid cells in BM)
Promyelocytes are blast equivalents only for APL
Threshold < 20% for genetically defined AML
[eg, t(8;21), t(15;17), inv(16)] Promonocytes are always blast equivalents
Myeloblast threshold may not be reached in Erythroblasts are blast equivalents only in acute pure erythroid leukemia
acute pure erythroid leukemia due to marked Distinction from MDS and MDS/MPN based largely on blast percentage
preponderance of erythroblasts (80%) and genotype
Flow cytometry is not a substitute for morphology in determining blast
percent; not all blasts are CD34+
Lineage of blasts Must confirm lineage of immature cells by Morphology, cytochemical stains, and multicolor flow cytometric IP
morphology, cytochemistry, and/or IP integrated into myeloid lineage determination and exclusion of ALL
Most relevant to AML, NOS classification May have to rely on IHC staining on core biopsy for inaspirable
for acute myelomonocytic, monocytic, specimens
megakaryoblastic leukemias
Cytogenetics/ Biologic parameters are integral part of WHO Recurrent cytogenetic abnormalities that define AML subtypes include
FISH/molecular classification of AML t(8;21), t(15;17), inv(16), t(9;11), t(6;9), inv(3), t(1;22)
A specific cytogenetic or molecular finding Molecular abnormalities that define AML subtypes include NPM1 and
defines many AML subtypes CEBPA mutations
ALL = acute lymphoblastic leukemia; AML = acute myeloid leukemia; AML, NOS = AML, not otherwise specified; APL = acute promyelocytic leukemia;
BM = bone marrow; FISH = fluorescence in situ hybridization; IHC = immunohistochemistry; IP = immunophenotype; MDS = myelodysplastic syndrome;
MPN = myeloproliferative neoplasm

424
[18.13] Clues and Caveats 16. Distinctive morphologic clues to AML with t(8;21)
1. Clinical, morphologic, cytochemical, immunophe- include low blast count, long, tapered Auer rods, and
notypic, and molecular genetic features should all be salmon-colored cytoplasmic granulation with a rim of
integrated into AML diagnosis. basophilia.
2. Hematopoietic failure (anemia, neutropenia, and 17. Distinctive morphologic clues to AML with inv(16)
thrombocytopenia) is an expected feature of all types include increased bone marrow eosinophils with mixed
of acute myeloid leukemia; the WBC count is highly eosinophil/basophil granules.
variable. 18. Distinctive clues to APL include marked leukopenia,
3. Exceptions to the 20% threshold for AML diagnosis profound thrombocytopenia, hypergranular
include cases with AML-defining cytogenetic promyelocytes, stacks of Auer rods, maturation block at
abnormalities [(eg, t(8;21), t(15;17), and inv(16)] and promyelocyte stage, intense myeloperoxidase positivity,
acute erythroid leukemia. and coagulopathy.
19. Distinctive morphologic and CBC features of
4. Promyelocytes are blast equivalents only in APL.
microgranular APL include leukocytosis, folded (sliding
5. Promonocytes (blast equivalents) have slightly folded
plate) nuclei, rare hypergranular promyelocytes,
nuclei, dispersed chromatin, nucleoli, and moderate
maturation block at promyelocyte stage, intense myeloper-
amounts of basophilic cytoplasm. oxidase positivity, and coagulopathy.
6. Promonocyte enumeration is critical in the distinction 20. There is substantial morphologic overlap between
between acute and chronic monocytic leukemias. microgranular APL and acute monocytic/
7. Erythroblasts are included in the blast percentage only in myelomonocytic leukemias.
cases of acute pure erythroid leukemia. 21. Three different scenarios are used to classify a case as AML
8. Maturing megakaryocytic lineage cells and micromega- with MDS-related changes:
karyocytes are not blast equivalents. a. History of MDS or MDS/MPN (but not linked to
9. Rapid diagnosis of APL is imperative due to risk of antecedent therapy)
hemorrhage. b. MDS-associated cytogenetic abnormality
10. Classic hypergranular APL is usually associated with a c. Multilineage (at least 2 lineages) dysplasia involving at
very low WBC, while marked leukocytosis is typical in least 50% of cells in each affected lineage.
microgranular APL. 22. The lack of nonspecific esterase positivity does not
11. Flow cytometric immunophenotyping is not the exclude a diagnosis of acute monocytic/myelomonocytic
optimal modality to diagnose APL; morphology and leukemia if morphology and flow cytometric immunophe-
cytochemistry are superior for diagnosis, which should notyping support the diagnosis.
be confirmed by genetic testing. 23. Recurrent AML-defining cytogenetic abnormalities
12. The biologic classification of AML includes cases defined prevail over other findings in AML classification, although
by specific cytogenetic or molecular features, as well as therapy-related myeloid neoplasm with an AML-defining
cases defined by clinical parameters: underlying Down cytogenetic abnormality should be classified as therapy-
syndrome, prior chemotherapy, or antecedent myeloid related AML with t(15;17), etc.
24. If a case of AML has complex cytogenetic abnormalities
neoplasm (eg, myelodysplasia).
that include an AML-defining cytogenetic abnormality
13. Therapy-related disorders include cases of AML,
[eg, t(8;21), t(15;17), etc.], it should be classified within
myelodysplasia, and hybrid myelodysplastic/myelopro-
the AML-defining cytogenetic abnormality group.
liferative diseases, but, due to similarities in outcome,
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18

Acute Myeloid Leukemia

in 2 clinical situations (1.prior cytotoxic chemotherapy

t18.1 Selected Risk Factors for AML

Inherited Genetic Disorders*

18: A CUTE MYELOID L EUKEMIA

t18.3 Acute Myeloid Leukemia: Diagnostic Steps*

18: A CUTE MYELOID L EUKEMIA

Rare AML lacks surface CD13 and CD33

18: A CUTE MYELOID L EUKEMIA

Erythroblasts are included in the blast percentage only in acute

18: A CUTE MYELOID L EUKEMIA

18: A CUTE MYELOID L EUKEMIA

18: A CUTE MYELOID L EUKEMIA

[18.5] Classification of AML Down syndrome Y myeloid leukemia associated

NPM1 or CEBPA AML with recurrent genetic

18: A CUTE MYELOID L EUKEMIA

AML t(15;17) Young adults Leukocytosismicrogranular Abnormal promyelocytes predominate (blasts

BM Biopsy Flow Cytometry Confirmatory Tests/Other

Hypercellular Blasts: CD34+, CD117+, CD13+, Favorable subtype

Micromegakaryocytes are present Blasts typically express CD41 and Cytogenetics

18: A CUTE MYELOID L EUKEMIA

BM Biopsy Flow Cytometry Confirmatory Tests/Other

18: A CUTE MYELOID L EUKEMIA

also occur after previous chemotherapy (therapy-related)

18: A CUTE MYELOID L EUKEMIA

f18.5 Partial karyotype showing chromosomes 15 and 17 illustrates t(15;17)

Activating mutations in NRAS, KRAS, KIT, or FLT3 are seen

18: A CUTE MYELOID L EUKEMIA

18: A CUTE MYELOID L EUKEMIA

t18.6 Characteristics of AML with Variant RARA Translocations

The differential diagnostic considerations vary between

AML with Variant MLL Translocations

18: A CUTE MYELOID L EUKEMIA

dysplasia is common as is basophilia i18.46, i18.47 [Oyarzo 2004,

18: A CUTE MYELOID L EUKEMIA

AML with t(1;22)(p13;q13); RBM15-MKL1

18: A CUTE MYELOID L EUKEMIA

18: A CUTE MYELOID L EUKEMIA

t18.7 Cytogenetic Abnormalities Sufficient to

18: A CUTE MYELOID L EUKEMIA

t18.8 Comparison of Clinicopathologic Features of 2 Types of Therapy-Related Myeloid Neoplasms*

Bone marrow Usually hypercellular, but may be hypocellular Hypercellular

than specifying subtypes [Vardiman 2008a]. Furthermore, therapy-

18: A CUTE MYELOID L EUKEMIA

a b Down Syndrome-Associated AML and Transient Abnormal

[sidebar 18.1] GATA1, Down Syndrome, and

18: A CUTE MYELOID L EUKEMIA

t18.9 Acute Myeloid Leukemia, Not Otherwise Specified (AML, NOS)

18: A CUTE MYELOID L EUKEMIA

[18.7.1] AML with Minimal Differentiation [18.7.3] AML with Maturation

18: A CUTE MYELOID L EUKEMIA

AMML with t(8;16)(p11;p13); MYST3-CREBBP

18: A CUTE MYELOID L EUKEMIA

[18.7.5] Acute Monocytic Leukemias

esterase stains may be negative or weak in up to 20% of cases

[18.7.6] Acute Erythroid Leukemia

18: A CUTE MYELOID L EUKEMIA

cells in at least 2 lineages are dysplastic, a diagnosis of AML