Annals of Diagnostic Pathology 10 (2006) 39 – 65

Review article
Advances in the pathologic diagnosis and biology of acute
myeloid leukemia
Sergej Konoplev, MD, PhD, Carlos E. Bueso-Ramos, MD, PhD4
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030-4095, USA

Abstract In a general surgical pathology practice, cases of acute myeloid leukemia (AML), including myeloid
sarcoma, are relatively rare; the diagnosis is very often difficult, however, and consequences of a
missed or improper diagnosis compromise patient care. Currently, accurate diagnosis of every case of
AML requires integration of the morphological features and results of cytochemical and
immunohistochemical stains, flow cytometric immunophenotyping, cytogenetics, and molecular
studies. This review focuses on a practical approach to diagnosis of AML according to current
standard of practice and discusses some of recent changes in the field of AML.
D 2006 Elsevier Inc. All rights reserved.
Keywords: Acute myeloid leukemia; Myeloid sarcoma; Cytogenetics; Molecular

1. Introduction the very beginning, the diagnosis is delayed and

appropriate therapy is compromised.
1.1. Why should a surgical pathologist care about acute
3. The disease is encountered relatively rarely in
myeloid leukemia?
extramedullary sites compared with other entities,
Acute myeloid leukemia (AML), including extramedul- and there is a tendency to forget about the possibility
lary tumor referred to as myeloid (granulocytic) sarcoma of AML when dealing with a specimen outside the
(MS), represents a serious problem for a surgical pathologist bone marrow.
for several reasons, including the following: 4. Acute myeloid leukemia demonstrates a wide range
of morphological features, competing in extramedul-
1. Acute myeloid leukemia is a medical emergency,
lary sites with melanoma for the title bgreat
and delay in making a proper diagnosis jeopardizes a
mimicker.Q Therefore, the bclassical pictureQ of MS
patient’s life.
may not be present, and thus the diagnosis of MS is
2. The therapy for AML is not universal; specific
missed in most cases.
therapies are effective for one AML subtype and
totally inappropriate for another, such as all-trans- 1.2. Practical approach
retionic acid (ATRA) for t(15;17), and thus it is not
To assure the best patient care possible, the practical
sufficient to diagnose bAML.Q Acute myeloid
approach to pathologic diagnosis of AML is summarized in
leukemias with recurrent cytogenetic abnormalities
Fig. 1 and discussed here. This discussion is followed by a
such as t(15;17), t(8;21), or inv(16) present not
detailed description of the current World Health Organiza-
uncommonly as extramedullary tumors. Many
tion (WHO) classification of AML [1,2].
modalities used for precise classification (eg,
1. Consider MS in the differential diagnosis of poorly
cytogenetics, flow cytometry) require fresh tissue,
differentiated or undifferentiated tumors. The first step in
and if a possibility of AML is not considered from
recognizing any entity is to remember its possibility, and this
rule applies very well to MS. Good pathology practice advises
4 Corresponding author. Division of Pathology and Laboratory that MS, like melanoma, be put in the differential diagnosis
Medicine, Hematopathology Program, The University of Texas MD
Anderson Cancer Center, Houston, TX 77030-4095, USA. Tel.: +1 713
of every surgical case with poorly differentiated morphology.
792 6328; fax: +1 713 794 1800. 2. Review the patient’s medical history. Knowing that a
E-mail address: cbuesora@mdanderson.org (C.E. Bueso-Ramos). patient has AML usually alerts a pathologist to the
1092-9134/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.anndiagpath.2005.10.001
40 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

possibility of MS, but no history of AML by no means
allows ruling out MS, as it may precede bone marrow
involvement. On the other hand, a report of hypoplastic
bone marrow (which may be diagnosed as aplastic anemia
elsewhere) gives an important hint, as MS may coexist with
hypoplastic bone marrow.
3. Always make touch imprints. Touch imprint technique
does not require much time or resources. Acute myeloid
leukemia blasts are recognized as such on Wright-Giemsa–
stained slides based on the fine chromatin, distinct nucleoli,
and cytoplasmic azurophilic granules that are positive for
myeloperoxidase (MPO) or nonspecific esterase. Granulo-
cytic precursors, megakaryocytes, and normoblasts are also
easily identified on touch imprints. Very often it is possible
to identify some unique morphological features of AML
with recurrent cytogenetic abnormalities, which allows
confirmatory ancillary studies to be expedited. Moreover,
unstained touch imprint slides may be used for ancillary
studies such as cytochemical stains, fluorescence in situ
hybridization (FISH), or polymerase chain reaction (PCR).
Pay attention to immature eosinophils and maturing
granulocytes because their presence is an important diag-
nostic clue pointing toward MS.
4. Do not rely on a limited panel of immunostains.
Practically all immunostains have cross-reactivity with
different entities, and no tumor reacts with all antibodies
with which it is said to react; therefore, a panel of antibodies
is needed to confirm the diagnosis of AML. This is
particularly true for MS. The panel we use includes MPO,
lysozyme, terminal deoxynucleotidyl transferase (TdT),
CD34, CD43, CD45 (LCA), CD68, and CD117. Because
Ewing sarcoma enters the differential diagnosis of cases of
MS, it is important to realize that CD99 expression is not
specific for Ewing sarcoma; CD99 expression was detected
in as many as 55% of MS cases and 43% of AML [3].
5. Be aware of hematopoietic growth factors. Hemato-
poietic growth factors (granulocyte colony-stimulating
factor, granulocyte macrophage colony-stimulating factor,
erythropoietin, thrombopoietin) are potent stimulants of
immature and mature hematopoietic cells, and changes
induced by these factors may be indistinguishable from a
picture of AML [4-6]. Be extremely cautious in diagnosing
AML if hematopoietic growth factors were given to a
patient in the last 4 weeks. Moreover, tumors can produce
hematopoietic cytokines and mimic leukemias. Speak to the
clinician, and if the patient’s clinical condition allows,
require another sample after a period without growth
factor(s). It is also important to be aware of generalized
infections that might cause leukocytosis and a prominent
left shift, a clinical picture referred to as leukemoid reaction.
Information regarding prior chemotherapy is crucial for
making a diagnosis of therapy-related AML.
6. Review the peripheral blood smear. A report by an
outside facility cannot substitute for internal review. The
degree of dysplasia can be subtle and is best identified on
peripheral blood smear. The presence and percentage of blasts
may be underestimated, especially in cases in which immature
cells are blast equivalents rather than classic blasts. In these
cases, monoblasts and promonocytes can be misclassified as
monocytes and neoplastic progranulocytes as myelocytes.
7. Use a systematic approach and secure material for
ancillary studies. See Fig. 1 for more information.
8. Use a systematic approach to the bone marrow sample.
Three important checkpoints include cellularity of aspirate
smear, percentage of erythroid precursors, and percentage of
blasts (or blast equivalents). We routinely make several

Touch imprints1 Gross exam 10 buffered

(at least 6: formalin2
Wright-Giemsa,
5 unstained)

Cell suspension Cell suspension Frozen tissue at Fresh tissue for

in 10 RPMI in 10 RPMI –80oC (at least molecular
medium for medium for 0.5x0.5x 0.5cm)5 study5
Flow cytometry3 cytogenetics4

Fig. 1. Diagram of workflow of a specimen suspected to be AML. 1Touch imprints can be used for cytochemical (MPO, butyrate), immunofluorescence
(TdT, POD test for AML M3), FISH, and molecular studies (cell scraping); 2Formalin fixed, paraffin-embedded tissue can be used for
immunohistochemical, DNA- and RNA-based molecular studies (T-cell receptor beta and gamma, IgH, JAK-2), and FISH; 3Some institutions have
adopted a process and hold policy when cells are processed immediately, but the actual immunophenotypical analysis is held waiting for a pathologist
decision. This approach secures a specimen and avoids unjustified flow cytometric studies; 4Conventional cytogenetics is the only way to obtain a full
karyotype, while all other techniques simply answer the question of whether a particular abnormality is present or absent. It does require viable cells; 5Fresh
and frozen tissue may be used for RNA and DNA extraction. RNA is used for RT-PCR tests for the t(15;17), t(8;21), inv(16), and t(9;22) abnormalities;
DNA for FLT3 and RAS mutation studies, T-cell receptor beta and gamma, IgH, and JAK-2. Some institutions prefer paraffin-embedded tissue to fresh or
frozen tissue for molecular studies.
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65
touch imprints of a bone marrow biopsy specimen and use
them for differential count if the aspirate smear is hypo-
cellular or of poor quality. If erythroid precursors dominate
( N50% of all nucleated cells), the blast percentage is
calculated on the basis of all non-erythroid cells. A similar
approach is used in rare cases of bone marrow involvement
by another malignant process (eg, chronic lymphocytic
leukemia), and blast percentage is reported as a fraction of
cells not involved in the known process. Otherwise, blast
count is reported as a fraction of all nucleated cells.
1.3. Background
Acute leukemias are clonal malignant disorders resulting
from genetic alterations in hematopoietic stem cells [7].
These alterations limit their ability to differentiate into
erythrocytes, granulocytes, and platelets and lead to the
proliferation of bleukemicQ cells or bblasts.Q Acute myeloid
leukemia, also referred to as acute nonlymphocytic leuke-
mia, is a heterogeneous group of disorders [1,2].
Approximately 11 000 cases of AML are diagnosed
annually in the United States. The overall annual incidence
of AML is 3.4 per 100 000 populations [8]. Although
leukemia is the most common malignancy of children
15 years and younger, most cases are diagnosed in older
adults. The median age at presentation for AML is 68 years
[8]. The incidence of AML increases with age being less
than 1 per 100 000 per year for persons younger than
30 years and 17 per 100 000 for persons 75 years of age [8].
The incidence of AML is higher in males than in females
and in whites than in African Americans [8,9]. The
incidence of AML is increasing in the elderly. Several
factors including improved diagnosis and longer life
expectancy, resulting in increased environmental exposures,
are probably responsible for this increase [10]. Acute
myeloid leukemia accounts for less than 15% of cases of
leukemia in children younger than 10 years and 25% to 30%
of cases between 10 and 15 years of age [8,11]; in adults,
AML accounts for 80% to 90% of cases of acute leukemia
[8]. The incidence of leukemia, along with myelodysplasia,
appears to be rising, particularly in the population older than
60 years [10]. In adults, AML is by far the most common
type of acute leukemia. The incidence of AML is slightly
higher in males and in populations of European descent [9].
Recent reports indicate that acute promyelocytic leukemia
(APL), a distinct subtype of AML, is more common among
certain populations of Hispanic background [12].
An increased incidence of AML is seen in patients
involving certain disorders with excessive chromatin fragil-
ity, such as Bloom syndrome, Fanconi anemia, and
Kostmann syndrome, and in individuals with Wiskott-
Aldrich syndrome or ataxia-telangiectasia. Other syndromes
such as Down (chromosome 21 trisomy), Klinefelter (XXY
and variants), and Patau (chromosome 13 trisomy) have also
been associated with AML [13,14].
Survivors of the atomic bombs in Japan had an increased
incidence of myeloid leukemias that peaked 5 to 7 years
41
after their exposure [15,16]. Therapeutic radiations appear
to impart minimal risk, but the risk may be significant if it
was administered with concurrent chemotherapy with an
alkylating agent.
There are two types of chemotherapy-related AML. The
bclassicQ alkylating agent type (eg, cyclophosphamide,
melphalan, nitrogen mustard) has a latency period of 4 to
8 years and often is associated with abnormalities of
chromosome 5 and/or chromosome 7 [17,18]. The other
type, associated with exposure to agents that inhibit the DNA
repair enzyme topoisomerase II (eg, etoposide, teniposide),
has a shorter latency period, usually 1 to 3 years, and is
associated with chromosome 11q23 abnormalities [19].
Drugs such as chloramphenicol, phenylbutazone, chlo-
roquine, and methoxypsoralen can result in bone marrow
damage that may later evolve into AML. Exposure to
benzene, a ubiquitous solvent used in several industries,
cigarettes, dyes, herbicides, and pesticides, has been
implicated as another potential risk factor [20,21].
Acute myeloid leukemia may also be secondary to the
progression of a myelodysplastic process or due to
progression of a chronic bone marrow bstem cellQ disorder,
such as polycythemia vera, chronic myelogenous leukemia,
or paroxysmal nocturnal hemoglobinuria.
1.4. Clinical features
The proliferation of abnormal leukemic cells and their
impact on normal hematopoiesis lead to the symptoms and
signs seen in AML.
Fatigue, bruising or bleeding, fever, and infection reflect
a state of bone marrow failure. Although the term leukemia
may suggest marked elevations in white blood cells counts,
pancytopenia is more common. More than 95% of patients
in whom AML is diagnosed have a hemoglobin level of less
than 12 g/dL. Only 10% of patients with newly diagnosed
AML present with leukocyte count greater than 100 000/lL
[22] and are at higher risk of tumor lysis syndrome and
leukostasis. Tumor lysis syndrome can be due to spontane-
ous or treatment-mediated cell destruction [23,24]. It is
characterized by hyperuricemia, renal failure, acidosis,
hypocalcemia, and hyperphosphatemia [23,24]. Leukostasis
may manifest as dyspnea, chest pain, headaches, altered
mental status, cranial nerve palsies, or priapism [25,26].
Leukostasis and tumor lysis syndrome are both oncologic
emergencies and require prompt recognition and manage-
ment. Disseminated intravascular coagulation is most
common in cases with the t(15;17) [27]. Disseminated
intravascular coagulation is typically seen at presentation or
during induction chemotherapy [28,29] and manifested by
diffuse oozing of blood with thrombocytopenia, hypofibri-
nogenemia, elevated fibrin split products, and deficiency of
coagulation factors [30].
Physical findings other than bleeding and related to
infection may include organomegaly, sternal tenderness,
retinal hemorrhages, and infiltration of gingivae, skin, soft
tissues, or meninges (more common with monocytic
42 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

Neutropenia is common in patients with MS; the marrow

may be hypocellular and often, but not invariably, reveals
increased blasts (Fig. 2). Myeloid sarcoma may be present at
diagnosis of AML or may precede the diagnosis. In
children, the presence of MS involving sites other than skin
was an independent favorable prognostic factor compared

Fig. 2. Hypocellular bone marrow. A patient presented with pancytopenia,

and a bone marrow specimen was hypocellular with no evidence of AML.
The patient was treated with antithymocyte globulin, cyclosporine,
granulocyte colony-stimulating factor, and erythropoietin without re-
sponse. One year later, a mass was detected in the psoas and iliac
region, and biopsy revealed MS. A bone marrow biopsy (shown here)
was obtained at that time; extensive workup including immunohisto-
chemical staining with MPO, lysozyme, CD34, and CD117 demonstrated
no evidence of AML.

variants M4 or M5 French-American British Cooperative

Group [FAB] classification) [31].
Myeloid sarcoma occurs in 2% to 14% of cases of AML,
more frequently in children than in adults [32- 40]. Myeloid
sarcoma most often involves soft tissues, skin, bone
(including periosteum), lymph nodes, and/or the orbit and
paranasal sinuses, but it has been reported in virtually all
anatomic locations [32-40]. Testicular mass is less common
than in ALL; to date only 37 cases have been reported [36].

Table 1
WHO classification of Acute Myeloid Leukemia (AML)
1. AML with recurrent cytogenetic abnormalities
AML with t(8;21)(q22;q22), (AML1/ETO)
AML with inv(16)(p13q22) or t(16;16)(p13;q22), (CBFb/MYH11)
APL (AML with t(15;17)(q22;q21), (PML/RARa and variants)
AML with 11q23 (MLL) abnormalities
2. AML with multilineage dysplasia
With prior MDS
Without prior MDS
3. AML and MDS, therapy related
Alkylating agent related
Topoisomerase II inhibitor related
4. AML, not otherwise categorized
AML, minimally differentiated
AML without maturation
AML with maturation
Acute myelomonocytic leukemia
Acute monoblastic/acute monocytic leukemia
Acute erythroid leukemia (erythroid/myeloid and pure erythroleukemia)
Acute megakaryoblastic leukemia Fig. 3. Myeloid sarcoma involving the myometrium. Note that
Acute basophilic leukemia neoplastic cells are streaming along muscle fascicles. (A) Hematoxylin
Acute panmyelosis with myelofibrosis and eosin (H&E); (B) CD117 immunohistochemical stain; (C) lysozyme
Myeloid sarcoma immunostain.
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65
with patients with MS involving skin or patients without
MS [39]. In adult patient population, patients with MS fare
worse compared with patients without MS [38].
1.5. Handling of the bone marrow or peripheral blood
specimen
Specimens of every case of AML must be handled
properly to optimize the diagnostic yield of the ancillary
tests. The specimens are most commonly peripheral blood
or bone marrow aspirates. A bone marrow core biopsy with
touch imprints and bone marrow aspirate clots also should
be obtained in all newly diagnosed cases. Specimens
intended for flow cytometry should be kept in test tubes
containing EDTA. A sterile sample for cytogenetic analysis
is collected with sodium heparin as the anticoagulant.
Samples for cytogenetics are cultured immediately or kept
at 48C for culture within 24 hours of collection. Specimens
kept at room temperature are acceptable for flow cytometric
analysis for up to 3 days.
For molecular studies, a variety of specimens can be used,
depending on which specific molecular tests are required.
For instance, detection of monoclonal rearrangements of the
T-cell receptor gene and the immunoglobulin heavy chain
gene through PCR assays can be achieved with fresh tissues,
air-dried smears, Wright-Giemsa–stained smears, and for-
malin-fixed, paraffin-embedded tissues. Because heparin
interferes with the results of PCR-based molecular studies,
EDTA is preferred in this situation and the samples should be
kept at 48C. RNA must be extracted within 24 hours after
Fig. 4. Acute myeloid leukemia involving the uterine cervix. (A) Low power demonstrates an ill-defined infiltrate extending to the surface of a vaginal cuff. (B)
High power shows blasts with monocytoid nuclei and mitoses. (C) CD68 staining. (D) Lysozyme immunostain.
43
collection. If it is not possible to extract DNA and RNA
immediately, fresh tissue (at least 0.5  0.5  0.5 cm) should
be frozen for DNA and RNA studies.
Bone marrow trephine biopsy tissues are most com-
monly fixed in 10% buffered formalin, decalcified in 5%
formic acid for 12 hours, embedded in paraffin, and cut
into sections 2 lm thick. If the bone marrow aspiration is
not successful (most often because of packed or fibrotic
bone marrow), certain alternative approaches may be taken
to optimize the results. Touch imprints of the core biopsy
are invaluable for cytological examination in this setting
and are also a useful source of materials for cytochemical,
immunocytochemical, and FISH studies. A variety of
cytogenetic defects can be evaluated by loss of heterozy-
gosity studies. These studies have an advantage over
conventional cytogenetic studies in that archival tissues
may be used. Allelic losses are more common in extra-
medullary sites (eg, spleen) than in bone marrow [42].
Immunohistochemical analysis applied to a paraffin-
embedded biopsy specimen is a useful alternative immuno-
phenotyping technique because many cell lineage–specific
markers that are resistant to routine fixation and processing
have become available. These markers are described in
detail later in this article.
1.6. Classification of AML
The current WHO classification of acute leukemias and
myelodysplastic syndrome (MDS) has evolved away from
the FAB classification, which is based on morphology, to
44 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

include not only morphology, but also clinical, immuno-

phenotypic, and cytogenetic features [1,2,43]. Besides
evaluation of percentage of immature cells and their
morphological, cytochemical, and immunophenotypic char-
acteristics, it is necessary to evaluate residual hematopoiesis
for the presence and degree of dysplasia, obtain the patient’s
history of chemotherapy, and learn from cytogenetic and/or

Fig. 6. Myeloid sarcoma involving the skin. The patient presented with a
scalp lesion diagnosed as MS. Extensive workup, including a bone
marrow biopsy, reveals no evidence of disease. The patient opted to wait
and, 3 months later, developed full-blown AML M5. (A) Myeloid
sarcoma in scalp (H&E, 20); (B) blasts showing monocytoid nuclei and
cytoplasm (H&E, 4000); (C) lysozyme immunostain (40).

molecular studies whether a patient has a recurrent

Fig. 5. Acute myeloid leukemia with inv(16) presented as lymphadenop-
athy. (A-B) A lymph node ([A] 20; [B] 1000). Note the interfollicular
pattern of involvement by blasts and prominent mitotic activity with tingle-
body macrophages creating a starry-sky pattern. (C) Bone marrow (400)
demonstrating sheets of blasts admixed with eosinophils.
cytogenetic abnormality.
The current WHO classification of AML includes
4 categories: AML with recurrent cytogenetic abnormalities,
AML with multilineage dysplasia, therapy-related AML and
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65 45

MDS, and AML not otherwise specified (NOS). Acute

myeloid leukemia NOS categories are usually reported in
terms of the FAB AML classification (Table 1).

2. Morphological features of AML

Acute myeloid leukemia comprises a group of diseases
whose cell morphology sometimes differs significantly from
one group to another. Therefore, common morphological

Fig. 8. Acute myeloid leukemia without maturation (AML M1 by FAB).

(A and B) hypercellular bone marrow with increased blasts; (C) immature
blasts with fine chromatin and high nucleus-cytoplasm ratio.

features of AML and special features of particular subtypes

are discussed here.
Fig. 7. Acute myeloid leukemia appearing as sarcoma in liver and lung. (A) 2.1. Myeloid sarcoma
Hypercellular bone marrow with sheets of immature cells and residual
hemopoiesis (H&E, 400); (B-C) liver biopsy with neoplastic spindle- The tumor usually represents a diffuse infiltrate
shaped cells ([B] H&E, 40; [C] H&E, 400). composed of a relatively uniform population of immature
46 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

cells, which may include all stages of myeloid differen-
tiation or be predominantly blastic such that few or no
cells have recognizable granulocytic differentiation. In
some anatomic sites, such as myometrium, neoplastic
cells may stream along muscle fibers (Fig. 3), whereas in
other sites, neoplastic cells may invade local structures
such as glands (Fig. 4). The presence of immature
eosinophils and maturing granulocytes is an important
diagnostic clue, especially in patients with no known AML.
Neoplastic cells are of moderate size with irregularly
shaped, angulated nuclei, irregular nuclear contours,
and fine chromatin (Fig. 5). The neoplastic cells may have
folded nuclear contours with smaller nucleoli and more
delicate chromatin suggestive of monocytic differentiation
(Figs. 4, 6). Mitotic figures may be sparse or as frequent
as 40 per 10 high-power fields. In cases with high
mitotic activity, tingle-body macrophages may be seen
giving a bstarry-skyQ appearance to a section (Fig. 5).
Single-cell necrosis (apoptotic bodies) may be identified in
poorly differentiated and blastic neoplasms. Geographic
areas of necrosis may be observed in some cases. In some
cases, marked desmoplastic reaction together with promi-
nent cellular atypia gives a sarcoma-like appearance to
MS (Fig. 7).
In the past, MS was subclassified on the basis of degree
of differentiation into blastic, immature, and differentiated
[33,41]. It is important to keep in mind that, in cases of MS
with bone marrow involvement, the morphology of the
blasts composing MS may differ from that of the blasts in
bone marrow.

Fig. 9. Morphological features of AML with the t(8;21) abnormality. (A) Hypercellular bone marrow with residual hematopoiesis. (B) Sheets of immature cells
with fine chromatin and 2 to 3 nucleoli form a solid sheet in the center and are intermixed with residual mature cells (left upper corner). (C-D) Immature blasts
with fine chromatin, 2 to 3 nucleoli, and high nucleus-cytoplasm ratio are intermixed with mature granulocytes. Long, slender Auer rod is present. (E)
Myeloperoxidase is positive in some blasts (MPO stain with Wright-Giemsa counterstain).
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65 47

2.2. Peripheral blood, bone marrow aspirate, and touch
imprint
2.2.1. Blasts
By the FAB criteria, acute leukemia is diagnosed when
a 200-cell differential reveals the presence of more than
30% blasts in a marrow aspirate. The WHO changed this
minimum criterion to 20%.
In addition, patients with the clonal, recurring cytoge-
netic abnormalities t(8;21)(q22;q22), inv(16)(p13q22) or
t(16;16)(p13;q22), and t(15;17)(q22;q21) should be consid-
ered to have AML regardless of the blast percentage. If the
marrow contains more than 50% normoblasts and pronor-
moblasts, the blast percentage is based only on the non-
erythroid cells; in these cases, the diagnosis is typically
acute erythroid leukemia, which can be confirmed if
glycophorin A is expressed on the blasts’ surface.
In AML, blasts are classified as myeloblasts, monoblasts,
erythroblasts, or megakaryoblasts.
Three major types of myeloid blast are reported in the
literature. The type I myeloblast has fine nuclear chromatin,
2 to 4 distinct nucleoli, and a moderate rim of pale to
basophilic cytoplasm without azurophilic granules (Fig. 8).
The type II myeloblast has nuclear and cytoplasmic features
similar to type I with the addition of up to 20 delicate
azurophilic granules in the cytoplasm (Fig. 9). The type III
myeloblast has numerous azurophilic granules in the
cytoplasm. Distinction between the type III blast and
promyelocyte is somewhat arbitrary and may be impossible.
One characteristic feature of myeloblasts in AML is the
presence of Auer rods, which are seen in 60% to 70 % of all
cases and represent abnormal azurophilic granules. In some
cases, leukemic cells contain numerous Auer rods and are
referred to as bfaggot cellsQ (Fig. 10). The faggot cells are
typical, but not unique, for cases with the recurring
cytogenetic abnormality t(15;17)(q22;q21). The long, slen-
der Auer rods with tapered ends are typical for cases with the
recurring cytogenetic abnormality t(8;21)(q22;q22) (Fig. 9).
The monoblast has a round, sometimes folded nucleus,
finely dispersed nuclear chromatin, variably prominent
nucleoli, abundant slightly basophilic cytoplasm with fine
granulation, and occasional vacuoles (Figs. 11, 12).
The erythroblast has a round nucleus, slightly condensed
nuclear chromatin, variably prominent nucleoli, and a
moderate amount of deeply basophilic cytoplasm (Fig. 13).
The megakaryoblasts vary significantly in their appear-
ance from one case to another, ranging from undifferentiated
blasts to mature-appearing megakaryocytes (Fig. 14). In

Fig. 10. Acute myeloid leukemia with the (15;17). (A) Hypercellular bone marrow. (B) High-power microscopy demonstrates immature cells with fine
chromatin and abundant pink cytoplasm. (C) Cell with numerous Auer rods referred to as faggot cell. (D) Myeloperoxidase is positive in all neoplastic
progranulocytes (compare with MPO positivity of AML M2).
48 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

Fig. 11. Morphological features of AML M4. (A) Hypercellular bone marrow. (B-C) Mixture of typical myeloblasts with monocytoid cells with twisted nuclei
and more abundant cytoplasm. (D-E) Some neoplastic cells are positive for MPO and some are positive for butyrate esterase, respectively.

some cases, the full range is present, whereas in others, there
is a predominance of a particular type. Cells are of moderate
size, with dispersed nuclear chromatin, distinct nucleoli, and
a moderate amount of light basophilic cytoplasm, with very
fine azurophilic granules frequently located to the perinu-
clear zone. The cytoplasm may be irregular and show
pseudopod formation (Fig. 14).
In some cases of AML, blasts exhibit any of several
unusual morphological features. Marked nuclear lobulation
is seen in cases with the recurring cytogenetic abnormality
t(15;17)(q22;q21). Multinucleation is seen in many types
of blasts, especially erythroblasts and megakaryoblasts.
Pseudo-Chédiak-Higashi-type giant granules result from
fusion of primary granules and are occasionally seen in
leukemic blasts and granulocytic precursors, especially in
cases with t(8;21). Erythrophagocytosis is seen most
commonly in cases of AML with monocytic differentia-
tion, especially cases with the translocation t(8;16). Some
myeloblasts have prominent vacuolization with an appear-
ance similar to the L3 blasts of ALL (Fig. 15).
2.2.2. Blast equivalents
To fulfill the diagnostic criterion of 20% blasts set
for AML, in some cases it is necessary to combine brealQ
blasts with so-called blast equivalents. The term bblast
equivalentsQ is specific to a particular type of AML. The
neoplastic promyelocyte considered a blast equivalent
in cases with the recurring cytogenetic abnormality
t(15;17)(q22;q21), or AML M3, has an eccentric, often
folded and lobulated nucleus with slightly condensed
nuclear chromatin, intense cytoplasmic granularity, and an
apparent Golgi zone (Fig. 10). The promonocyte considered
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65 49

Fig. 12. Morphological features of AML M5. (A) Hypercellular bone marrow with osteosclerosis. (B-C) Neoplastic cells have twisted nuclear shapes with
irregular nuclear contour, indentations and grooves, and abundant eosinophilic cytoplasm. (D-E) Cells are negative for MPO and strongly positive for
butyrate esterase, respectively.

a blast equivalent in cases of AML with monocytic
differentiation (Fig. 12).
2.2.3. Dysplasia in myeloid cells
In addition to evaluating blasts and blasts equivalents,
pay close attention to cells appearing as mature or
maturing for several reasons. These cells may be part of
a neoplastic clone and demonstrate unusual morphological
features, such as giant neutrophils or giant platelets, or
contain Auer rods. Chunky basophilic granules in maturing
eosinophilic precursors give a very important hint of AML
with recurring cytogenetic abnormalities inv(16)(p13q22)
or t(16;16)(p13;q22) (Fig. 16). Significant dysplasia may
be a hint of a history of previous chemotherapy and, in the
absence of such a history, warrants classification of the
case as AML with multilineage dysplasia (Fig. 17).
Dysplastic features are also seen in cases of therapy-
related AML (Fig. 18). By definition, the dysplasia in
AML with multilineage dysplasia must be severe, that is,
present in at least 50% of cells of at least 2 lines. The
criteria for dysplasia are as follows:
! granulocytic dysplasia: agranular or hypogranular
polymorphonuclear cells, hyposegmented nuclei
(pseudo Pelger-Huet), or pseudo-Chédiak-Higashi-
type giant granules;
! megakaryocytic dysplasia: micromegakaryocytes,
multiple separated nuclei, or large mononuclear forms;
! erythroid dysplasia: karyorrhexis, megaloblastoid
aspect, multinuclearity, nuclear fragments, nuclear
budding, internuclear bridging, cytoplasmic vacuoli-
zation, or aberrant periodic acid–Schiff (PAS) posi-
tivity (Fig. 16C).
2.2.4. Dysplasia using flow cytometry immunophenotyping
Dysplastic features can be assessed by using flow
cytometry immunophenotyping [44]. The proposed criteria
for myeloid cells are abnormal granularity, abnormal decrease
in CD45 expression, presence of HLA-DR or lack of CD11b,
50 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65
Fig. 13. Morphological features of AML M6. (A) Hypercellular bone marrow with marked dysplastic erythroid and megakaryocytic hyperplasia; (B) aspirate
smear. Note deep blue cytoplasm and presence of cytoplasmic vacuoles. (C) PAS positivity in neoplastic erythroid precursors. (D) Iron stain shows a ringed
sideroblast.
abnormal relationship between CD13 and CD16, expression
of CD56 on a subpopulation of myeloid cells, lack of CD33
expression, asynchronous shift to the left, presence of
lymphoid antigens, or CD34 on myeloid cells [44]. For
monocytic cells, the abnormalities include abnormal granu-
larity, abnormal CD11b or HLA-DR expression, loss of CD13
or CD16, presence of CD56, lack of CD33 or CD14, presence
of CD34, or lymphoid antigen expression on monocytes [44].
2.3. Bone marrow biopsy and clot section
The bone marrow sections in AML are generally
hypercellular and in most cases are extensively replaced
by leukemic cells. Approximately 5% of adult AML cases
present with hypocellular bone marrow, which may pose a
diagnostic challenge. Another diagnostic challenge is focal
involvement by leukemia with a significant proportion of
sections spared. Immunohistochemical stains for MPO,
CD34, and CD117 are helpful to highlight the few foci of
myeloblasts (see bImmunohistochemistryQ section).
Myeloblasts may be distributed uniformly throughout the
bone marrow interstitium or may be present in a large focal
aggregate. In most cases, blasts are relatively uniform and
have a round to oval, sometimes indented nucleus, 1 to 4
variably prominent nucleoli, and a moderate amount of
cytoplasm. In some cases, as in those with the recurring
cytogenetic abnormality t(15;17)(q22;q21), blasts have
abundant, deeply eosinophilic cytoplasm with distinctly
granular appearance (Fig. 10). In some cases with recurring
cytogenetic abnormality inv(16)(p13q22) or t(16;16)
(p13;q22), increased and/or abnormal eosinophilic precur-
sors are apparent on bone marrow biopsy or clot sections
(Fig. 16). In cases with monocytic differentiation, blasts are
large and have abundant amphophilic to lightly eosinophilic
cytoplasm and round to oval, sometimes very convoluted
(bmonocytoidQ) nuclei with single or multiple, usually
eosinophilic nucleoli, which may be very prominent (Fig.
12). In cases with erythroid differentiation, erythroblasts
have abundant basophilic cytoplasm and nuclei with distinct
nucleoli and nuclear chromatin, which is slightly coarser
than that of myeloblasts. In cases with megakaryocytic
differentiation, megakaryoblasts may demonstrate abnor-
mal differentiation, forming micromegakaryocytes in small
clusters or broad sheets (Fig. 14). Micromegakaryocytes
show nuclear cytoplasmic asynchrony characterized by fine
nuclear chromatin and a rim of eosinophilic cytoplasm.
There may be increased reticulin fibers.
3. Cytochemical features of AML
An important aspect of the classification of acute
leukemia is the cytochemical reactivity pattern of the blasts.
Cytochemical stains include MPO (or, to less extent, Sudan
black B), nonspecific esterase (a-naphthyl butyrate or
a-naphthyl acetate), and PAS. Some laboratories also use
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65 51

Fig. 14. Morphological features of AML M7. (A-B) Bone marrow biopsy demonstrates hypercellular marrow with a mixture of immature cells and increased,
clustering megakaryocytes at different stage of differentiation ([A] 200; [B] 500). (C-D) Blasts are medium sized with basophilic cytoplasm forming
characteristic projections. (E) Electron micrographs showing cytoplasmic projections. Note the platelet peroxidase reaction product deposited in the perinuclear
envelope of the blast.

naphthol ASD chloroacetate esterase. If 3% or more of the 4. Ancillary laboratory investigation

blasts stain positive for MPO or Sudan black B SBB, the
diagnosis is AML. If the blasts are MPO negative, but stain
for butyrate or nonspecific esterase, acute monocytic
leukemia (a variant of AML) is diagnosed.
A minority of MPO-negative and Sudan black B–
negative cases are AML, and may include minimally
differentiated leukemia (M0), acute monocytic leukemia
(M5), and megakaryocytic leukemias (M7) that require
electron microscopy study (Figs. 14, 19) or flow cytometry
immunophenotypic analyses (Figs. 20, 21) for characteriza-
tion [45].
4.1. Flow cytometry
Immunophenotyping by use of flow cytometry has
dramatically improved the accuracy of diagnosis of AML.
It is strongly recommended, however, that immunologic
marker study results be interpreted in conjunction with
morphological studies. Only the typical immunophenotypic
features of AML and some of the pertinent information
related to the differential diagnosis of AML are presented
here. Various T-cell, B-cell, and myeloid-associated markers
are available for flow cytometric immunophenotypic anal-
52 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

Fig. 15. Acute myeloid leukemia M4 with blasts containing numerous vacuoles. (A) Peripheral blood smear; (B-D) bone marrow aspirate smear.

ysis. Table 2 summarizes the immunophenotypic profile of
immature myeloid cells. Table 3 shows the European Group
for the Immunological Classification of Leukemias (EGIL)
scoring system of lineage specificity for different markers
proposed in 1995, later revised in 1998 [46,47]. For
instance, two markers, CD3 and cytoplasmic MPO, are
highly specific for T-cell and myeloid-cell lineages, respec-
tively. Myeloid antigens are CD13, CD33, CD117, CD14,
CD64 (the latter two are monocytic markers), glycophorin A
(an erythroid marker), and CD41 (a megakaryocytic
marker). Nevertheless, the specificity of these markers is
not absolute.
Flow immunophenotypic displays of two typical AML
cases are illustrated in Figs. 20, 21. For more details on flow
cytometric immunophenotyping in leukemia, please refer to
appropriate review articles [48-50].
4.1.1. Immunohistochemistry
The antigen retrieval technique allows routinely fixed,
paraffin-embedded bone marrow biopsy sections to be
used for antigenic characterization. When fresh bone
marrow aspirate and peripheral blood samples are not
available, this method is useful in classifying acute
leukemias. The following markers are often included in
the panel:
1. progenitor-associated markers: CD34, TdT;
2. myeloid-associated markers: MPO, lysozyme;
CD117, CD68, CD15, CD33;
3. erythroid-associated markers: hemoglobin A, glyco-
phorin A;
4. megakaryocyte-associated markers: factor VIII,
CD41, CD61;
5. T cell–associated markers: CD3, CD5, CD4, CD8;
6. B cell–associated markers: CD20, CD22, CD79a,
Pax-5, immunoglobulin kappa and lambda, CD10.
In cases of MS without previous or concurrent AML,
CD45 antibody is useful to establish the hematopoietic
origin of the tumor.
4.1.2. Electron microscopy
With improvements in laboratory investigations, the role
of electron microscopy has diminished. Demonstration of
platelet peroxidase is useful in documenting the diagnosis of
acute megakaryocytic leukemia (M7). Characteristically, the
platelet peroxidase reaction product is deposited in the
perinuclear envelope and endoplasmic reticulum, but not in
the Golgi regions (Figs. 14, 19).
4.2. Cytogenetic and molecular markers
Table 4 summarizes frequency of recurrent chromo-
somal abnormalities in pediatric and adult patient pop-
ulations. Molecular study of many recurring cytogenetic
abnormalities has revealed genes that may be involved in
leukemogenesis. A well-accepted concept states that, for
an acute leukemia to develop, at least two broad mutations
are necessary, referred to as class I and class II mutations.
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65
Fig. 16. Morphological features of AML with inv(16). (A-B) Hypercellular
bone marrow with numerous blasts and immature eosinophils. (C)
Myeloblasts intermixed with characteristic immature eosinophils with
chunky basophilic granules.
Class I mutations give a proliferative and/or survival
advantage; these include RAS and FLT3 mutations. Class
II mutations complement class I mutation and impair
myeloid differentiation.
Both inv(16) and t(8;21) belong to class II mutations and
involve subunits of the transcription factor complex core-
binding factor (CBF ). The inv(16) abnormality results in
53
fusion of the CBFb (CBFB) gene on the q arm and the
myosin heavy chain (MYH11) gene on the p arm. The 8;21
translocation involves the CBFa (CBFA) gene on chromo-
some 21, called the AML1 (also RUNX1) gene, joining the
ETO gene on chromosome 8. Like the t(15;17) gene
product, the AML1/ETO protein acts to block transcription
of CBFA-CBFB–controlled genes.
Another example of class II mutation is involvement of
the myeloid-lymphoid (or MLL) gene located on 11q23. The
MLL gene has two regions that encompass multiple zinc
fingers and at least two additional potential DNA-binding
motifs. Abnormalities in the MLL gene are relatively common
in patients with AML who do not have 11q23 rearrangements
on cytogenetic analysis. MLL gene locus at 11q23 is
involved in more than 30 leukemia-associated translocations
[52,53]. The most common translocation is t(4;11)
(q21;q23), which gives rise to a chimeric MLL/AF4 fusion
gene. Prognosis of AML with 11q23 rearrangements is poor.
The t(15;17) translocation does not fit readily into the
class I-class II mutation concept, as it has features of both. It
encodes a chimeric protein, PML/RARa, which is formed
by the fusion of the retinoic acid receptor-a (RARa) gene
from chromosome 17 and the promyelocytic leukemia
(PML) gene from chromosome 15. The RARa gene encodes
a member of the nuclear hormone receptor family of
transcription factors. After binding retinoic acid, RARa
can promote expression of a variety of genes. The t(15;17)
translocation juxtaposes PML with RARa in a head-to-tail
configuration that is under the transcriptional control of
PML. The PML-RARa fusion protein tends to suppress
gene transcription and blocks differentiation of the cells.
Pharmacological doses of the RARa ligand ATRA (tretinoin)
relieve the block and promote differentiation.
The presence of the t(15;17) also may be detected by a
unique immunofluorescence method using antibody specific
for promyelocytic leukemia (PML) protein. Normally, PML
is compactly located at multiple nuclear domains known as
PML oncogenic domains (PODs), whose structures are dyna-
mically regulated and disrupted in t(15;17) cases (Fig. 22).
Besides conventional cytogenetic studies, FISH may
be used to detect the above-mentioned abnormalities
(Figs. 23, 24), as may reverse transcriptase–PCR. Both
techniques may use archival formalin-fixed, paraffin-
embedded tissue; however, FISH studies using paraffin-
embedded tissue are more technically difficult and the
results not straightforward to interpret. Please see review
articles for more details [54-57].
4.2.1. Gene expression profiling
Oligonucleotide or complementary DNA microarray
technology is being established as an alternative and
extension to conventional karyotyping and FISH in the
diagnosis of leukemia subtypes. Several groups have
demonstrated that the sensitivity and specificity of micro-
array technology is as high as 100% for some types of AML
with recurrent cytogenetic abnormalities and more than 90%
54 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65
Fig. 17. Morphological features of AML with multilineage dysplasia. (A) Sheets of blasts; (B-F) marked dysplasia in all 3 lineages. Note that MPO stain is
absent in mature granulocytes, another sign of dysplasia (F).
for other types [58]. It appears unlikely, however, that
microarray technology will completely replace other tech-
niques in the near future.
4.3. Prognostic indicators
The prognosis of an individual with AML depends on
several parameters, including age, cytogenetic findings,
preceding MDS and/or exposure to chemotherapeutic
agents, and coexisting morbidity. Acute myeloid leukemia
with recurrent cytogenetic abnormalities t(8;21), inv(16), or
t(15;17) is associated with a better prognosis, whereas AML
with 11q23, complex karyotype, preceding MDS, or
previous chemotherapy is associated with a worse prognosis.
Tools for further discrimination of prognosis in this
patient group are becoming available. It has been estab-
lished, for example, that patients who have a normal
karyotype but also have duplication of the FLT3 [59,60]
or MLL [61,62] gene have considerably poorer prognoses
than those described above. The approximately 10% of
patients who have a normal karyotype and a mutation in the
CEBPA gene and no FLT3 duplication have a prognosis
similar to that of patients with CBF AML [63]. Increased
expression of the BAALC gene has been shown to worsen
prognosis in patients with a normal karyotype [64].
Identification of such genes will provide new therapeutic
btargetsQ as well as further refinement of prognoses. Once
covariates such as those noted above are specified, neither
the presence of dysplasia [65] nor morphological diagnosis
(eg, AML RAEB-t or RAEB or, among AML patients, FAB
subtype) affects prognosis [66].
4.3.1. Minimal residual disease
Since disease recurs in most patients who are in complete
remission (CR), detection of minimal residual disease in
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65 55

detection is, in principle, relevant to all patients.

However, the inability to establish an aberrant
immunophenotype in every patient might be prob-
lematic. Furthermore, because it is not logistically
feasible to do a complete screen for an aberrant
immunophenotype at every time point, screening
once treatment begins focuses on the aberrant
immunophenotype detected at diagnosis. Hence, the
possibility that the immunophenotype could change
between diagnosis and relapse could also limit
applicability [71].
3. Molecular. Polymerase chain reaction screening to
detect minimal residual disease is currently applica-
ble to APL, as described in that section. On the other
hand, the applicability of such analysis to CBF AML
is unresolved [73].
For more details on methods for detecting MDR in AML,
please see the comprehensive review by Raanani and Ben-
Bassat [74].

5. Differential diagnosis
In a general surgical pathology practice, cases of AML,
including MS, are relatively rare, but the diagnosis is very
often difficult.
5.1. Hematologic malignancies

5.1.1. Acute lymphoblastic leukemia

Distinguishing between AML and ALL on morpholog-
ical basis only is not reliable, especially in cases of AML
Fig. 18. Morphological features of therapy-related AML. (A) Bone marrow
biopsy showing foci of immature cells and dysplastic megakaryocytes. (B)
Bone marrow aspirate smear showing blasts and dysplastic features in
erythroid and granulocytic lineages.

patients who appear to be approaching CR or who are in CR

has received considerable attention. Detection methods may
be grouped as follows:
1. Cytogenetic. It has been found that among patients
presenting with abnormal cytogenetic findings,
cytogenetic analysis 21 days after beginning therapy
is predictive of relapse-free survival, independent of
the proportion of blasts in the marrow on day 21 or
initial cytogenetic results [67]. Others have noted
that the presence of residual abnormal metaphases at
the time of CR is predictive of shorter relapse-free
survival time [68,69].
2. Immunophenotypic. Aberrant immunophenotypes
(eg, asynchronous surface antigen expression or
lymphoid-associated markers such as CD2, CD5, or
CD19) suggestive of the presence of morphologically
Fig. 19. Electron micrograph of a myeloblast. In this case, less than 3% of
undetectable AML may be useful either during the blasts demonstrate weak MPO positivity by cytochemistry. Electron
first course of therapy or in CR [70-72]. Unlike cyto- microscopy demonstrated MPO positivity in blasts. Note dense-staining
genetic or molecular methods, immunophenotypic peroxidase-positive primary granules.
56 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65
Fig. 20. Typical flow cytometry immunophenotype of a case with t(8;21). Neoplastic cells are positive for CD34, CD45, CD117, and MPO. Note coexpression
of a lymphoid marker, CD19, on CD34-positive cells.
with blasts showing little or no differentiation. To complicate
the issue, the leukemic cells in AML M0 as well as AML M7
are negative for MPO, some AML cases demonstrate
aberrant expression of TdT and other lymphoid-associated
markers, and some cases of ALL demonstrate aberrant
expression of myeloid-associated markers. It is therefore
mandatory to use a panel of basic histochemical and
immunophenotypic markers in all cases of acute leukemia
to assign an appropriate lineage. The components of such a
panel vary from one institution to another. At MD Anderson
Cancer Center, we routinely use MPO, a-naphthyl butyrate
esterase, PAS, naphthol ASD chloroacetate esterase (by
histochemical methods), POD (by an immunofluorescence
method), and, by flow cytometry, a panel of markers
that includes CD3, CD4, CD5, CD7, CD8, CD10, CD13,
CD14, CD19, CD33, CD34, CD64, CD117, MPO, TdT, and
HLA-DR. The use of this panel allows us to assign a lineage
of differentiation (and thus make a definitive diagnosis of
AML or ALL) in most cases and recognize cases of
ambiguous leukemia.
5.1.2. Biphenotypic leukemia
Biphenotypic acute leukemias have been described in the
literature as mixed lineage or hybrid acute leukemia. The
EGIL summarized the defining criteria, which are based on
the number and degree of specificity of the markers
(lymphoid and myeloid) expressed by the leukemic cells
(Table 3) [46].
Cases of acute leukemia with two distinct populations of
cells expressing different markers are called bbilineage
leukemiaQ or bmixed-lineage leukemia.Q For cases with a
single population of cells expressing the markers of two
lineages, the term bbiphenotypic leukemiaQ is used. It is
possible sometimes to see two distinct (by morphological
criteria) blast populations in the same case, one of small size
with a high nucleus-cytoplasm ratio (resembling lympho-
blasts) and the other larger with more abundant cytoplasm
with or without granulation (resembling myeloblasts). The
following cases have been found to have a high incidence of
structural chromosome abnormalities: the Ph chromosome,
rearrangements involving 11q23, and complex cytogenetic
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65
Fig. 21. Flow cytometric immunophenotype of an AML M5 case. Neoplastic cells are positive for CD13, CD33, CD45, CD117, and MPO and negative for
CD34 and HLA-DR.
abnormalities [75]. A flow cytometric panel with an optimal
number of markers and the use of well-defined criteria for
the diagnosis of biphenotypic acute leukemia are critical in
making the distinction from AML.
5.1.3. Blast phase of chronic myeloid leukemia
The blast phase may be an initial presentation of chronic
myeloid leukemia, and distinguishing it from AML may be
difficult. The presence of Ph chromosome and/or BCR/ABL
fusion gene transcripts is not a reliable criterion, as some
cases of AML with these features have been reported [76],
including cases in which BCR/ABL fusion gene transcripts
were acquired by an MDS clone [77]. To complicate the
issue, the blast karyotype in blast phase is often very
complex. Some authors believe that the only reliable way to
distinguish these entities is to wait until CR and then test
maturing hematopoietic elements for the BCR/ABL fusion
gene transcript, which would be present in CML and absent
in AML. Fortunately, the short-term approach to these two
entities is rather similar. At MD Anderson, we make a
57
diagnosis of AML for cases of AML with Ph and/or
BCR/ABL fusion gene transcripts, raising the possibility of
CML blast phase in a comment. In our opinion, the presence
of typical micromegakaryocytes and increases in the number
of eosinophils and/or basophils increase the chances of
CML, but do not make it certain.
5.2. CD4+CD56+ hematodermic neoplasm
The CD4+CD56+ hematodermic neoplasm, also known
as blastic natural killer (NK) cell lymphoma and designated
as such in a current WHO classification, represents a
relatively recently described entity with aggressive behavior.
Originally thought to belong to the NK lineage, the
neoplastic cells derive from plasmacytoid dendritic cells
[78]. The disease may be limited to skin involvement only,
may spread to a regional lymph node, or may be systemic
with involvement of the bone marrow and peripheral blood
[78]. Histological analysis reveals that medium-sized
lymphoid-appearing cells with scant cytoplasm, a slightly
irregular-shaped nucleus with fine chromatin, and one to
58 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

Table 2 Table 4
Immunophenotypic profiles of immature myeloid elements Recurrent cytogenetic abnormalities in children and adults with AML [51]
Cell type Characteristic immunophenotypic features/comments Abnormalities Fusion genes Frequency (%)
Myeloblast wCD45, CD34, CD33a, CD15, CD13a, vCD11c, Children Adults
CD4, HLA-DR, MPO t(8;21)(q22;q22) AML1-ETO 10-15 8-12
Promyelocyte CDllc, CD13, CD15, CD33, CD45, MPO, loss of inv(16)(p13q22) CBFB-MYH11 6-12 8-12
HLA-DR, and acquisition of strong CD15 and t(15;17)(q22;q21) PML-RARa 8-15 8-10
CD11c associated with maturation. Gradual loss
of CD33 also characterizes successive maturation
stages. include small cell lung carcinoma, Merkel cell tumor, and
Monoblast wCD45, vCD34, CD33, CD15, CD13, CD11c, rhabdomyosarcoma in adults and Ewing sarcoma/primitive
vCD4, HLA-DR
neuroectodermal tumor (PNET) and rhabdomyosarcoma in
Promonocyte HLA-DR, CD13, CD33, CD14, CD4, CD15,
CD11c, CD45 children. The morphological distinction between AML and
Erythroblast Glycophorin A, hemoglobin A, CD45 all other types of small round cell tumors is difficult when
Megakaryoblastb HLA-DR, vCD34, CD41b, CD13, vCD33, the neoplasms are initially detected in the bone marrow.
CD61, CD45, CD31. Progressive maturation Routine Wright-Giemsa–stained preparations may show
characterized by loss of CD34 and acquisition of
overlapping cytological features, although these cases will
CD42 and von Willebrand factor
be negative for MPO and TdT.
v indicates variable antigen expression; w, weak antigen expression.
a As a rule, cytomorphology alone is not sufficient to
Rare AML lacks surface CD13 and CD33.
b
False-positive CD41 expression by flow cytometry may be secondary distinguish these entities, but in conjunction with relevant
to platelet adherence to blasts. clinical information (eg, patient’s age), morphology is often
helpful in predicting the type and the primary site of the
several medium or small nucleoli infiltrate the dermis. tumor. Immunohistochemical, ultrastructural, and genetic
Occasionally, large and small cells and/or an associated and molecular studies are also helpful when the primary site
inflammatory infiltrate can be seen in the background. is not evident from radiological studies or when laboratory
Mitotic figures are rare. The epidermis is always spared. studies (eg, ectopic secretion of calcitonin by small cell
In lymph nodes, the involvement starts in the medulla carcinoma or other amine and peptide hormones) are not
and advances to the sinuses and cortical interfollicular areas. sufficient for making a diagnosis.
The neoplastic cells are positive for CD4, CD43, CD45, Usually, little stromal fibrotic reaction is induced by
CD45RA, and CD56 and, in about 50% of cases, for CD68 nonhematopoietic small round cell tumors metastatic to the
[78]. The cells are negative for markers of B-cell lineage bone marrow before initiation of chemotherapy or irradia-
(CD19, CD20, CD23, CD24, CD79A, and immunoglobu- tion. Thus, the marrow involved by these tumors is easily
lin), T-cell lineage (CD2, CD3, CD5, CD7, CD8, b-F1, and aspirated. A bone marrow core biopsy and imprints made
d-TCR1), NK cell lineage (CD16 and CD57), and myelo- from needle biopsy should accompany all aspirates because
monocytic cell lineage (CD13, CD14, CD15, CD33, and these two marrow-sampling techniques, when performed
CD117), with rare exceptions [78]. together; maximize the odds of detecting metastases. In
addition, the histological appearance of the tumor in the
5.3. Nonhematologic malignancies biopsy core is sometimes sufficient to distinguish between
In addition to lymphoma and acute leukemia, pathologic acute leukemia and a metastatic process.
entities characterized as small round cell tumors may As discussed previously, the diagnosis of AML requires
well-prepared and stained aspirate smears, along with an
Table 3 appropriate workup with various ancillary laboratory
Scoring system for defining acute biphenotypic leukemiasa [46] studies. Care should be exercised when interpreting
Scoring points B cell T cell Myeloid nonspecific esterase results because some carcinomas or
2 CytCD79a (mb-1) CD3 (m/cyt) MPOb rhabdomyosarcomas may give positive reactions. Although
CytCD22 Anti-TCR metastatic small cell tumors tend to present as cell
CytIgM aggregates, diffuse noncohesive cells are common on bone
1 CD19 CD2 CD117
CD20 CD5 CD13
marrow aspirate smears.
CD10 CD8 CD33
CD10 CD65 5.3.1. Melanoma
0.5 TdT TdT CD14 It is well known that the morphology of metastatic
CD24 CD7 CD15 melanoma varies significantly and that metastases may
CD1a CD64
present without any history of primary skin involvement.
Cyt indicates cytoplasmic; m, membrane; TCR, T-cell receptor.
a This also applies to bone marrow. Cells are usually
Biphenotypic acute leukemia is established when the score from two
separate lineages is greater than 2. moderately pleomorphic with large nuclei, focally promi-
b
Myeloperoxidase demonstrated by cytochemical or flow cytometric nent nucleoli, and abundant eosinophilic cytoplasm that
studies. contains fine granular brown pigment. Cells usually grow
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65 59

Fig. 22. Promyelocytic leukemia protein oncogenic domain test, also referred to as POD. This is an immunofluorescence technique using antibody specific for
PML. Normally, PML is compactly located at multiple nuclear domains known as PODs, whose structures are dynamically regulated and disrupted in t(15;17)
cases. (A) Normal coarse staining pattern. (B) A fine, dispersed staining pattern in a case with the t(15;17).

diffusely or in nodular aggregates. Occasionally, small cells
with scant cytoplasm or spindly cells may be present or may
even be predominant. In some cases, cells contain no
pigment (Fig. 25). Growth in a discrete rounded aggregate
with a nevoid appearance is helpful. Melanin may be
misinterpreted as lipochrome pigment or hemosiderin.
S-100 antibodies are considered more sensitive than
HMB-45 for detection of melanoma cells. Other useful
markers include melan-A (Mart-1), tyrosinase, and neuron-
specific enolase (NSE).
One recently published case of metastatic melanoma
presenting as pancytopenia in a 3-year-old boy brought out
an important point, as melanoma cells were positive for CD56
and CD117 on flow cytometric immunophenotype analysis
[79]. That observation stresses again the importance of not
relying on a limited panel of immunophenotypic markers.
5.3.2. Rhabdomyosarcoma
Rhabdomyosarcoma may present as disseminated disease
with no certain primary lesion and in some cases is restricted

Fig. 23. The LSI PML/RARa Dual Color Translocation Probe. A normal
cell has a two orange and two green signal pattern. In an abnormal cell
containing a PML/RARa fusion, one green (RARa), one magenta (PML),
and one closely adjacent or fused green/magenta (yellow) signal pattern is
observed. Fluorescence in situ hybridization can be performed on an
archive smear as shown in this picture.
Fig. 24. The LSI CBFB (Core Binding Factor Beta subunit) Dual Color,
Break Apart Rearrangement Probe. Nucleus without inv(16) will have two
fused red/green (yellow) signals. The inv(16) results in separate red and
green signals appearing on opposite arms of the inverted 16 chromosome
creating an adjacent or fused red/green signal on the q arm of one of the
16 chromosomes and a green signal on the other arm of 16, while the
16 chromosome homolog will only contain the red signal on one arm.
60 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

Fig. 25. Involvement of bone marrow in a patient with malignant melanoma. (A) Bone marrow clot packed by immature cells with a similarity to blasts
or immature plasma cells. (B-D) Neoplastic cells in the bone marrow aspirate. Although cells are very similar to blasts, note that nuclear chromatin is
more condensed that nuclear chromatin of a typical myeloblast. Binucleation is also not typical for AML, but is very typical for multiple myeloma. (E)
HMB-45 stain.

to bone marrow [80]. Almost all reported cases of
rhabdomyosarcoma with bone marrow involvement, includ-
ing cases presenting in bone marrow, were alveolar
rhabdomyosarcoma [80]. The diagnosis of acute leukemia
is suspected because of the presence of undifferentiated cells
in the bone marrow and signs of acute systemic process
(fever, anorexia, disseminated intravascular coagulation). In
fact, in one report, 5 of 10 cases of rhabdomyosarcoma with
bone marrow involvement had a referring diagnosis of acute
leukemia [81]. By morphology, malignant cells may be
indistinguishable from blasts (Fig. 26). Immunohistochem-
ical studies for the muscle markers (desmin, myoglobin,
myosin, muscle-specific actin [HHF-35], and more specific
MyoD1 and myogenin) usually aid in the diagnosis. It is
important to bear in mind that some undifferentiated cases
of rhabdomyosarcoma may be nonimmunoreactive with all
markers but vimentin. Molecular and cytogenetic studies are
helpful. Most alveolar rhabdomyosarcoma cases have the
translocation t(2;13)(q35;q14); a smaller subset have the
translocation t(1;13)(p36;q14) [82]. Most cases of embryo-
nal rhabdomyosarcoma have allelic loss in chromosomal
region 11p15 [83].
5.3.3. Ewing sarcoma/PNET
The pelvic bones are the presenting sites of Ewing
sarcoma/PNET in 12% to 18% of patients. As such, a bone
marrow aspirate and biopsy may be the first evidence of this
tumor in a patient. The cytology of the tumor cells on the
aspirate smear and biopsy section is usually not sufficient
for a definite diagnosis. The use of immunoperoxidase
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65 61

detected by immunologic methods. Such bone marrow

involvement seems to be associated with poorer patient
survival rates [85]. On marrow aspirate smear, these
tumor cells present as clusters of large tumor cells with
immature chromatin and mitosis. The malignant tumor
cells show immunoreactivity with antibodies against NSE,
neurofilaments, chromogranin, synaptophysin, low-molec-
ular-weight cytokeratin, CD57, epithelial membrane anti-
gen (EMA), TTF-1, and Bcl-2 and are negative for CD99
and 34b12.

5.3.5. Germ cell tumor

Occasionally, germ cell tumors enter the differential
diagnosis for MS. The morphology can be extremely similar
(Fig. 27), and to complicate the issue, some cases of
seminoma are positive for CD117 and CD57 and negative
for cytokeratin. CD45 is usually negative, and immunore-
activity for placental alkaline phosphatase (PLAP) and
a-fetoprotein often helps distinguish seminoma from MS.
In addition to PLAP and CD57, embryonal sarcomas are
usually positive for CD30 and cytokeratin.

Fig. 26. (A-B) Rhabdomyosarcoma involving bone marrow. Note the

different staining characteristics of cytoplasm of malignant cells, while the
nuclear chromatin is very similar to that of myeloblasts.

stains for leukocyte common antigen (CD45), TdT, CD43,

CD34, CD99, MPO, NSE, and lysozyme is critical in this
situation. Ewing sarcoma/PNET is positive for vimentin,
CD99, NSE, and, in some cases, cytokeratin and negative for
other markers. Molecular and cytogenetic studies are useful
to demonstrate the fusion of the EWS gene, located on 22q12,
with one of the members of the ETS family. Two recurrent
translocations, t(11;22)(q24;q12) and t(21;22)(q22;q12),
account for more than 95% of all cases [84], whereas other
translocations such as t(7;22), t(17;22), and t(2;22) and
inversion 22 are rare. Some authors believe that virtually all
cases of Ewing sarcoma/PNET have some form of EWS/ETS
gene fusion [84].
It is very important to realize that the expression of CD99
(MIC2) is by no means specific for Ewing sarcoma. In
addition to Ewing sarcoma/PNET and ALL, CD99 expres-
sion has been detected in as many as 55% of MS cases and
43% of AML [3].
Fig. 27. Seminoma involving a lymph node. (A) Medium power
5.3.4. Small cell lung cancer (400). Sheets of neoplastic cells with residual lymphoid tissue. (B) High
Fifty to seventy percent of patients with small cell power (1000). Neoplastic cells are indistinguishable morphologically
cancer of the lung have bone marrow involvement from monoblasts.
62 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65

5.3.6. Poorly differentiated carcinoma

Poorly differentiated carcinoma enters the differential
diagnosis for MS (Fig. 28). The morphology of immature
cells assessed on touch imprint preparation could be
extremely helpful. Histochemical and immunoperoxidase
studies for markers of myeloid or monocytic differentiation
(MPO, naphthol ASD chloroacetate esterase, lysozyme,
CD68) and for cytokeratin are useful in distinguishing these
two entities. For male patients, immunohistochemical stains
for prostate-specific antigen, prostatic acid phosphatase,
CD57, a-methylacyl-CoA racemase (P504S), EMA, carci-
noembryonic antigen (CEA), B72.3, and 34bE12 are useful
in the diagnosis of metastatic prostate carcinoma. For female
patients, a-lactalbumin, low-molecular-weight cytokeratin,
EMA, CEA, B72.3, and BCA-225 are used to rule out breast
carcinoma. TTF-1, surfactant apoprotein A, and low- and
high-molecular-weight cytokeratins are useful for lung
primary tumors.
5.4. Megaloblastic anemia
Rarely, megaloblastic anemia can be morphologically
indistinguishable from AML M6. Both conditions share
hypercellular bone marrow and bizarre morphology of

Fig. 29. Megaloblastic anemia due to severe vitamin B12 deficiency. (A)
Hypercellular bone marrow. (B) Aspirate smear shows sheets of immature
erythroblasts. Compare the morphology of erythroid precursors in this case
with AML M6b (Fig. 13).

erythroid precursors (Fig. 29). Increased percentage of

myeloblasts helps in establishing the diagnosis of AML
M6a, but this feature is absent by definition in AML M6b
(so-called pure erythroid leukemia). The presence of
hypersegmented granulocytes and a range of megaloblastic
erythroid precursors with nuclear/cytoplasmic asynchrony is
a typical feature of megaloblastic anemia, but in severe
cases, there is no maturation in both myeloid and erythroid
series. The serum level of folic acid and vitamin B12 and
pertinent clinical history including exposure to several
medications are crucial.

Fig. 28. Metastatic involvement of bone marrow in a patient with cervical
carcinoma. (A-B) Note the similarity of neoplastic cells to AML.
6. Summary
With our present knowledge, the optimal management
of individual cases of AML requires that all cases be
studied by morphological, cytochemical, cytogenetic,
immunologic, and molecular techniques. Other techniques,
such as gene expression profiling in AML, are important
in the advancement of knowledge but have not yet had a
major impact on patient management. Future studies on the
clinical importance of the expression of various markers
will further aid clinicians in treating AML and in
preventing refractory disease.
 S. Konoplev, C.E. Bueso-Ramos / Annals of Diagnostic Pathology 10 (2006) 39 – 65
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