2. OVERVIEW
CLASSIFICATIONS PROPOSED FOR ACUTE
MYELOID LEUKEMIA
2008 WHO CLASSIFICATION- NEW FEATURES
MORPHOLOGY,CYTOCHEMISTRY AND
IMMUNOPHENOTYPING OF AML
3. THE PHENOTYPIC DIAGNOSIS
Virchow was the first to use the term
“Leukemia”
The term "myeloid" was coined by Franz Ernst
Christian Neumann in 1869, - first to recognize
white blood cells were made in the bone myelos =
(bone) marrow) as opposed to the spleen.
1900, the myeloblast- characterized by Otto
Naegeli, who divided the leukemias into myeloid
and lymphocytic
4. ACUTE MYELOID LEUKEMIA
A group of relatively well-defined hematopoietic
neoplasms involving precursor cells committed
to the myeloid line of cellular development (ie,
those giving rise to granulocytic, monocytic,
erythroid, or megakaryocytic elements).
5. Classification is the language of medicine
An effort so that everyone speaks the same
language
All diseases must be classified before being studied
and treated
Bhargava R, Dalal BI. Two steps forward, one step back:
4th WHO classification of myeloid neoplasms (2008). Indian J
Pathol Microbiol 2010;53:391-4
6. The purpose of any classification- identify distinct
biological entities which differ in aetiology,
mechanisms of leukaemogenesis,
clinicopathological features, and prognosis.
Optimal treatment for such entities differs, their
recognition is not only of scientific interest but is
also essential for optimal care of patients.
7. CLASSIFICATIONS
(1) by morphology and cytochemistry supplemented by
immunophenotyping, as proposed by the French-
American-British (FAB) group
(2) by morphology, immunophenotyping and
cytogenetics as proposed by the MIC groups
(3) by immunophenotyping alone, as proposed by the
European Group for the Immunological
Classification of Leukemias (EGIL)
(4) by the nature of the stem cell or progenitor cell in
which the leukaemogenic mutation occurred.
Classification of acute leukaemia: the need to incorporate
cytogenetic and molecular genetic information
Barbara J Bain Clin Pathol 1998;51:420-423
8. FAB
In 1976 - FAB- a series of papers concerning the
classification of acute leukaemia.
Initially classification was based on cytology and
cytochemistry.
Subsequently - immunophenotyping - acute
lymphoblastic leukaemia and facilitated the
diagnosis of acute megakaryoblastic leukaemia
and acute myeloid leukaemia with minimal
evidence of myeloid differentiation (MO AML).
9. FAB- DEFINING A BLAST CELL
Whether immature myeloid
cells containing small
numbers of granules are
classified as blasts is a
matter of convention.
Myeloblasts rather than
promyelocytes.
Type I blasts lack granules
and have a diffuse
chromatin pattern, a high
nucleocytoplasmic ratio and
usually prominent nucleoli.
Type II blasts resemble type
I blasts except for the
presence of a few
azurophilic granules and a
somewhat lower
nucleocytoplasmic ratio.
10. Type II blast cells may contain Auer rods rather
than granules; less often they contain large
rectangular crystals or large inclusions (pseudo-
Chédiak–Higashi inclusions).
Other groups have proposed accepting a type III
blast, which has more than ‘scanty’ granules but
otherwise has no features of a promyelocyte
Galton DAG (1962) Contributions of chemotherapy to the study of
leukaemia. In: The Scientific Basis of Medicine, Annual Reviews.
British Postgraduate Medical Federation, Athlone Press, London, pp.
152–171
11. No azuriphilic primary
granules.
No Auer rod
Few (<20) azuriphilic
primary granules.
Auer rods may be seen.
>20 azuriphilic primary
granules without a Golgi
zone.
Auer rods may be seen.
12. Two groups of acute leukaemia, 'lymphoblastic' and
myeloid
Dysmyelopoietic syndromes that may be confused
with acute myeloid leukaemia are also considered.
The FAB classification requires that peripheral
blood and bone marrow films be examined and that
differential counts be performed on both.
Br J Haematol. 1976 Aug;33(4):451-8.
Proposals for the classification of the acute leukaemias. French-
American-British (FAB) co-operative group.
Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick
HR, Sultan C.
13. Eight subtypes, M0 through to M7- based on the
type of cell from which the leukemia developed and
its degree of maturity.
30%* of the total nucleated cells in the marrow-
blast cells
If the bone marrow shows erythroid
predominance (erythroblasts ≥50% of total
nucleated cells) and at least 30% of non-erythroid
cells are blast cells (lymphocytes, plasma cells and
macrophages also being excluded from the
differential count of nonerythroid cells)
If the characteristic morphological features of
acute promyelocytic leukaemia are present
14. FOR
Some morphological categories recognised by the
FAB group, specifically acute hypergranular
promyelocytic leukaemia and its variant form
(M3 and M3 variant AML) and L3 acute
lymphoblastic leukaemia (ALL), were
subsequently shown to indeed be biological entities
Introduced objectivity
Improved uniformity of diagnosis
Common language for people studying and treating
acute leukemias
15. AGAINST
30% or more myeloblasts and promyelocytes in
marrow for diagnosis
Ambiguity in the interpretations of descriptions
Did not recognize the significance of
myelodysplastic changes in acute myeloid
leukemias or cytogenetic abnormalities
16. REVISED CLASSIFICATION-1986
Distinguish between MI and M2
M4 with eosinophilia added
Defined the number of marrow cells to be counted to
diagnose acute leukemia
Definition of erythroleukemia (M6) and its distinction
from refractory anemia or refractory anemia with an
excess blast cells (RAEB).
The Revised French-American-British Classification of Acute Myeloid
Leukemia: Is New Better? CLARA D. BLOOMFIELD, M.D.; and
RICHARD D. BRUNNING, M.D.
Ann Intern Med. 1985;103(4):614-616. doi:10.7326/0003-4819-103-4-614
Acute basophilic leukemia was proposed as a ninth
subtype, M8, in 1999.
Duchayne E, Demur C, Rubie H, Robert A, Dastugue N (1999).
"Diagnosis of acute basophilic leukemia". Leuk Lymphoma
17. MIC CLASSIFICATION
MIC groups- classification of acute leukaemia on
the basis of the FAB morphological classification
supplemented by immunophenotype and
cytogenetics - a morphological, immunological,
cytogenetic (MIC) classification.
18. FOR AGAINST
Cases with atypical
cytogenetic findings or
failed cytogenetic can
be included
Without cytogenetic
anomaly but with
molecular abnormality
Morphological and
cytogenetic similarity-
differ at molecular level
No single definable
molecular lesion
Accumulations of
mutations of
oncogenes and cancer
supressor genes
Eg- AML with alkylating
drugs, AML in elderly
19. THE EUROPEAN GROUP FOR THE IMMUNOLOGICAL
CLASSIFICATION OF LEUKEMIAS (EGIL)
Acute leukaemia be classified on the basis of
immunophenotype alone.
This classification has the strength that it suggests
standardised criteria for defining a leukaemia as
myeloid, T lineage, B lineage, or biphenotypic.
20. Criteria for distinguishing biphenotypic leukaemia
from AML with aberrant expression of lymphoid
antigens, and from ALL with aberrant
expression of myeloid antigens.
However, a purely immunologic classification has
the disadvantage that discrete entities may fall into
one of two categories
For example - some cases of AML of FAB M2
subtype associated with t(8;21)(q22;q22) would be
classified as "AML of myelomonocytic lineage“
while others would be classified as "AML with
lymphoid antigen expression," depending on
whether or not a case showed aberrant expression
of CD19.
21. 2001- WHO
A Classification of Tumors of the Hematopoietic
and Lymphoid Tissues as part of the 3rd edition of
the series, WHO Classification of Tumors.
Genetic information with morphologic,
cytochemical, immunophenotypic, and clinical
information into diagnostic algorithms for the
myeloid neoplasms
22. Morphologic, genetic, and clinical data is a
major theme of the WHO classification
In the WHO classification, the blast threshold for
the diagnosis of AML is reduced from 30% to 20%
blasts in the blood or marrow.
Patients with the clonal, recurring cytogenetic
abnormalities t(8;21)(q22;q22), inv(16)(p13q22) or
t(16;16)(p13;q22), and t(15;17)(q22;q12) should
be considered to have AML regardless of the blast
percentage
23.
24. 2001 CLASSIFICATION OF AML
AML with recurrent genetic abnormalities
AML with multilineage dysplasia
AML and myelodysplastic syndromes, therapy-related
AML not otherwise categorized
AML of ambiguous lineage
25. 20% to 29% blasts - similar clinical features,
response to therapy and survival times—as
those with 30% or more blasts.
Identical profiles of proliferation and apoptosis
Poor-risk cytogenetic abnormalities and increased
expression of multidrug-resistance glycoproteins
26. 20% to 29% blasts - similar clinical features,
response to therapy and survival times—as
those with 30% or more blasts.
Identical profiles of proliferation and apoptosis
Poor-risk cytogenetic abnormalities and increased
expression of multidrug-resistance glycoproteins
27. In addition, data from the International MDS Risk
Analysis Workshop indicate that RAEBT is not an
indolent disease.
In that study, 25% of patients with 20% to 30%
blasts evolved to AML in 2 to 3 months, 50% in 3
months, and more than 60% developed AML within
1 year.
WHO proposal- 20% to 29% blasts and
myelodysplasia will be classified as AML with
multilineage dysplasia
28. The blast percentage, assessment of degree of
maturation and dysplastic abnormalities - 200-cell
leukocyte differential performed on a peripheral
blood smear and a 500-cell differential
performed on marrow
Monoblasts and promonocytes and the
megakaryoblasts - “blast equivalents”
(APL)- blast equivalent is the abnormal
promyelocyte.
29. Erythroid precursors (erythroblasts) – Not included
in the blast count except - “pure” erythroleukemia.
Dysplastic micromegakaryocytes are also excluded
from the blast percentage.
The percentage of CD34 cells should not be
considered a substitute for a blast count from
the smears or an estimate from the bone
marrow biopsy.
Although CD34 hematopoietic cells generally are
blasts, not all blasts express CD34.
Geller RB, Zahurak M, Hurwitz CA, et al. Prognostic
importance of immunophenotyping in adults with acute
myelocytic leukaemia: the significance of the stem-cell
glycoprotein CD34 (My10). Br J Haematol. 1990;76:340-
347.
30. 2008 WHO CLASSIFICATION
Expanded the genetic disease subtypes of AML-
addition of 3 entities- t(9;11), t(6;9),inv(3),t(1;22)
Two provisional- AML with NMPI or CEBPA
mutation
AML with multilineage dysplasia changed to AML
with MDS related changes
Proliferations associated with Down’s syndrome
and blastic plasmacytic dendritic cell neoplasm
31.
32.
33.
34. CLINICAL FEATURES
Hepatosplenomegaly: Most AML t(1;22) and A
Baso. Some AML inv(3) or t(3;3)..
DIC : APL, AML t(9;11)
CNS: Common in A Monoblastic and A
Monocytic,APL with systemic relapse and inv(16)
Adenopathy: AML NPM1. Rare in AML inv(3) or
t(3;3).
35. Hb : Low in many AML. High in AML CEBPA
Thrombocytopenia: many AML. Very common in
AML t(6;9) and AML t(1;22).
Thrombocytosis: highest count of all in AML
NPM1. Common in AML inv(3) or t(3;3). Rarely in A
Mega (more commonly thrombocytopenia).
Platelet morphology: Occasional giant
hypogranular platelets in AML inv(3) or t(3;3).
Other- High LDH in AML CEBPA
36. MORPHOLOGY
First step in the evaluation
PBS, Aspirate, Trephine biopsy, Particle crush
smears
Critical for requesting ancillary studies
Standard Romanowsky stains
Wright unsuitable- granules suboptimal staining
Diagnosis of AML with <20% blasts
37. MORPHOLOGIC EVALUATION
Identification of blasts and blast equivalents
Differentiation of blasts and promoncytes from
mature monocytes
Identification of blast cell features characteristic of a
recurrent cytogenetic abnormality
38. The percentage of blasts - the percent blasts as a
component of all nucleated marrow cells, with the
exception of acute erythroleukemia
If a myeloid neoplasm is found concomitantly with
another hematologic neoplasm—for example,
therapy-related AML and plasma cell myeloma—
the cells of the nonmyeloid neoplasm should be
excluded when blasts are enumerated.
39. NON BLAST MORPHOLOGY
Monocytosis in AML inv(16) or t(16;16),AML
t(9;11), AML with inv(3), AML w M.
Topoisomerase therapy related
Increase in eos - AML inv(16) or t(16;16), AML
t(8;21), AML w M and AML t(8;21),
Abnormal eos: AML inv(16) or t(16;16)
Basophilia common in AML t(6;9), A Baso, AML w
M, t-AML/t-MDS/MPN
40. DYSPLASIA
MULTILINEAGE
AML t(6;9), inv(3) or t(3;3)
Cases of AML with MDS.
AML NPM1
t-AML/t-MDS/MPN related with alkylating drugs
Granulocyte dysplasia- AML with maturation,
AMLt(8;21)
Increased megakaryocytes with dysplasia- Inv(3)
Intense fibrosis- Acute megakaryoblastic leukemia
41.
42.
43.
44. AML characterized by a multitude of chromosomal
abnormalities and gene mutations- marked
differences in responses and survival following
chemotherapy
A detailed understanding of the molecular changes
associated with the chromosomal and genetic
abnormalities in AML - rationale for therapy design
and biomarker development
45. AML WITH RECURRENT GENETIC
ABNORMALITIES,”
30% of patients with AML
Strong correlation between the genetic findings
and the morphology
The genetic abnormality can usually be predicted
from the microscopic evaluation of the blood and
marrow specimens.
Distinctive clinical findings and a favorable
response to appropriate therapy
46. AML with morphologic features suggestive of a
specific genetic abnormality- the pathologist should
issue a report that indicates the case may belong
to a particular genetic category but that more
data are required to prove it.
The report should indicate what data are needed
and whether the studies are in progress or if a new
specimen is necessary.
A carefully worded report that informs the
clinician of what more needs to be done to
accurately complete the diagnosis.
47.
48.
49.
50.
51. Most common
structural
aberration in AML
5-12% of AML and
one third of cases
of AML with
maturation
Predominantly in
younger patients
Myeloid sarcomas
at presentation
52.
53. Auer rods(long and
sharp with tapered
ends in neutrophils)
Promyelocytes,
myelocytes and
neutrophils with
variable dysplasia
cytoplasm has a waxy,
orange appearance
and lacks a granular
texture in
Romanowsky-stained
specimens
54.
55. c-KIT mutations - adverse prognostic features
Transcripts of the RUNX1-RUNX1T1 detected by
RT-PCR even when they have been in remission
for many years
IPT: CD19 + and CD34 positive blasts in 2/3 of
cases
Co-expression of lymphoid and myeloid
molecules is a well-known feature of acute
myeloblastic leukemia (AML) with t(8;21).
Subset is CD 56 positive- significantly shorter CR
duration and survival
56.
57. AML WITH INV(16)(P13.1Q22) OR
T(16;16)(P13.1;Q22); CBFB-MYH11
10-20% of AML
Morphological features of acute
myelomonocytic leukemia with bone marrow
eosinophilia (AML-M~EO).
May be overlooked on cytogenetics
Improved prognosis compared with other AMLs,
similar to the t(8;21) AMLs
higher WBC and blast counts vs t(8;21)
58.
59.
60. All stages of
eosinophils in marrow
with no significant
maturation arrest
Immature eosinophils
granules at
promyelocyte and
myelocyte stage-
Large, purple violet
obscuring cell
morphology
61. CYTOCHEMISTRY
NSE faintly positive
The abnormal eosinophils are weakly positive for
chloracetate esterase.
The blast cells express most of the myeloid-
associated antigens, and some cases aberrantly
express CD2.
62.
63. APL WITH T(15;17)(Q24.1;Q21.1);
PML-RARA
Chromosomal translocation involving the retinoic
acid receptor-alpha gene on chromosome
17(RARA).
In 95% of cases of APL, (RARA) is involved in a
reciprocal translocation with the promyelocytic
leukemia gene (PML) on chromosome 15, a
translocation denoted as t(15;17)(q22;q21).
This rearrangement is seen in 13 percent of newly
diagnosed AML and is highly specific for APL
Typical, hypergranular and hypogranular
64. The t(15;17)(q22;q21), resulting in a PML/RARα
fusion, is the most commonly identified abnormality,
but other translocations of RARα on chromosome
17 with PLZF at 11q23, NuMA at 11q13 and NPM at
5q35 may occur.
Cases that lack an RARα translocation or have a
translocation involving PLZF do not respond to all-
trans-retinoic acid and require a different
therapeutic approach
65.
66. ZBTB16-RARA
VARIANT OF APML
Predominance of cells
that lack the
characteristic bi-lobed
or folded nuclei with
many granules.
Increased number of
pseudo-Pelger-Huet
cells with strong MPO
reactivity.
67.
68. 11Q23 REARRANGEMENTS IN AML
The MLL gene on 11q23 is involved in a number of
translocations with different partner chromosomes.
The most common translocations -
t(9;11)(p21;q23) and the t(11;19)(q23;p13.1);
Strong association between AML M5/M4 and
deletion and translocations involving 11q23.
Two clinical subgroups of patients - one is AML in
infants with MLL rearrangement in about 50% of
cases; the other group is "secondary leukemia"
(sAML) potentially after treatment with DNA
topoisomerase II inhibitors.
69. M5a in half cases, M4
(20%), M1 or M5b
(10% each), M2 (5%)
DIC, extramedullary
myeloid sarcoma and
tissue infiltration
(gingiva, skin)
Prognosis may be
superior to other AML
with 11q23
translocations
70. ACUTE MYELOID LEUKEMIA WITH T(6;9)
(P23;Q34)
0.7-1.8% of AML
Chimeric fusion gene between DEK (6p23) and
CAN/NUP214 (9q34).
Any FAB morphology (except APM). Most common
AML with maturation, acute myelomonocytic or
megakaryoblastic.
Considered by some to be a separate disease
entity because of its distinct clinical and
morphologic features and poor prognostic
implication.
Associated with a high frequency of FLT3 gene
mutations
71. t(6;9) AML and FLT3-ITD mutations - higher
white blood cell counts, higher bone marrow
blasts, and significantly lower rates of complete
remission
>20% blasts, multilineage dysplasia with frequent
ringed sideroblasts and basophilia (>2% in marrow and
peripheral blood)
74. AML WITH INV(3)
May present de novo or arise from a prior MDS.
M4 was the most common type (11 cases, 37%),
followed by M0 (6 cases, 20%), M2 (6 cases, 20%),
M1 (3 cases, 10%), M5 (1 case, 3%), and M7 (1
case, 3%).
Normal or increased platelet counts
A subset of cases has less than 20% blast cells at
the time of diagnosis- with features of chronic
myelomonocytic leukaemia.
75. Atypical megakaryocytes in a background of
multilineage dysplasia
De novo acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): a
clinicopathologic and cytogenetic study of an entity recently added to the WHO
classificationJianlan Sun1, Sergej N Konoplev, Xuemei Wang, Wei Cui, Su S Chen, L
Jeffrey Medeiros and Pei Lin,Modern Pathology (2011) 24, 384–389;
doi:10.1038/modpathol.2010.210; published online 26 November 2010
76. Acute
myelomonocytic and
dysplastic
granulocytes
acute myeloid
leukemia without
maturation showing a
dysplastic granulocyte
erythroid dysplasia;
Trephine biopsy
section, numerous
dysplastic, and
micromegakaryocytes
(arrows) are present in
a background of
increased immature
cells
77. Blasts are increased in
the marrow aspirate.
Dysplastic changes
can also be noted in
the myeloid and
erythoid series.
Dysplastic
megakaryocytes
cluster together.
78. After the publication of the 3rd edition of the
classification in 2001- not only chromosomal
rearrangement or numerical abnormalities but also
mutated genes- establish the leukemic process and
influence its morphologic and clinical features
Discovery of new mutations in leukemogenesis
paved the way for the genetic characterization of
many cases of cytogenetically normal AML.
Identification of new leukemic entity and powerful
prognostic indicators
James Vardiman, University of Chicago Medical Center, Chicago, USA
79.
80. AML WITH MUTATED
CEBPA(CCAAT/ENHANCER BINDING PROTEIN
A (CEBPA) GENE )
CEBPA - transcription factor- development and
differentiation of granulocytes from hematopoietic
precursors.
Newly diagnosed AML - 5% to 9%, with
predominance for the M1 and M2 FAB morphologic
subtypes.
Independent prognostic marker- lower relapse
rates and improved overall survival.
81. PATHOLOGIC FEATURES OF LEUKEMIC
CELLS IN AML WITH MUTATED CEBPA
Normal karyotype
Many Auer rods seen in blasts in peripheral
blood smear or bone marrow aspirate (Auer
rods are abnormal, needle-shaped or round,
light blue or pink-staining inclusions found in
the cytoplasm of leukemic cells.)
Aberrant CD7 expression on blasts
82. AML WITH MUTATED NPM1
Nucleophosmin is a nucleolar protein found
mutated and rearranged in a number of
haematological disorder
Distinctive biological and clinical
characteristics_support its inclusion as a provisional
entity in the new WHO classification.
High WBC counts
Approximately 10% of therapy-related AML are
NPM1-mutated.
Blasts- Myelomonocytic or monocytic with dysplasia
in atleast 2 cell lines
83. NPM1 staining pattern
most commonly
observed - leukaemic
cells show both
cytoplasmic and nuclear
positivity for
nucleophosmin
the totality of leukaemic
cells show an aberrant
cytoplasmic expression
of nucleophosmin
Cells showing nucleus-
restricted positivity in
the centre of the field
represent normal
residual haemopoietic
cells.
84. When the 2008 WHO classification was being
prepared, the significance of an NPM1 mutation in
the setting of morphologic dysplasia in an AML
patient with NK was still unclear.
Thus, the new WHO classification presently
recommends that cases with overlapping features
should be diagnosed as “AML with MD-related
changes,” additionally annotating the presence of
NPM1 mutation.
85.
86.
87. AML WITH MDS RELATED CHANGES
>20% blasts in blood or marrow
Dysplasia in 2 or more cell lines, generally
including megakarocyes
Dysplasia > 50% cells of atleast 2 lines in a pre
treatment specimen
De novo or following MDS or MDS/MPN
When MDS precedes MPN- AML evolving from
MDS
88. Previous, well-documented, history of MDS or
MDS/myeloproliferative neoplasm
Myelodysplasia-related cytogenetic abnormalities
Multilineage dysplasia (ie, detection of dysplasia in
50% or more of cells in 2 or more myeloid lineages
in bone marrow and/or peripheral blood smears).
Absence of both
Prior cytotoxic therapy for unrelated disease
Recurrent cytogenetic abnormality
89. DYSGRANULOPOIESIS
Hypogranulosis
Hyposegmented (i.e.,
pseudo-Pelger Huet
forms), or abnormally
segmented nuclei.
MPO may be aberrant,
because patients may
develop an acquired
MPO deficiency as part
of the dysplastic
process.
97. The three-ball
pawnbroker's symbol
that these
megakaryocytes
resemble is ancient
and may have derived
from the insignia of the
Medici family or the
symbol of Saint
Nicholas of Myra.
98. AML WITH MDS, THERAPY RELATED
-Alkylating agent/radiation
-Topoisomerase II inhibitor related
99.
100.
101. AML, NOT OTHERWISE
SPECIFIED (NOS)
2008 WHO classification - AML, not otherwise
specified (NOS) [Arber 2008c, Vardiman 2009].
Cases that fulfill general criteria for AML but lack:
a) an AML recurrent cytogenetic or molecular
abnormality, b) a link to prior chemotherapy,
c) multilineage dysplasia involving the majority of
cells, d) MDS-type cytogenetic abnormalities,
e) an association with Down syndrome, or
f) a history of MDS or MDS/MPN
102. AML, MINIMALLY DIFFERENTIATED
No evidence of myeloid differentiation by
morphology on light microscopy or
cytochemistry
Associated dysplastic features in erythroid and
megakaryocyte lineages may provide indirect
evidence that a leukaemia is myeloid, not
lymphoid.
Immunohistochemistry, flow cytometry, or EM
cytochemistry to characterize as myeloid
Cell or origin- Stem at earliest stage of myeloid
maturation
103. PROOF OF MYELOID
DIFFERENTIATION
The demonstration of ultrastructural features of
cells of granulocytic lineage, e.g. characteristic granules
The demonstration of MPO activity ultrastructural
cytochemistry
The demonstration of MPO protein by
immunocytochemistry with an anti-MPO monoclonal
antibody.
The demonstration of other antigens characteristic
of myeloid cells by the use of monoclonal antibodies -
CD13, CD14, CD15, CD33, CD64, CD65 and CD117
(but without expression of platelet-specific antigens,
which would lead to the case being categorized as AML
M7).
Messenger RNA (mRNA) for MPO has also been
104. Medium sized,round to
slightly indented nuclei,
dispersed chromatin
Agranular cytoplasm with
varying basophilia
No Auer rods
1-2 nucleoli
M0 AML is associated with
adverse cytogenetic
abnormalities and poor
prognosis
105. CYTOCHEMISTRY
Fewer than 3% of blasts are positive for MPO, SBB
and CAE
Maturing myeloid cells may show peroxidase
deficiency or aberrant positivity for both
chloroacetate and non-specific esterase
106. IPT
The blasts must not express CD79a and must express
at least two myelomonocytic antigens such as CD13,
CD33, CD15, or CD117 to be classified as myeloblasts.
IPT- most specific lymphoid markers – CD3 and CD22 –
not expressed in M0 AML
Expression of less specific lymphoid-associated
antigens such as CD2, CD4, CD7, CD10 and CD19, in
addition to CD34, human leucocyte antigen DR (HLA-
DR) and terminal deoxynucleotidyl transferase (TdT).
CD7 is more often expressed than in other FAB
categories of AML
DDX- ALL, acute megakaryoblastic leukemia,
Biphenotypic leukemia, leukemic phase of large cell
lymphoma
107. ACUTE MYELOID LEUKEMIA
WITHOUT MATURATION-M1
15-20% of AML
Significant number of blasts(>90% of non erythroid
cells) without evidence of maturation to
neutrophils
<10% of non-erythroid cells in the bone marrow
belong to the maturing granulocytic component
(promyelocytes to neutrophils).
MPO/SBB positivity(>3% of cells) and/or Auer
rods
108. Typically markedly
hypercellular marrow,
but normocellular and
hypocellular cases
occur
Very immature cells
with few azurophilic
cytoplasmic granules
Variation in size
Irregular/indented
nuclei with folding
Prominent nucleoli
Auer rods
109. CYTOCHEMISTRY
Minimum of 3% of blasts that are positive for MPO or
SBB.
Hayhoe and Quaglino - SBB reaction is a more
sensitive marker of early granulocyte precursors
than MPO.
Positive for CAE, - less sensitive than either MPO or
SBB
Weak or negative reaction for non-specific esterases.
In the case of (ANAE) and (ANBE) the reaction is
usually negative, whereas in the cases of naphthol AS-D
acetate esterase (NASDA) there is usually a weak
fluoride-resistant reaction.
The PAS reaction- usually negative, but may show a
weak diffuse reaction with superimposed fine granular
positivity.
111. Auer rods give
positive MPO and
SBB reactions and
occasionally weak
PAS reactions.
The reaction for CAE is
usually weak or
negative except in M2
AML associated with
t(8;21)) in which Auer
rods are often
positive for CAE
112. MOLECULAR DESCRIPTION
● Associated with t(8;21)
● FLT3 ITD in 22%
● FLT3 mutations associated with HLA-DR negative
patients
113. ACUTE MYELOID LEUKEMIA WITH
MATURATION (FAB AML M2)
10% of AML cases; 5% of childhood leukemias
Bone marrow maturing granulocytic component
(promyelocytes to polymorphonuclear
leucocytes) >10% of non-erythroid cells
Bone marrow monocytic component
(monoblasts to monocytes) <20% of non-
erythroid cells and other criteria for M4 not met
114. S In contrast to M1 AML,
blasts are often
predominantly type II.
Auer rods frequent(70%)
Promyelocytes,myelocyte
s and neutrophils>10% of
cells
Neutrophils-hyper and
hyposegmentation,abnor
mal nuclear shape
Erythroid and
megakaryocyte
precursors - dyspoiesis
and a frank panmyelosis
is present in some cases
115. A leukemic blast
population composed
mostly of granulated
promyelocytes vs
acute promyelocytic
leukemia.
The latter often shows
bilobed nuclei and a
more homogeneous
blast population
compared with cases
of AML with
differentiation.
116. Maturation - along the neutrophil pathway but
eosinophilic or basophilic maturation occurs in a
minority. - M2Eo or M2Baso.
Increased basophils/mast cells-
deletion/translocation 12p(11-13) or t(6;9)
Erythrophagocytosis- t(8:16) (p11:p13)
AML with eosinophilia - M1/M2 t(8;21)
Leuk Lymphoma.1995;18 Suppl 1:61-3. AML M1 and M2 with eosinophilia
and AML M4Eo: diagnostic and clinical aspects.Löffler H, Gassmann
W, Haferlach T.
118. The cytochemical reactions in M2 AML - same as M1
AML- reactions are stronger and a higher
percentage of cells are positive with MPO and SBB
stains.
CAE - more often positive in M2 than in M1 AML and
reactions are stronger.
Auer rods show the same staining characteristics as
in M1 AML but are more numerous.
A population of neutrophils- which lack SBB and MPO
activity.
This may be demonstrated cytochemically or by means
of an automated differential counter based on the
peroxidase reaction, which shows a low mean
peroxidase score and an abnormally placed neutrophil
119. ACUTE PROMYELOCYTIC
LEUKEMIA
5-8% of AML cases
Median age 35-40 years, but can occur at any age
Typical and microgranular APL- DIC
Microgranular APL- Very high leucocyte count
with rapid doubling time, The WBC is usually
higher in M3V than in M3 AML.
120. MORPHOLOGY
Examining an adequate bone marrow aspirate
important
The specimen may clot during attempted
aspiration- associated hypercoagulable state,
In acute hypergranular promyelocytic leukaemia,
the predominant cell is a highly abnormal
promyelocyte.
In the majority of cases, blasts are fewer than 30%
of bone marrow nucleated cells
121. Hypergranular APL, the
most typical form,
showing promyelocytes
with cytoplasm packed
with purple
granules(120-1000nm).
Bilobed/kidney shaped
nuclei
Auer rods, sometimes
stacked in bundles of
faggots
122. A minority of cells -
agranular, have sparse
granules or have fine red
or rust-coloured dust-like
granules.
Cells that lack granules -
lakes of hyaline pink
material in the cytoplasm
may also be seen.
Basophilic
differentiation
No dysplasia
123. MICROGRANULAR
APL
Fine dustlike
granulation(100-
400nm) in the
cytoplasm
Most of the cells
show bilobed or
folded nuclei, a –
DDX acute
myelomonocytic
leukemia.
Few of the cells -
typical features of
abnormal
promyelocytes
the prognosis of
M3 variant is
somewhat worse than
that of M3 AML
124. Hyperbasophilic form, -
cells with high N/C ratio,
and strongly basophilic
cytoplasm with either
sparse or no granules.
Conspicuous
cytoplasmatic budding is
usually present, recalling
the feature of
micromegakaryocytes
Leukemia.1994 Sep;8(9):1441-
6. Acute promyelocytic
leukemia: morphological
aspects.Castoldi GL, Liso
V, Specchia G, Tomasi P
125. AUER RODS OF PROMYELOCYTIC
LEUKAEMIA
Auer rods in promyelocytic leukaemia differ from
those in M1 and M2 AML
Larger Auer rods
Hexagonal arrangement of tubular structure,
specific periodicity- 250mm vs 6-20 of other AML
Microfibrils and stellate configurations of rough
endoplasmic reticulum - characteristic of M3 AML,
particularly M3 variant
126. MPO- strongly
positive, reaction
product covering
cytoplasm and
nucleus
MPO positivity -
granules, Auer rods,
perinuclear space and
some rough
endoplasmic reticulum
profiles
127. Strong SBB and CAE positivity
NSE- positive in 25%(The reaction is weaker than in
monocytes)
The PAS reaction usually shows a cytoplasmic ‘blush’ –
a fine diffuse or dust-like positivity
M3V AML usually shows similar cytochemical
reactions but sometimes the reactions are weaker
Cases with basophilic differentiation show
metachromatic staining with toluidine blue.
The acid phosphatase reaction is strongly positive.
Strong lysozyme activity of granules and Auer rods,
M3 variant AML activity - weak to moderately strong
128. Auer rods in M3 AML are SBB, MPO and CAE
positive, whereas in other categories of AML they
are usually negative with CAE
Weakly PAS positive.
SBB, MPO and CAE - core of the rod may be left
unstained and occasionally the core is ANAE
positive on a mixed esterase stain
129.
130. ACUTE MYELOMONOYCTIC
LEUKEMIA
Proliferation of neutrophil and monocyte precursors
5-12% of AML
> 20% blasts, Neutrophils and their precursors,
monocytes and their precursors each > 20% of
marrow cells
Distinguishes from AML with or without maturation
in which some monocytes may be present
Peripheral blood- monocytes more mature than
those in marrow
131. Goasguen JE, Bennett JM, Bain BJ, Vallespi T, Brunning R, Mufti GJ for the International
Working Group on Morphology of Myelodysplastic Syndrome (IWGM-MDS).
Morphological evaluation of monocytes an their precursors. Haematologica
2009;94:994-997. doi 10.3324/haematol.2008.005421
132.
133.
134. CYTOCHEMISTRY
3% of cells – MPO positivity, Monoblasts- MPO
negative, Promonocytes-scattered positivity
Monocytes, monoblasts and promonocytes-
NSE positive
If morphological criteria met- NSE negativity does
not exclude diagnosis
Double staining with NSE and naphthol ASD
chloracetate esterase or MPO- dual positive cells
DDX- Acute monocytic leukemia and AML with
maturation
135.
136.
137. IPT
Variably express myeloid markers- CD13, CD33
Markers of monocytic differentiation- CD14, CD 4,
CD11c,CD11b, CD64, CD36 and lysozyme
Residual population of less differentiated
myeloblasts- CD34
138. ACUTE MONOBLASTIC AND ACUTE
MONOCYTIC LEUKEMIA
80% of cells are of monocytic lineage- Monoblasts,
promonocytes and monocytes
Minor neutrophil component< 20%
5-8% of AML, young individuals
Infancy- abnormalities of 11q23
Weakness, bleeding and a diffuse erythematous
skin rash.
High frequency of extramedulary infiltration of the
lungs, colon, meninges, lymphnodes, bladder and
larynx and gingival hyperplasia.
Serum and urinary muramidase levels are often
extremely high.
139.
140. MORPHOLOGY AND
CYTOCHEMISTRY
Intense NSE activity
10-20% weak or negative NSE
Monoblasts- MPO negative, Promonocytes-
scattered positivity
Biopsy-Monoblast- positive with antibody to
lysozyme
Promonocytes- CD68 and lysozyme positive
143. ACUTE ERYTHROID LEUKEMIA
Predominant erythroid population
Two subtypes based on presence and absence of
significant myeloid population
ERYTHROLEUKEMIA
> 50% erythroid precursors in nucleated cell
population
>20% myeloblasts in non erythroid population
PURE ERYTHROID LEUKEMIA(extremely rare)
Neoplastic proliferation of immature cells - erythroid
population(>80% of marrow cells) with no
significant myeloid component
144.
145. 1% of AML cases
Children to >age 90, usually adults, male
predominance
20% of therapy related AML but only 1% of de novo
AMLDi
Guglielmo syndrome: nonspecific clinical findings
of anemia, thrombocytopenia, variable neutropenia
Hemophagocytic lymphohistiocytosis: recurrent
and specific complication
Peripheral smear may have prominent erythroblasts
152. Block like staining with
PAS
Negative for SBB,
MPO
Reactive with alpha
napthyl esterase and
acid phosphatase
IHC- HB, glycophorin
153. IPT
Myeloblasts- CD33, CD13, CD117 with or without
CD34 and HLA DR
Erythroblasts- Glycophorin A, Haemoglobin A
Gero antibody, CD36 and carbonic anhydrase 1
154. ACUTE MEGAKARYOBLASTIC
LEUKEMIA
Acute leukemia where >50% of blasts are of
megakaryocyte lineage
Up to 10% of AML in children, 5% or less of adult
AML
In 1963, Szur and Lewis - pancytopenia, low
percentages of blast cells, and intense
myelofibrosis but an absence of teardrop red cells,
splenomegaly, leukocytosis, and thrombocytosis,
the usual features of primary myelofibrosis.
They designated the syndrome malignant
myelosclerosis
IPT- megakaryoblasts indicated the cases were
variants of AML rather than of primary myelofibrosis
155. ADULTS CHILDREN
Pancytopenia with low
blast counts in the
blood, myelofibrosis,
an absence of
hepatosplenomegaly,
poor response to
chemotherapy,and
short clinical course.
Markedly elevated LDH
Acute myelofibrosis
Leukocytosis
Complete remission
and long term survival
Germ cell tumors in
young boys.
Hepatosplenomegaly-
prominent abdominal
masses- t(1;22)
156. Micromegakaryocytes-
small cells, with mature
cytoplasm and one or
two round nuclei with
condensed chromatin,
Megakaryblastic
fragments, dysplastic
platelets
Circulating
hypogranular
neutrophils
157. Medium to large
blasts(12-18micromm)
Round slightly irregular
to indented nuclei
Fine reticular chromatin
1-3 nucleoli
Large and small blasts
in the same patient
Agranular, basophilic
cytoplasm with
pseudopod formation
or blebs
158. ACUTE MEGAKARYOBLASTIC LEUKEMIA
WITH T(1;22)(P13;Q13); RBM15-MKL1
acute
megakaryoblastic
leukaemia occurring in
infants and young
children.
Prominent collagenous
and reticulin fibrosis
Small and large
megakaryoblasts ,
micromegakaryocytes,
undifferentiated blasts,
159. Blasts may be
clustered and spindled
and form intertwining
bundles resembling
metastatic disease
cytoplasmic platelet
shedding may be a
useful clue to the
diagnosis in these
cases.
160.
161. CYTOCHEMISTRY
Consistently negative for MPO and SBB
They are alpha naphthyl butyrate esterase
negative
Variable alpha naphythyl acetate esterase activity
usually in scattered clumps or granules in the
cytoplasm inhibited by flouride (vs diffuse
cytoplasmic positivity)
PAS staining also varies from negative to focal or
granular positivity, to strongly positive staining.
Acid phosphatase – localised positive
162. IMMUNOPHENOTYPING
expression of platelet glycoproteins CD41 and
CD61, and less frequently CD42b.
positive for CD36. with aberrant expression of
CD7.
The myeloid markers CD13 and CD33 may be
positive, but CD34, CD45, HLA-DR and anti-MPO
are negative.
163. ACUTE BASOPHILIC LEUKEMIA
Primary differentiation is to basophils
May represent trasformation of an undetected
Philadelphia chromosome positive CML
Symptoms related to hyperhistaminemia,skin
Pinvolvement, organomegaly, lytic lesions
Poorly differentiated acute basophilic leukemias -
AML minimally differentiated without confirmation
by electron microscopy.
164. Medium sized blasts
with round to bilobed
nuclei, dispersed
chromatin and
prominent nucleoli
Basophilic
cytoplasm with
coarse basophilic
granules which may
stain with
metachromatic
stains
Vacuolation of
cytoplasm
Sparse mature
basophils
Erythroid dysplasia
165.
166. DDX-
● AML- t(6;9)
● ALL with coarse granules
● CML in blast crisis
● Mast cell leukemia
APL with basophilic differentiation
Diagnosis: may require EM
● Enzyme cytochemistry: MPO+ by EM but
negative by light microscopy
acid phosphatase+;
variable PAS+ in blocks and lakes;
Sudan Black B-, non-specific esterase-
167. LEUKEMIAS WITH BASOPHILIA
ACUTE BASOPHILIC LEUKEMIA Blasts with coarse granules and
vacuoules
MPO -, TB +, PAS +, AB+ ,CD117 -
ACUTE MYELOID LEUKEMIA
T(6;9)
Multilineage dysplasia
Occasional ringed siderblasts
ALL WITH COARSE GRANULES At least three clearly defined
azurophilic granules (Each 0.5 microns or
greater in diameter) in
more than 5% blasts and are negative for
myeloperoxidase ,SB and NSE
CML IN BLAST CRISIS bcr/abl, t(9;22)(q34;q11).
History of CML
ACUTE MAST CELL LEUKEMIA peroxidase and PAS stains -,
chloroesterase strongly +, TB and AB
+,CD 117 +
168. ACUTE PANMYELOSIS WITH
MYELOFIBROSIS
Acute panmyeloid proliferation with accompanying
fibrosis of the bone marrow
Pancytopenia without splenomegaly
The prognosis is usually poor.
Aspiration- suboptimal specimen
Marked increase in reticulin fibres> collagen
171. Megakaryocytes - loose
clustering, dislocation
towards the endosteal
border and appearance
of atypical microforms
with compact nuclei.
increase in reticulin
fibrosis.
CD34, CD13, CD33 and
CD117 positive and
negative for
megakaryocytic lineage
and myeloperoxidase.
172. DDX
Acute megakaryoblastic leukemia(no prominent
changes in granulocytes or erythroid cells)
Metastatic tumour with desmoplastic reaction
Chronic idiopathic myelofibrosis
(more immature cells, dispersed chromatin in
hypolobated or monolobate megakaryocytes, no
splenomegaly)
AML with MDS related changes with fibrosis(lacks
high percentage of blasts
173. MYELOID SARCOMA
A tumour mass of
myeloblasts or immature
myeloid cells
Extramedullary site/bone
Precede/concurrent with
AML/MDS/MPN
Relapse of leukemia in
remission
FAB classification (1976) –
does not specify
WHO classification (2001).
Included under ‘AML not
otherwise categorized’
• WHO Classification (2008).
Separate entity in
classification of AML
Lymphoid tissue: lymph node,
tonsil, spleen, thymus
Subperiostal bone: skull, orbit
(classical presentation),
sternum, ribs, vertebrae, pelvis,
etc.
Skin
Soft tissue: muscle,
mediastinum, etc.
Mucosae: gastrointestinal tract,
urinary tract, mouth, larynx,
etc.
Different organs: breast,
kidney, lung, testis, ovary,
uterus (cervix), genital tract,
prostate
Central nervous system and
174. A blastic variant with
predominance of
myeloblasts,
An immature variant with
a mix of myeloblasts and
promyelocytes,
A differentiated variant
with promyelocytes and
more mature
granulocytes
Intracytoplasmic auer
rods-MDS
Monoblastic-11q23
Predominantly
erythroid/megakaryocyte
s/trilineage
haematopoesis- MPN
FAB M2
specific cytogenetic
abnormalities (e.g. t(8;21)
or inv(16))
Myeloblasts express T-
cell surface
markers, CD13, or CD14
high peripheral WBC
counts
175. diffuse monotonous
infiltrate.
medium-sized to large
blastic cells with ovoid
vesicular nuclei with
medium-sized or large
centrally located nucleoli
and dispersed chromatin.
cytoplasm scant to
moderate.
The mitotic count can be
high.
There may be apoptotic
bodies phagocytosed by
histiocytes (tingible body
macrophages).
176. CYTOCHEMISTRY IPT
MPO and naphthol
ASD CAE positive
Monoblastic- NSE
positive
CD13,CD33,CD117,M
PO
CD14, CD116, CD11c-
monoblastic
CD 43 but not CD3
177. TRANSIENT MYELOPROLIFERATIVE DISORDER
10% of newborns with Down’s syndrome
Resembles congenital acute leukemia
Within first days of life with numerous blasts in
peripheral blood, more than those in marrow
High rate of spontaneous resolution; usually
resolves in 2-14 weeks in neonates
20-30% progress to AML-M7 [FAB] (acute
megakaryoblastic leukemia) within 3 years
Phenotypically normal neonates - mosaic for
trisomy 21
180. AML M7 IN DOWN’S SYNDROME
Most common type of leukemia in Down’s
syndrome
Down Syndrome (DS): 150x increased risk of AML
compared to non-Down children age 0-4 years;
70% are AML M7 compared to 3-6% in non-Down
children
DS children ages 0-3 years: ALL vs AML risk is
1:1.2 compared to 4:1 for non-DS children
Trisomy 8
Leukemogenesis of AMkL - somatic mutations
involving the GATA1 gene
GATA1 is a chromosome X linked transcription
factor which is essential for erythroid
andmegakaryocytic differentiation.
182. Young children with
DS and AML with
GATA1 mutation -
better response to
chemotherapy and
better prognosis
compared to non-DS
with AML
Older children with
GATA1 mutation
have poorer
prognosis - AML in
non-DS children
SBB, MPO and Tdt
negative
PAS- scattered
granular positive
May express CD7
183. BLASTIC PLASMACYTIC DENDRITIC
CELL NEOPLASM
The tumor was initially described in 1995 as an
acute agranular CD4-positive natural killer (NK) cell
leukemia
Based on the blastic appearance and CD56
expression, the term "blastic NK cell lymphoma"
was used.
Subsequently, the term "agranular CD4+CD56+
hematodermic neoplasm/tumor" was coined based
on the immunophenotype and a predilection for skin
involvement
BPDCN - derived from plasmacytoid dendritic cells
(type 2 dendritic cells)blastic plasmacytoid dendritic
cell neoplasm- 2008 WHO classification of tumors
of the hematopoietic and lymphoid tissues
184. monomorphic, poorly
differentiated,
intermediate-sized blasts,
resembling those seen in
the skin
Bone marrow
involvement is present in
over 80 percent of the
patients and diffuse
involvement is common.
The tumor cells may
show microvacuoles
along the cell membrane
("pearl necklace"
appearance) and
pseudopod-like
185. The tumor cells
express CD4 and
CD56
CD123 (interleukin-3 α-
chain), BDCA-
2/CD303 (blood
dendritic cell antigen-
2), TCL1, and SPIB
TdT- 40%
CD7 (a T cell antigen)
and CD33 (a myeloid
antigen) are also
expressed relatively
frequently
186. BIPHENOTYPIC ACUTE LEUKEMIA
A single clone of leukemia cells express markers of
2 lineages- myeloid and lymphoid.
Two populations of cells or cytologically uniform
187.
188.
189.
190. REFERENCES
WHO Tumours of haematopoetic and lymphoid tissues
Leukemia Diagnosis- Barbara Bain
Acute Myeloid Leukemia: Importance of Ancillary
Studies in Diagnosis and Classification Daniel A. Arber,
MD
Acute Myeloid leukemia- Kathryn Foucar, Karyn
Reichaard
World Health Organization (WHO) classification of the
myeloid neoplasms James W. Vardiman, Nancy Lee
Harris and Richard D. Brunning
Acute Myeloid Leukemia Diagnosis in the 21st Century
Bryan L. Betz, PhD; Jay L. Hess, MD, PhD (Arch Pathol
Lab Med. 2010;134:1427–1433)
Current concerns in haematology 2: Classification of
acute leukaemia B Bain, D Catovsky J Clin Patlol