CLINICAL INTERNSHIP - SEMINAR 1

Oral Reporting
OUTLINE

TOPIC: UNDERSTANDING THE DIFFERENCE OF ACUTE MYELOCYTIC

& LYMPHOCYTIC LEUKEMIA

GROUP 3 – BATCH FAGELSON:

Aporbo, Roy James
Conol, Lestle Mae
Enrique, Jephunneth Klint
Fabiola, Kristine Danielle
Maghinay, Laiza Mitch
Sambrana, Christine Mae
Yase, Jevie Lynn

I. OBJECTIVES:
At the end of this lesson the students would be able to;
 COGNITIVE: Comprehensively describe Acute Leukemia, according to the WHO and
FAB Classification System; and Outline the broad types of acute leukemia based on its
certain clinical and diagnostic features.
 AFFECTIVE: Differentiate Acute Leukemias based on a systematic approach from
Preliminary Evaluation, Bone Marrow Aspirate Examination, Cytochemical Staining,
Immunologic Analysis, and other Advance Laboratory Studies.
 PSYCHOMOTOR: Choose and Describe treatment options available for patients
suffering from such Hematopoietic Malignancy.

II. INTRODUCTION:
The broad term "Leukemia" is derived from the ancient Greek words "leukos", meaning
white, and "haima", meaning blood.

ACUTE LEUKEMIA refers to the rapid, clonal proliferation in the bone marrow of lymphoid
or myeloid progenitor cells known as Lymphoblasts and Myeloblasts, respectively. This
results to suppression of normal hematopoiesis in which blasts are seen in the peripheral
blood of the patient, when examined. Unfortunately, most cases of such malignancy have
unknown causes, however, certain causes where also tracked down through multiple clinical
studies, including:
 Progressive Mutations or "Multiple Hits" Mutations (leads to proliferative advantage,
in addition to mutations that hinder differentiation) - In detail these mutations transforms
normal Hematopoietic Stem Cells (HSC's) into Leukemic Stem Cells (LSC's) that initiate,
proliferate, and sustain leukemia.
 Genetic Changes (toxin-induced genetic changes)
 Environmental Exposures (lead, benzene, radiation)
 Familial Predispositions (Heredity) (familial cancer predisposition syndromes)

The same with most other cancer types, leukemia can also be treated using Alkylating
Agents and other forms of Chemotherapy, which consequently induces DNA damage that
can lead to another complication of Therapy-related Leukemias.
III. QUICK RECAP – DEVELOPMENT & MAINTENANCE OF HEMATOPOIETIC TISSUES:
In application of the “Monophyletic Theory – suggests that all blood cells are derived from a
single progenitor stem cell called Pluripotent HSC’s” & “Stochastic – random hematopoietic
differentiation & Instructive Model – bone marrow microenvironment influenced
hematopoietic differentiation” of hematopoiesis; the formed elements of the blood: Red
Blood Cells, Granulocytes, Monocytes, Platelets, and Lymphocytes have a common origin
from Hematopoietic Stem Cells (HSC’s), which are pluripotent cells with self-renewal
capacity that sit at apex of a hierarchy of bone marrow progenitors. These HSC’s give rise to
cells referred to as Multipotent Progenitors, which are more proliferative than HSCs but
have a lesser capacity for self-renewal.
Eventually, these progenitors become committed and commence an inexorable journey
down a road that leads to terminal differentiation and death in which proliferation becomes
more rapid in response to multiple growth factors and cytokines.

Figure 1 Differentiation of Hematopoietic Cells.

IV. CLASSIFICATION SCHEMES FOR ACUTE LEUKEMIAS:
As early as 1970 the FRENCH-AMERICAN-BRITISH (FAB) CLASSIFICATION SCHEME
of acute leukemias was devised based on Morphology & Cytochemical Stain Reactions
this was used to distinguish lymphoblasts from myeloblasts, which has the following distinct
characteristics and features:

Table I: Lymphoblasts vs. Myeloblasts.

LYMPHOBLASTS MYELOBLASTS

Blood Picture

Figure 2.1 Lymphoblast Figure 2.2 Myeloblast

2-3 times the normal 3-5 times the normal
Diameter
lymphocyte diameter lymphocyte diameter
Nucleoli Inconspicuous nucleoli Two or more prominent nucleoli
Chromatin Coarse chromatin Uniform fine chromatin
Cytoplasm Scant blue cytoplasm Moderate gray cytoplasm
Other Features *Deeper staining than myeloblasts *Possibly contain Auer rods

In recent advances with Flow Cytometry and Genetic/Molecular Studies hematologist and
pathologists are now moving toward a more precise classification of many of the leukocyte
neoplasms based on Recurring Chromosomal & Genetic Lesions found in many patients.
This paved the way for the development of the WORLD HEALTH ORGANIZATION (WHO)
CLASSIFICATION SCHEME of acute leukemias that melded the older schemes with the
newer schemes recognizing nearly all of the tumors of hematopoietic and lymphoid tissues.
Thus far the FAB & WHO still remains the two major classifications of acute leukemias that
broadly divide it into: Acute Lymphoblastic Leukemia (ALL) & Acute Myeloid Leukemia
(AML) [alternate names: Acute Non-lymphoblastic Leukemia, Acute Myelogenous
Leukemia, Acute Myeloblastic Leukemia, or Acute Granulocytic Leukemia]; which is based
on the two major lineages of hematopoiesis: Lymphoid & Myeloid Lineage.

V. ACUTE MYELOID LEUKEMIA:

INTRODUCTION:
Acute Myeloid Leukemia (AML) is the most common type of leukemia in adults (less
common in children) in which incidence increases with age it has nonspecific clinical
presentation but reflects decreased population of normal bone marrow elements.

Laboratory Findings:
 WBC Count: Between 5-30 x109/L, although it may range from 1-200 x109/L (this usually
increases in presentation of the malignancy).
 Peripheral Blood Picture: Myeloblasts are seen (true to 90% of patients).
 Bone Marrow Picture: Hypercellular and >20% blast cells seen.
 Hyperphosphatemia (due to cell lysis).
 Hypocalcemia (due progressive bone destruction).
 Hypokalemia
 Hyperuricemia (caused by increased cellular turnover).
 Hyperuricosuria (most probably due to Tumor Lysis Syndrome that most of the time
develops during induction chemotherapy, which is caused by the breakdown products of
dying cancer cells, causing acute uric acid nephropathy and renal failure. This is also
contributory to the latter four findings of AML).
 Anemia, Thrombocytopenia, and Neutropenia.
 These give rise to the clinical findings of Pallor, Fatigue, Fever, Bruising, & Bleeding.
 Disseminated intravascular coagulation (DIC) & other Bleeding disorders may also be
present.
 Malignant cell infiltration (mucosal sites and skin).

Other Clinical Findings:

 Splenomegaly (true to 50% of patients).
 Lymph node enlargement (rare).
 Cerebrospinal fluid involvement (rare and does not seem to be an ominous sign as in
ALL, thus they only have few symptoms related to the central nervous system).

OVERVIEW OF AML BASED ON THE FAB & WHO CLASSIFICATION SCHEME:

FRENCH-AMERICAN-BRITISH (FAB) CLASSIFICATION OF AML:
This classification scheme was proved to be useful in the standardizing the Morphological
Classification of acute myeloid leukemia, which also puts emphasis on Cytometric
Phenotype, and Cytochemical Reactions. In a way this classification schema made the
diagnostic process for acute myeloid leukemia more objective and unambiguous as
possible, although ambiguity still exists.

Table II: FAB Classification of AML based on Cellular Morphology.

TYPE CHARACTERISTICS
M0 Myeloid Without Cytologic Maturation: Morphologically undifferentiated
leukemic blasts with myeloid immunophenotype.
M1 Myeloid Without Maturation: Marrow leukemia cells are primarily myeloblasts
with no azurophilic granules.
M2 Myeloid With Maturation: Leukemia cells show prominent maturation beyond
myeloblast stage.
M3 Promyelocytic: Abnormal, hypergranular promyelocytes dominate; Auer rods
easily found; increased incidence of DIC; strong MPO staining.
M3m Microgranular Variant Of M3: Indistinct granules; rare to occasional Auer rods,
nucleus often reniform or bilobed; increased incidence of DIC; strong MPO
staining.
M4 Myelomonocytic; Both monocytic (monocytes and promonocytes) and myeloid
differentiation (maturation beyond myeloblast Stage).
M4eo M4 With Bone Marrow Eosinophilia: Similar to M4 with marrow eosinophilia
(abnormal and immature); associated with abnormal 16q karyotype.
 M5a Monocytic, Poorly Differentiated: Monoblasts predominate, typically with
abundant cytoplasm and single distinct nucleoli.
M5b Monocytic, Well Differentiated: Predominantly promonocytes in marrow and
more pronounced maturation in blood.
M6 Erythroleukemia: Dysplastic erythroblasts with multinucleation, cytoplasmic
budding, vacuolation, and megaloblastoid changes.
M7 Megakaryoblastic: Wide range of morphology; cytoplasmic projections
sometimes present; electron microscopy or immunocytochemical stains
necessary for diagnosis.

Table III: FAB Classification of AML based on Cytometric Phenotypes.

TYPE IMMUNOLOGIC SURFACE MARKERS
M0 (+) to at least one: CD13, CD33, or CD117; (-) to lymphoid antigens.
M1 (+) to at least two: CD13, CD33, or CD117; (-) to lymphoid antigens.
M2 (+)CD13, CD33, & CD117; may also become (+) to: CD19, CD34, & CD56.
M3 (+) CD13 & CD33; (-) CD34 & HLA-DR.
M4 (+) CD11b, CD11c, CD14, CD64, & CD4.
M5 (+) CD64, CD56, CD4, CD13, CD33, & HLA-DR.
M6 (+) CD13, CD33, CD34, CD117, HLA-DR, & Glycophorin A.
M7 (+)CD13, CD33, CD41, CD42b, CD61, CD36, & Factor VIII.

Table IV: FAB Classification of AML based on Cytochemical Reactions.

CYTOCHEMICAL STAINS & REACTIONS
TYPE
MPO SBB CAE NSE AcP PAS CrP
M0 - - - - - - -
M1 + + +/- - - +/- -
M2 + + + - -/+ -/+ -
M3 + + + +/- + +/- -
M4 + + + + + +/- -
M5 +/- +/- - + - +/- -
M6 -* -* -* +/- - + -
M7 - - - +/- + + -

WORLD HEALTH ORGANIZATION (WHO) CLASSIFICATION OF AML:

The overview presents the mended classification of acute myeloid leukemia, using the two
major classification schemes, namely the WHO & FAB:
 Acute myeloid leukemia with Recurrent Genetic Abnormalities:
 AML with t(8;21)(q22;q22.1);RUNX1/ RUNX1T1
 AML with inv(16)(p13.1q22) or t(16;16) (p13.1;q22);CBFB-MYH11
 AML with t(15:17);PML-RARA
 AML with t(9;11)(p22;q23);KMT2A (MLL)- MLLT3
 AML with t(6;9)(p23;q34.1); DEK-NUP214
  AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2, MECOM (RPN1-EVI1)
 AML with t(1;22)(p13.3;q13.3) RBM15-MKL1
 AML with BCR-ABL1
 AML with gene mutations
 AML with mutated NPM1
 AML with biallelic mutation of CEBPA
 AML with mutated RUNX1
 Acute myeloid leukemia with Myelodysplasia-related Changes
 Therapy-related Myeloid Neoplasms
 Acute myeloid leukemia, not otherwise specified:
This category did not easily fit into the WHO subtypes, thus it utilizes the FAB
classification based on Morphology, Cytometric Phenotyping, & Cytochemical Reactions.
 Myeloid Sarcoma
 Myeloid proliferations associated with Down Syndrome
 Blastic Plasmacytoid Dendritic Cell Neoplasm
 Acute leukemias of Ambiguous Lineage

DISCUSSION ON SOME CLINICALLY SIGNIFICANT AML TYPES:

• Acute Myeloid Leukemia with Recurrent Genetic Abnormalities:
 AML with t(8;21)(q22;q22.1);RUNX1/ RUNX1T1
It is found to occur in 5% of AML cases that is
predominantly seen in children and young
adults.
It has characteristic myeloblasts with
dysplastic cytoplasm, Auer rods, and some
maturation that is similar to the FAB M2
classification showing leukemia cells with
prominent maturation beyond the myeloblasts
stage.
Various anomalies, such as pseudo–Pelger- Figure 3.1 Bone Marrow Aspirate from a
Patient with Acute Myeloid Leukemia
Huët cells and hypogranulation, can be seen; with t(8;21).
and possible eosinophilia.
It has a favorable prognosis, but it may be affected by other underlying
abnormalities, such as Monosomy 7 – Myelodysplasia & Leukemia Syndrome-1
(M7MLS1).

 AML with inv(16)(p13.1q22) or t(16;16) (p13.1;q22);CBFB-MYH11

It accounts for approximately 5% to 8% of all
AML cases that occurs at all ages, but it is
found predominantly in younger patients.
Myeloblasts, monoblasts, and promyelocytes
are seen in the peripheral blood and bone
marrow.
Additionally, eosinophilia with dysplastic
changes may be seen in the bone marrow’
and it is classified as FAB M4eo.
This AML type has a higher incidence of
becoming extramedullary, with the central
nervous system (CNS) being its common site Figure 3.2 Peripheral Blood Film from a
Patient with Acute Myeloid Leukemia
for relapse. with inv(16).
 Its remission rate is good, especially with therapy containing high-dose cytosine
arabinoside, but only one half of patients are cured.

 AML with t(15:17)(q22;12);PML-RARA

It comprises 5% to 10% of all AML cases that
occurs in all age groups, but is seen most
commonly in young adults.
This AML type is highly associated with
maturation arrest at the promyelocyte stage as
a result of the genetic aberration.
Abnormal promyelocytes with heavy
granulation, sometimes obscuring the nucleus,
abundant cytoplasm, and bundles/stacks of Figure 3.3 Peripheral Blood Films from
Auer rods (Faggot Cells) may be seen; these a Patient with Acute Myeloid Leukemia
features suit the FAB M3 classification. with t(15;17) or Promyelocytic Leukemia
In its one variant that is classified as FAB – FAB M3 classified.
M3m indistinct granules can be seen together
with rare to occasional Auer rods, and
promyelocytes have nucleus that are often
reniform or bilobed.
This AML type is treated differently from all
other AML types that targets differential
induction of malignant promyelocytes using
all-trans-retinoic acid (ATRA) and arsenic
trioxide.
However, other genetic translocation variants Figure 3.4 Peripheral Blood Films from
may not respond well with the said therapy. a Patient with Acute Myeloid Leukemia
with t(15;17) or Promyelocytic Leukemia
– FAB M3m classified.

 AML with t(9;11)(p22;q23);KMT2A (MLL)- MLLT3

It is a rare AML type that only accounts 6% of all
AML cases that typically occurs in children
associated with gingival and skin involvement
and/or DIC.
This AML type presents with an increase in
monoblasts and immature monocytes that is
classified as FAB M5a/M5b depending on the
predominant cells found and rate of
differentiation.
Figure 3.5 Bone Marrow Aspirate of a
The blasts are large with abundant cytoplasm Patient with Acute Myeloid Leukemia
and fine nuclear chromatin cells that may have with t(9;11) Abnormalities, FAB M5a
motility, with pseudopodia seen frequently. classified.
Granules and vacuoles can be observed in the blasts.

 Acute Myeloid Leukemia with Myelodysplasia-related Changes:

It is an AML type that typically occurs in elderly patients, which incorporates
leukemia with at least 20% Blasts, Multilineage Dysplasia (>50% Blasts), History of
Myelodysplastic Syndrome or Myeloproliferative Neoplasm, and/or Myelodysplastic-
associated cytogenetic abnormality (e.g. -7/ del(7q) and -5/del(5q)).
Most often patients present clinically with severe pancytopenia.
 Therapy-related Myeloid Neoplasms (t-MN’s):
This AML type accounts for 10% to 20% of all AML cases that is common among
patients receiving therapies such as, Alkylating Agents, Radiation, or Topoisomerase
II Inhibitors.
However, It may also become a complication to patients with non-malignant
conditions but are receiving intensive treatment and care requiring Cytotoxic
Therapy, which in effect poses neoplastic/dysplastic damage to tissues like that of
the bone marrow.

 Acute Myeloid Leukemia, not otherwise specified:

This category does fit into the WHO subtypes, thus it utilizes the FAB classification
based on Morphology, Cytometric Phenotyping, & Cytochemical Reactions.
(Refer to Tables: II, III, & IV)

 Myeloid Sarcoma:
It is an extramedullary type of myeloid tumor commonly affecting the Lymph Nodes,
Skin, Sinuses, Brain, and Periosteum of Bones.
Three types of myeloid sarcoma are generally recognized and are based on the
degree of maturation, namely:
 Blastic Myeloid Sarcoma; predominantly composed of myeloblasts.
 Immature Myeloid Sarcoma; predominantly composed of myeloblasts and
promyelocytes.
 Differentiated Myeloid Sarcoma; predominantly composed of neutrophils.
It is very important to remember that unlike extramedullary hematopoiesis (liver,
spleen, & other fetal sites) is not a compensatory mechanism, rather a uncontrolled
spread of a neoplastic proliferation.

 Myeloid Proliferations associated with Down Syndrome:

Uniquely, approximately 10% of newborns with Down syndrome present with
transient abnormal myelopoiesis, which is morphologically indistinguishable from
AML, research states that it is most probably associated with GATA1 mutations.
Furthermore, it is also said that individuals with down syndrome have fifty-fold
increased incidence of AML during its first five years.

 Blastic Plasmacytoid Dendritic Cell Neoplasm:

Blastic plasmacytoid cell neoplasm is a rare clinically aggressive tumor derived from
precursors of plasmacytoid dendritic cells.
It presents with skin lesions and may ultimately progress to involve peripheral blood
and bone marrow.

 Acute Leukemias of Ambiguous Lineage:

Even in the advent of advance molecular and genetic studies, some other types of
acute leukemia still lacks clear lineage, showing characteristics of both myeloid and
lymphoid, or both B and T lymphoid lineages.
The hallmark of these rare leukemias of ambiguous lineage is that the overall
findings are insufficient to classify as myeloid or lymphoid (undifferentiated) or the
antigen expression is truly ambiguous and shows significant coexpression across
lineages.
In general, its ambiguity holds such classification of acute leukemia together.
VI. ACUTE LYMPHOID LEUKEMIA:
INTRODUCTION:
Acute Lymphoid Leukemia (ALL) is mostly a disease of children and adolescents,
accounting for 25% of childhood cancers and up to 75% of childhood leukemia that peaks
around ages between 2 and 5 years of age.
In general, acute lymphoid leukemia is categorized according to the malignant cell lineage
involved in the lymphohematopoietic maturation, namely: B Cell – ALL and T Cell – ALL.
These major types of ALL present similar and different clinical symptoms associated with all
other abnormal laboratory findings, such as:

Table V: Laboratory & Clinical Findings of B Cell – ALL vs. T Cell – ALL.
B CELL - ALL T CELL – ALL
Laboratory Findings  Anemia (causes fatigue)  Large Mediastinal Mass
 Neutropenia & Infection (compromises regional
(causes fever) anatomic structures)
 Thrombocytopenia (causes
mucocutaneous bleeding).
 Lymphadenopathy
 Splenomegaly
 Hepatomegaly
 Osseous Pain (due to
progressive bone damage)
 Infiltration of malignant
cells (e.g. meninges,
testes, or ovaries).
Figure 4: Large Medial Mediastinal Mass.

 Anemia
 Often less severe
Leukopenia
 Thrombocytopenia
 Organomegaly
 Osseous Pain

OVERVIEW OF ALL BASED ON THE FAB & WHO CLASSIFICATION SCHEME:

FRENCH-AMERICAN-BRITISH (FAB) CLASSIFICATION OF ALL:
The French-American-British (FAB) classification of acute lymphoid leukemia separates
ALL into three morphological groups that have characteristic and distinct morphological
features that are made as basis for designating FAB-ALL classification.

Table VI: FAB Classification of Acute Lymphoid Leukemia (ALL).

FEATURES L1 L2 L3

Bone Marrow
Picture
 Cell Size Predominantly small Large heterogeneous Large homogeneous
Nuclear Homogenous in any Heterogeneous Finely stippled, and
Chromatin one case homogeneous
Nuclear Shape Regular, occasional Irregular, clefting and Regular (round to
clefting indentation common oval)
Nucleoli Inconspicuous One or more, often One or more,
large prominent
Cytoplasm Scanty Variable; often Moderately abundant;
moderately abundant strongly basophilic
Cytoplasmic Variable Variable Prominent
Vacuolation

WORLD HEALTH ORGANIZATION (WHO) CLASSIFICATION OF ALL:

The World Health Organization (WHO) classification of ALL is based on whether the clonal
lymphoblast population is of Bor T-lymphocyte lineage and whether the leukemic population
has the attributes of an “early lymphocyte” or “lymphoblast”.
These attributes are identified through immunophenotyping, involving the antigens
expressed by cells at a specific phase during maturation, which in the same cells follows
and expresses the same antigens, regardless of malignancy.
Thus, provides the basis for the WHO-ALL Classification, as presented below:

Table VII: WHO Classification of ALL based on Immunophenotypic Characteristics.

SUBTYPE PHENOTYPE
Precursor B – Cell HLA-DR, CD34, TdT, CD19, CD20(+/-), CD10
Cytoplasmic CD22
Pre B – ALL HLA-DR, TdT(+/-), CD19, CD20(+/-), CD10, cIgM
Cytoplasmic CD22
B – ALL (Intermediate/Common) HLA-DR, CD19, CD20, CD22, CD10 (4), slg
Precursor T – ALL HLA-DR(+/-), CD1, CD2, cCD3, dual CD4/CD8, CD5,
CD7(+/-), CD10(+/-), CD34(+/-), TdT

Even if most hematologists and pathologists would rely strictly on immunophenotypic

studies for the classifications of ALL, both FAB and WHO classification can be mended
together to make the diagnostic process for acute lymphoid leukemia more objective and
comparative.

Table VIII: Comparison of the WHO & FAB Classification of ALL.

WHO – IMMUNOLOGIC FAB – MORPHOLOGIC
Precursor B-Cell ALL
 Early-Pre-B-Cell ALL L1, L2 Morphology
 Pre-B-Cell ALL L1, L2 Morphology
B-Cell ALL L3 Morphology
T-Cell ALL L1, L2 Morphology
DISCUSSION ON SOME CLINICALLY SIGNIFICANT ALL TYPES:
 Precursor B-Cell ALL (Early-Pre-B-Cell & Pre-B-Cell):
This ALL type is seen predominantly in the pediatric age group (Birth to 16/18 years old),
which peaks at ages between 3 to 5 years of age, however, it may occur also occur at
any age of life.
As describe, it exhibits L1 and L2 morphology based on the FAB classification in which
such lineage and maturation was identified using its immunophenotypic characteristics.
Similar to AML, utilizing advancements in molecular and genetic assays the WHO
classification added that various genetic aberrations are also associated with specific
B-Cell ALL types, most commonly due to translocation mutations, such as:
 B-Cell ALL with t(12;21): TEL-AML1
It is generally considered the most common translocation in precursor B-Cell ALL
that accounts for approximately 16% to 28% of all B-Cell ALL cases.
It primarily affects children between 2 and 5 years of age; and rarely found in
adults (Good prognosis in pediatric ALL).
This translocation is more commonly detectable by RI-PCR than by routine
cytogenetic analysis

 B-Cell ALL with t(1;19): TCF3-PBX1

It still commonly diagnosed in pediatric patients (Poor prognosis in pediatric ALL)
This translocation is detectable in many cases by both RT-PCR and FISH.

 B-Cell ALL with t(9;22): BCR-ABL1

It is considered a high-risk B-Cell lineage ALL that is seen in about one quarter of
adult ALL cases, but is uniformly fatal in all age groups.
This translocation may also be detected by RT-PCR and FISH.

 B-Cell ALL with t94;11): AF4-MLL

It is the most common form of ALL known to occur/affect infants (Poor prognosis
in pediatric patients).
This translocation may be detected by RT-PCR, FISH using a specific probe,
Flow Cytometry.

All other types of B-Cell ALL associated with genetic abnormalities, include the following:
 B-Cell ALL with t(v;11q23.3); KMT2A (MLL) rearranged
 B-Cell ALL with hyperdiploidy
 B-Cell ALL with hypodiploidy
 B-Cell ALL with t(5;14)(q31.1;q32.1);IGH/IL3
 B-Cell ALL with BCR-ABL1-like
 B-Cell ALL with iAMP21
  Mature B-Cell ALL:
It is commonly known as Burkitt’s Leukemia/Lymphoma.
It is rare and most typically represents the leukemic phase of Burkitt lymphoma in
patients with bulky disease.
Uniquely, even if in the same B-cell lineage it was not classified as a subtype of
precursor B-Cell ALL due to the lack of the immunophenotypic characteristics of an
early B cell, namely the TdT is negative; it also has a FAB-L3 classification.
Patients with Burkitt’s leukemia are treated differently from patients with pre-B ALL,
which has a relatively good prognosis.
The prognosis for Burkitt’s leukemia (B-cell ALL) has traditionally been worse than
for most precursor B-cell ALL, but has improved with newer targeted therapies.

 Precursor T-Cell ALL:

This ALL type accounts for 15% to 25% of all ALL cases that occurs more often in
older children, affecting more males than females (Poor patient prognosis than those
with common pre-B-cell ALL). In the same, it is classified based on the FAB –
morphological characteristics and WHO – immunophenotypic characteristics.
Clinically, patients with T-Cell ALL present with a mediastinal mass, a high white
blood cell count, hepatosplenomegaly, and early meningeal involvement.
Interestingly, this ALL type is often confused with Lymphoblastic Lymphoma in
which patients with such condition also present with mediastinal mass; it also tests
positive for TdT and shows evidence of the T cell lineage.
Differential diagnosis of the two is based on a clinical criteria of presentation in which
T-Cell ALL is diagnosed in “Patients who present with prominent marrow and
peripheral blood involvement with mediastinal mass”, while Lymphoma is
diagnosed in “Patients who present with mediastinal involvement, but no marrow
or peripheral blood involvement”.

VII. LABORATORY EVALUATION OF ACUTE LEUKEMIA

A. PRELIMINARY EVALUATION
Clinically, a highly Systematic Approach is applied when diagnosing a patient. It
primarily considers Patient Demographics, Clinical History, and Physicality, which would
jumpstart the physician’s diagnosis; this ultimately applies to all cases of giving medical
interventions.
In the case of acute leukemia, physicians would initially request for the following
laboratory tests:
 Complete Blood Count (CBC) with Platelet Count;
 Typically Decreased.
 White Blood Cell (WBC) Differential Count; &
 Variable, ranging from significantly decreased to markedly elevated.
 Peripheral Blood Smear Examination
 Reveals Blast Cells or other Immature/Abnormal Cells.
KEY TAKEAWAYS:
EDTA anticoagulated blood may cause subtle morphological artifacts of
nucleated cells and platelets; and when specimen is left for longer than 30
minutes it may show Artifactual Vacuolation of Monocytes and Neutrophils,
 Nuclear Shape Changes and Swelling, as well as Degranulation of Platelets.
Thus, most of the time when a patient is suspected for a case of leukemia
physician’s would prefer Non-anticoagulated Fingerstick Blood sample as
it will not show these abnormalities.
Furthermore, as a medical technologist it is very important to keep in mind
these possible changes, especially if an EDTA-anticoagulated peripheral
blood smear is available for review.

B. BONE MARROW ASPIRATE EXAMINATION – EVALUATION OF MORPHOLOGY

Although a diagnosis of acute leukemia may be established by a peripheral blood
examination, well-prepared smears of Bone Marrow Aspirate Material is the
specimen of choice for establishing diagnosis for acute leukemias that is based on the
Morphology & Population Of Blast Cells seen and Cytochemical Criteria.

BONE MARROW COLLECTION SITES include the following:

 Posterior Superior Iliac Crest;
 Preferred for both adults & children that
provides adequate red marrow and is isolated
from anatomic structures that are subject to
injury.
 This site is used for both aspiration and core
biopsy.
 Anterior Superior Iliac Crest of the Pelvis;
 The same advantages with the posterior
superior iliac crest, only it is thicker; most of
Figure 5 The Posterior Superior Iliac
the time it is the preferred site for patients who
Crest – Bone Marrow Collection Site.
can only lie supine.
 Sternum below the angle of Lewis at the second Intercostal Space;
 It provides ample red marrow and cannot be used for core biopsy; furthermore, it
is prone to pericardial penetration, resulting to damage to the heart and great
vessels.
 Anterior Medial Surface of the Tibia;
 It can be used as a site for children younger than 2 years old, but only for
aspiration procedures.
 Spinous Process of Vertebrae, Ribs, & other Red Marrow containing Bones;
 Available, but is rarely used due to injury, or unless it is the suspected site of a
lesion detected on imaging.

BONE MARROW BIOPSY/ASPIRATION NEEDLES include the following:

 Disposable Sterile Jamshidi Bone Marrow Biopsy and Aspiration Needle
 Adult-size: 11-gauge x 4 inch biopsy/aspiration needle
 Children-size: 14-gauge x 6 inch biopsy/aspiration needle
 It has an Obturator, Core Biopsy Tool, and Stylet.
 Westerman-Jensen Bone Marrow Biopsy Needle
 Usually 14- to 18- gauge aspiration needle.
 It has an Obturator, Core Biopsy Tool, and Stylet.
 Snarecoil Bone Marrow Biopsy Needle
 Usually 8- to 11- gauge aspiration needle.
 It has a coil mechanism at the needle tip that allows for capture of the bone
marrow specimen without needle redirection
 It is very important to remember that arrangements
should be done first prior to collecting a bone marrow
specimen including, alignment of special studies (e.g.
Cytochemistry and Flow Cytometry) and special
handling procedures as requested by the physician.
Most of the time during the procedure the following
are noted:

1. The aspirate should have the presence of bone

marrow spicules, which indicates accession to the
bone marrow cavity and correct aspiration
procedure. If spicules are not present, another
aspiration may be necessary.
2. Bone marrow smears should be made
immediately to avoid clotting.
3. Special studies require direct aspiration into a
heparin coated syringe; Sodium Heparin being the Figure 6 Examination of Bone Marrow
anticoagulant of choice (e.g. Cytogenetics). Aspirate for the presence of Spicules.

The WHO classification system for acute leukemias requires a minimum blast cell
count of 20% in the peripheral blood/200 leukocytes or bone marrow/500 nucleated cells
for confirmation of the diagnosis of acute leukemia in comparison to the FAB
classification system which requires a blast cell count of 30%.

RE-PRESENTATION: Table I: Lymphoblasts vs. Myeloblasts.

LYMPHOBLASTS MYELOBLASTS

Blood Picture

Figure 2.1 Lymphoblast Figure 2.2 Myeloblast

2-3 times the normal 3-5 times the normal
Diameter
lymphocyte diameter lymphocyte diameter
Nucleoli Inconspicuous nucleoli Two or more prominent nucleoli
Chromatin Coarse chromatin Uniform fine chromatin
Cytoplasm Scant blue cytoplasm Moderate gray cytoplasm
Other Features *Deeper staining than myeloblasts *Possibly contain Auer rods
KEY TAKEAWAYS:
 A very helpful morphological feature is the Auer rods, the presence of which excludes
ALL. These are cytoplasmic inclusions that result from an abnormal fusion of primary
granules and are pathognomonic for a myeloproliferative process, particularly AML.
They can be found in “Bundles” or “Cigar-shaped Rods” termed as ―Faggot Cells‖.
 In general the bone marrow aspirate examination would help determine what
proliferative lineage is behind the malignancy of the patient, just by looking at the
Morphology & Population Of Blast Cells, which in most times should coincide with the
results that were acquired during preliminary evaluation.
 As invasive and complex as it is this procedure/intervention still does not lead to an
accurate diagnosis, however, it would help narrow down the patient’s case to make a
definitive diagnosis.

C. CYTOCHEMICAL STAINING
Most of the time, especially in the Philippines, bone marrow aspirate examination is
usually requested simultaneously with Cytochemical Staining, which are used to
identify chemical components of cells (e.g. Enzymes and Lipids) that help define
granulocytic and monocytic differentiation and continue to be useful to distinguish AML
from ALL. These special stains include the following:
Table IX: Summary of Cytochemical Reactions Useful in Diagnosing Acute Leukemia.
SPECIAL STAIN SITE OF ACTION CELLS STAINED REACTION
Myeloperoxidase Mainly primary Late myeloblasts,
granules (Peroxidase); granulocytes;
Auer rods monocytes less
intensely

Sudan Black B Phospholipids: sterols, Late myeloblasts,

neutral fats granulocytes;
monocytes less
intensely

Specific esterase Cytoplasm Neutrophilic

(Naphthol AS-D granulocytes; mast cells
chloroacetate)

Nonspecific esterase Cytoplasm Monocytes; focal

Alpha-naphthyl acetate staining in T cells;
(ANAE) ANAE also + in
megakaryocytes

Nonspecific esterase Cytoplasm Monocytes

Alpha-naphthyl butyrate
(ANBE)

Periodic acid—Schiff Glycogen and related Lymphocytes,

substances (e.g. granulocytes,
mucoproteins, megakaryocytes
glycoproteins,
glycolipids, and
polysaccharides).
 COMMENTS:
 Myeloperoxidase;
 It is useful in differentiating AML from ALL, because peroxidase is not present in
lymphocytes or their precursors.
 It is more specific for granulocytic differentiation than the Sudan black B stain.
 Sudan Black B;
 It generally parallels the myeloperoxidase staining pattern that is useful to
differentiate AML from ALL, however, advantageously it can be used for
specimens that are not fresh, because its reactivity does not diminish with time.
 It is less specific than MPO.
 Specific esterase (Naphthol AS-D chloroacetate) - NASDA;
 It is commonly referred to as ―Chloroacetate esterase (CAE)‖
 It roughly parallels the peroxidase and SBB stains, although it is not as sensitive.
 Its most important use is in demonstrating myeloid differentiation in paraffin-
embedded tissue sections.
 Nonspecific esterase (NSE) Alpha-naphthyl acetate (ANAE);
 It is most sensitive for monocyte differentiation.
 It is positive in monocytes and their precursors as well as in macrophages.
 It is also positive in megakaryocytes and platelets.
 Nonspecific esterase (NSE) Alpha-naphthyl butyrate (ANBE);
 It is most specific for monocyte differentiation.
 It is positive in monocytes and their precursors as well as in macrophages
 Periodic acid—Schiff;
 It is not very useful for characterizing acute leukemia and it is no longer
commonly used for classification.
 However, it is most helpful in supporting diagnosis of erythroleukemia vs.
pernicious anemia, showing a strong PAS positive reaction.

Table X: Acute Leukemia Cytochemical Reaction Chart

CONDITION MPO SBB NASDA ANAE ANBE
ALL - - - -/+ (focal) -/+ (focal)
AML + + + - -
AMML + + + + (diffuse) + (diffuse)
AMoL - -/+ - + (diffuse) + (diffuse)
Megakaryocytic
- - -- + (localized) -
Leukemia

KEY TAKEAWAYS:
 Almost in the same ground with bone marrow aspirate examination, cytochemical
stains as demonstrative as it is, it still does not present definitive diagnosis of acute
leukemia, they are most of the time used as clinical support for the diagnosis of a
specific leukemia type.

D. IMMUNOLOGIC ANALYSIS
Indispensably, immunologic methods are frequently used in the diagnosis and
classification of acute leukemia, which makes use of antibodies to detect markers
associated with a certain cell lineage and maturation stage. In clinical practice, there are
essentially two techniques used to detect antigens on cell surface, namely:
 FLOW CYTOMETRY
 It requires a cell suspension and is best done on peripheral blood or bone marrow
aspirate specimen.
 Main Principle: It is based on Light Scatter (Forward Scatter indicates relative size
of the cell, while Side Scatter indicates the complexity/granularity of the cell) and
emission of Fluorescence, using fluorescent-labeled antibodies/probes.

IMMUNOHISTOCHEMISTRY
 It is typically done on paraffin sections of the core biopsy, clot section made from the
aspirate, or on paraffin sections of other biopsy material.
 Main Principle: It uses Labeled Antibodies (e.g. Enzyme or Fluorescent Labels) to
detect tumor/cell antigens in formalin-fixed or frozen tissue sections of biopsy
material.

RE-PRESENTATION:Table III: FAB Classification of AML based on Cytometric Phenotypes

TYPE IMMUNOLOGIC SURFACE MARKERS
M0 (+) to at least one: CD13, CD33, or CD117; (-) to lymphoid antigens.
M1 (+) to at least two: CD13, CD33, or CD117; (-) to lymphoid antigens.
M2 (+)CD13, CD33, & CD117; may also become (+) to: CD19, CD34, & CD56.
M3 (+) CD13 & CD33; (-) CD34 & HLA-DR.
M4 (+) CD11b, CD11c, CD14, CD64, & CD4.
M5 (+) CD64, CD56, CD4, CD13, CD33, & HLA-DR.
M6 (+) CD13, CD33, CD34, CD117, HLA-DR, & Glycophorin A.
M7 (+)CD13, CD33, CD41, CD42b, CD61, CD36, & Factor VIII.

RE-PRESENTATION: Table VII: WHO Classification of ALL based on Immunophenotypic

Characteristics.
SUBTYPE PHENOTYPE
Precursor B – Cell HLA-DR, CD34, TdT, CD19, CD20(+/-), CD10
Cytoplasmic CD22
Pre B – ALL HLA-DR, TdT(+/-), CD19, CD20(+/-), CD10, cIgM
Cytoplasmic CD22
B – ALL (Intermediate/Common) HLA-DR, CD19, CD20, CD22, CD10 (4), slg
Precursor T – ALL HLA-DR(+/-), CD1, CD2, cCD3, dual CD4/CD8, CD5,
CD7(+/-), CD10(+/-), CD34(+/-), TdT

KEY TAKEAWAYS:
 Surface marker analysis has begun to replace conventional cytochemical methods for
lineage determination in acute leukemia. It offers a more precise and sensitive way of
classifying acute leukemias, which could highly influence the physician’s diagnosis.

E. ADVANCE LABORATORY STUDIES

Various advance laboratory studies for the diagnosis of acute leukemia has also been
elucidated in recent years, involving extensive studies of genes and other cytogenetic
abnormalities. These advances include the following:
 FLUORESCENCE IN SITU HYBRIDIZATION (FISH);
 Purpose: It is a laboratory technique used to detect and locate a specific DNA
sequence on a chromosome.
 Principle: In this technique, the full set of chromosomes from an individual is affixed
to a glass slide and then exposed to a probe, which is a small piece of purified DNA
tagged with a fluorescent dye that binds to its matching sequence in the
chromosome. If it does find a match fluorescence can be seen under a fluorescence
microscope.

POLYMERASE CHAIN REACTION (PCR);

 Purpose: It is a laboratory technique used to make multiple copies of a segment of
DNA using primers for rapid and precise detection of many specific cytogenetic
abnormalities at the molecular level.
 Principle: It is based on the use of DNA polymerase which is an in vitro replication of
specific DNA sequences to generate tens of billions of copies of a particular DNA
fragment (the sequence of interest, DNA of interest, or target DNA) from a DNA
extract (DNA template).

KEY TAKEAWAYS:
 These advance laboratory studies paved the way for specific chromosomal
identification of leukemia types, which became basis of leukemia associated with
known genetic aberrations. These methods by far offers the most accurate way of
diagnosing common cases of acute leukemia, however, the complex pathology of
leukemia itself still limits such studies, as causes of the other types of leukemia still
remains unknown.

VIII. TREATMENT OF ACUTE LEUKEMIA:

Therapy for acute leukemia (AL) is among the most complex of any anticancer programs
with the following goals:
1. Eradicate the leukemic clone.
2. Reconstitute normal hematopoiesis; and
3. Prevent any emergence of resistant leukemic subclones.

GENERALIZED TREATMENT OPTIONS:

 Chemotherapy;
THREE (3) PHASES OF ANTI-LEUKEMIC THERAPY:
1. Induction;
 It is designed to attain complete remission as quickly as possible; its success is
the best predictor of long-term disease free survival.
 It is usually very intense and lasts about 1 month.
2. Consolidation;
 It is designed to reduce the number of disease cells left in the body.
 It is also intense, lasting approximately 4 to 8 months.
3. Maintenance;
 It usually starts after a patient stays in remission after induction and consolidation
therapy.
 It is designed to destroy any leukemic cells that may remain in order to maintain
a disease-free state.
  It is less intense than the other two phases and may last 2 to 3 years.
 Prophylactic CNS Chemotherapy & CNS Radiation
 Tyrosine Kinase Inhibitors;
 It block the action of tyrosine kinase to send growth signals to cells and eventually
stopping the proliferation and division of cancer cells.
 Immunotherapy
 Targeted Therapy;
 Epigenetic Therapy
DNA methytransferase inhibitors; this inhibits DNA methylation.
Histone deacetylase (HDAC) inhibitors; this inhibits histone deacetylation.
 FLT3 Inhibitors
 Stem Cell Transplantation
 Radiation Therapy;
 This treatment uses beams of intense energy to kill cancer cells. Radiation therapy
most often uses X-rays.
 Intensive and Supportive Care
 Similarly in both conditions AML or ALL the same supportive care may be given such
as the following:
Transfusions
Antimicrobials (usually due to diminished immune capacity, in which patient is
prone to infection at any case)
Hydration & Urine Alkalization
Psychological Support

REFERENCES:

Harmening, D. M. (2009). Clinical Hematology and Fundamentals of Hemostasis (5th ed.).

Keohane, E. M., Otto, C. N., & Walenga, J. M. (2020). Rodak's Hematology Clinical Principles
and Aplications (6th ed.).

McPherson, R. A., & Pincus, M. R. (2017). Clinical Diagnosis and Management by Laboratory
Methods (23rd ed.).

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