Pamantasan ng Lungsod ng Maynila

(University of the City of Manila)

College of Medicine
Department of Pathology

CASE 2
Chronic Myeloid Leukemia (CML)

GROUP 4
3B 2020

BAMBA, Vivian Claire Approach to Diagnosis, Case slides

BATAC, Misha Approach to Diagnosis, Case slides
BAUTISTA, Lizette Differential Diagnosis, Case slides, Diagnostics, Working
Diagnosis
BRIONES, Abigail Loren Differential Diagnosis, Pathophysiology
CONCEPCION, Joshua Normal Histology, Salient Features, Tx & Management
CORDOVA, Robelle Differential Diagnosis,Working Diagnosis, Powerpoint
Presentation
LARRACAS, Jesamar Differential Diagnosis, Clinical Correlation, Working
Diagnosis
MACAM, Andrea Gaile Approach to Diagnosis, Case slides
MILLA, Franclene John Differential Diagnosis, Case slides, Microscopic Features
ONTINGCO, Andrea Joy Differential Diagnosis, Epidemiology
PALOMA, April Jeraldine Differential Diagnosis, Prognosis and Staging
SANTOS, Lyndsay Angeli Approach to Diagnosis, Case presentation
 CASE

A 69-year-old female was admitted to a hospital because of CBC result after an annual
executive check-up. She was otherwise asymptomatic. She denied having fevers, chills, night
sweats, weight loss and other constitutional symptoms. Physical examination showed no
hepatosplenomegaly and no lymphadenopathy. The patient was referred to a hematologist by
the attending physician and a bone marrow aspirate was subsequently requested.

TEST RESULT UNIT REFERENCE

Complete Blood Count

Hematocrit L 36.1 % [38.0-47.0]

Hemoglobin L 11.6 g/dL [12.0-16.0]

RBC Count L 3.7 10​6​/uL [4.2-5.4]

WBC Count H 62.5 10​3​/L [4.5-11.0]

Platelet Count H 415 10​3​/uL [150-400]

Red Cell Indices

MCV 97.3 fL [80.0-100.0]

MCH 31.3 pg [27.0-32.0]

MCHC 32.1 g/dL [31.0-35.0]

RDW-CV H 21.8 % [11.6-13.7]

MPV 9.4 fL [7.8-11.0]

Differential Count

Neutrophils * % [50.0-70.0]

Eosinophils * % [1.0-5.0]

Lymphocytes * % [20.0-40.0]

Monocytes * % [2.0-8.0]

Basophils * % [0.0-1.0]

Flagging from the hematology analyzer referred to the pathologist by the medical technologist.
Flag(s)
● WBC Abn Scg (WBC Abnormal Scattergram)
● Leuko + (Increased WBC count)
● Bl/Abn Ly? (300) (Possible blast or abnormal lymphocyte)
● Left shift? (300)
● Thrombo+ (Increased platelet)

1
 SALIENT FEATURES

NORMAL HISTOLOGY
NORMAL PERIPHERAL BLOOD SMEAR

Source: E​ roschenko, V. P., & Di Fiore, M. S. (2013). DiFiore's atlas of histology with functional
correlations. Lippincott Williams & Wilkins.
Erythrocytes ​or red blood cells are the most abundant elements in a blood smear.
Erythrocytes are anucleated and are uniform in size, measuring approximately 7.5 μm in
diameter. Meanwhile, ​platelets ​(blue arrow) are the smallest formed elements in a human
blood smear. They are nonnucleated cytoplasmic remnants of large-cell megakaryocytes that
appear as basophilic and exhibits a light blue peripheral zone and a dense central zone
containing purple granules. ​Neutrophils ​(black circle) are one of the polymorphonuclear
granulocytes that contain cytoplasmic granules and lobulated nuclei. It constitutes of
approximately 60-70% of WBC. The neutrophil cytoplasm contains fine violet or pink granules.
Its nucleus consists of several lobes connected by narrow chromatin strands. Immature
neutrophils contain fewer nuclear lobes. Agranular leukocytes (​lymphocytes​) (green circle)
have few or no cytoplasmic granules and exhibit round to horseshoe-shaped nuclei. The vary
in size from cells smaller than erythrocytes to cells almost twice as large. Smaller
lymphocytes have densely stained nucleus that occupies most of the cytoplasm, which
appears as a thin basophilic rim around the nucleus. While larger lymphocytes have
basophilic cytoplasm that is more abundant, and the larger and paler nucleus may contain
one or two nucleoli. Lymphocytes constitute approximately 20 to 30% of the blood leukocytes.

2

Source: E​ roschenko, V. P., & Di Fiore, M. S.
(2013). DiFiore's atlas of histology with functional
correlations. Lippincott Williams & Wilkins.
Source: E​ roschenko, V. P., & Di Fiore, M. S.
(2013). DiFiore's atlas of histology with functional
correlations. Lippincott Williams & Wilkins.
Source: E​ roschenko, V. P., & Di Fiore, M. S.
(2013). DiFiore's atlas of histology with functional
correlations. Lippincott Williams & Wilkins.
Eosinophils are identified in a blood smear by
their cytoplasm, which is filled with distinct,
large, eosinophilic granules. Its nucleus is
typically is bilobed, but a small third lobe may
be present. Eosinophils constitute
approximately 2 to 4% of the blood
leukocytes.
Basophils have also granules but they are not
as numerous as in eosinophils. They are
more variable in size, less densely packed,
and stain dark blue or brown. Although the
nucleus is not lobulated and stains pale
basophilic, it is usually obscured by the
density and number of granules. The
basophils constitute less than 1% of the blood
leukocytes and are therefore the most
difficult to find and identify in a blood smear.
Monocytes are the largest agranular
leukocytes in a blood smear. Its nucleus
varies from round or oval to indented or
horseshoe-shaped and stains lighter than the
lymphocyte nucleus. The nuclear chromatin is
finely dispersed and the abundant cytoplasm
is lightly basophilic with few fine granules.
Monocytes constitute approximately 3 to 8%
of the blood leukocytes.
3
NORMAL BONE MARROW ASPIRATE

Source: Anderson, S. C., & Poulsen, K. (2013). Atlas of hematology. Lippincott Williams & Wilkins.

Bone marrow are your major organ for hematopoiesis at birth and serves also as a lymphoid
organ. Pluripotent stem cells develop in this organ into myeloid blasts or lymphoblasts. In a
bone marrow aspirate of a normal elderly human, it will show that there is 20–50% cellularity,
instead of a 30-80% cellularity in a younger adult therefor, ​hematopoietic tissue is replaced by
fat.

Note a large darker clump of cells (lower

Source: Pathpedia (n.d.). Histology images of
bone marrow. Retrieved from
https://www.pathpedia.com/education/eatlas/histol
ogy/bone_marrow/Images.aspx?5
arrow) enmeshed in stromal cells and other
connective tissue cells that is called a
“spicule.” A spicule is bordered by marrow
cells that are more thinly spread out
(arrowhead) where morphologic examination
of the marrow cells is performed. The spicule
is too thick to be adequate for morphologic
examination. Note also fat cells (thin long
arrow) on the aspirate smear.

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 Marrow aspirate smear shows normal
hematopoietic elements with a myeloid to
erythroid ratio of about 3:1. The ME ratio
indicates the proportion of myeloid elements
including all their precursors in relation to the
number of nucleated erythroid precursors. In
normal adults there are about 3-4 myeloid
elements to every single nucleated erythroid
element.
Source: Pathpedia (n.d.). Histology images of
bone marrow. Retrieved from
https://www.pathpedia.com/education/eatlas/histol
ogy/bone_marrow/Images.aspx?5

In a normal bone marrow smear at high

magnification, we can note the presence of
hematopoietic cells such as megakaryocytes
(black arrow), erythroid precursors (red
arrow), and myeloid precursors (green arrow).
Adipose cells can also be seen (yellow
arrow). Sometimes, osteoclasts, endothelial
cells or capillaries can be also seen in a bone
marrow aspirate.
Source: Luca, D.C. 2012. Bone marrow -
nonneoplastic - Normal - General . Retrieved from
http://www.pathologyoutlines.com/topic/bonemarr
ownormalbonemarrow.html

5
 Case Slide Slide Description
Peripheral Blood Smear 1

The red blood cells are mostly hyperchromic

with anisocytosis. Howell-Jolly bodies are
also present and platelets are also seen.

Myeloblast- ​nucleus is round to oval, with 2-4 visible

nuclei; the cytoplasm is slightly basophilic and the N:C
ratio is around 1:1 to 1:0.5
Metamyelocyte- with an indented or kidney-shaped
nucleus; cytoplasm is clear pink with few granules
Band- ​nucleus is band-shaped or contains rudimentary
lobes that are connected by thick band; cytoplasm is
abundant, pink, and may contain granules
Neutrophil- ​nucleus is segmented (3-4) connected by
a thin thread of chromatin; cytoplasm is abundant, pink

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Peripheral Blood Smear 2

​ Myelocyte Metamyelocyte Band

​ Neutrophil Hypersegmented Neutrophil

The red blood cells are mostly hyperchromic,

with anisocytosis. Platelets are also seen.

Myelocyte- ​nucleus is round or oval, nuclear chromatin

may show coarse clumping; blue-pink cytoplasm with
red-violet granules
Metamyelocyte- with an indented or kidney-shaped
nucleus; cytoplasm is clear pink with few granules
Band- ​nucleus is band-shaped or contains rudimentary
lobes that are connected by thick band; cytoplasm is
abundant, pink, and may contain granules
Neutrophil- ​nucleus is segmented (3-4) connected by
a thin thread of chromatin; cytoplasm is abundant, pink
Hypersegmented Neutrophil - ​Neutrophil with six or
more segments

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Peripheral Blood Smear 3

Myeloblast
Promyelocyte Myelocyte

Band Neutrophil
Metamyelocyte

The red blood cells are mostly hyperchromic,

with anisocytosis. Platelets are also seen.

Myeloblast- ​nucleus is round to oval, with 2-4 visible

nuclei; the cytoplasm is slightly basophilic and the N:C
ratio is around 1:1 to 1:0.5
Promyelocyte- ​nucleus is round to oval and is often
eccentric, cytoplasm is evenly basophilic and with
granules
Myelocyte- ​nucleus is round or oval, nuclear chromatin
may show coarse clumping; blue-pink cytoplasm with
red-violet granules
Metamyelocyte- with an indented or kidney-shaped
nucleus; cytoplasm is clear pink with few granules
Band- ​nucleus is band-shaped or contains rudimentary
lobes that are connected by thick band; cytoplasm is
abundant, pink, and may contain granules
Neutrophil- ​nucleus is segmented (3-4) connected by
a thin thread of chromatin; cytoplasm is abundant, pink

Bone Marrow Aspirate 1

Bone marrow aspirate is hypercellular with

absence of monotony. There is presence of
large cells around scanty fat cells.

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Bone Marrow Aspirate 2

The bone marrow aspirate is hypercellular

with marked myeloid predominance. There is
an increased megakaryocytes in clusters with
some micromegakaryocytes (“dwarf” forms)
and large, multilobulated forms. Eosinophils
and basophils are increased in number.
Scattered sea blue histiocytes and adipose
cells are also seen. No fibrosis can be
appreciated in the case slide due to the
resolution of the image. M:E ratio cannot be
determined with the slide’s given
magnification.

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 PERTINENT POSITIVES PERTINENT NEGATIVES
Clinical Features
● 69 y/o ● Asymptomatic
● Female ● No fever
● No chills
● No night sweats
● No weight loss
● No hepatosplenomegaly
● No lymphadenopathy
Laboratory findings
● Normocytic normochromic anemia (Low
hematocrit, hemoglobin, RBC count)
● Leukocytosis (High WBC)
● Thrombocytosis (High Platelet Count)
● Anisocytosis (Inc RDW)
● Abnormal WBC Scattergram
● Left shift

Histologic Features (Peripheral Blood Smear)

● Hyperchromic erythrocytes with
anisocytosis
● Presence of Howell-Jolly bodies in
erythrocytes
● Presence of Platelets
● Presence of Myeloblasts
● Presence of Promyelocyte
● Presence of Myelocytes
● Presence of Metamyelocytes
● Presence of Band Neutrophils
● Presence of Neutrophils
● Presence of hypersegmented
Neutrophils
Histologic Features (Bone Marrow Aspirate)
● Hypercellular bone marrow ● Absence of cellular monotony
● High myeloid to erythroid ratio ● Absence of bone marrow spicules
● Presence of large cells
● Presence of adipose cells
● Presence of myeloblast
● Presence of promegakaryocyte
● Presence of megakaryocytes
● Presence of Dwarf megakaryocytes
● Presence of sea blue histiocyte

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APPROACH TO DIAGNOSIS

Fig 1. ​Approach to Diagnosis

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 The patient is a 69-year old female who underwent her annual physical check-up. There
was no noted fever, chills, night sweats or weight loss and upon physical examination, there
was no hepatosplenomegaly and lymphadenopathy. However, on her complete blood count, it
was discovered that she has an elevated white blood cell count (62.5x10​3​/L). This may be
associated with infections and conditions such as blood dyscrasias and malignancies. However,
since the differential count was not given, we cannot assess the proportion of each white blood
cell type in relation to one another. Consequently, we cannot determine the etiologic agent that
may have caused the leukocytosis for this patient. The patient’s RBC (3.7x10​6​/uL), hemoglobin
(11.6 g/dL) and hematocrit (36.1%) were also found to be decreased. The hemoglobin’s primary
function within the erythrocyte is to carry oxygen to and carbon dioxide from the tissues.
Hematocrit, on the other hand, is the volume of packed RBCs that occupy a given volume of
whole blood. Hemoglobin is a more reliable indicator of anemia compared to hematocrit
because hematocrit can be influenced by the size of the RBCs. Overall, these three parameters
indicate that this patient is anemic. The platelet count (415x10​3​/uL) was elevated, indicating
thrombocytosis. Platelets play an important role in the formation of blood clots. This can either
be a secondary or reactive thrombocytosis which may be due to malignancies, infections or iron
deficiency, or essential thrombocytosis which can be traced back into conditions involving the
bone marrow (Rodak, Fritsma, Keohane, 2012).

The results of the red cell indices revealed the patient to have normocytic normochromic
anemia and an elevated red cell distribution width (RDW). An elevated RDW signifies variation
in RBC size or anisocytosis which may be complemented by the results of the peripheral blood
smear (Rodak, 2012). Anisocytosis with normal RBC size may be present in anemias (e.g. Iron
deficiency or megaloblastic), chronic liver disease and myelodysplastic syndromes (Kasper ​et
al.​, 2015).

The hematology analyzer also generated flags notably for left shift and an abnormal
WBC scattergram. ​Flagging indicates that the technologist should review the peripheral blood
smear for abnormal cells or mark it for further investigation (Rodak, 2012). A shift to the left
indicates the presence of immature forms of leukocytes such as neutrophil band cells,
metamyelocytes and myelocytes in the peripheral blood smear which generally reflects early or
premature release of myeloid cells from the bone marrow (Kasper et al., 2015). This is observed
in most bacterial infections and in some neoplasms. If left shift is observed, the blood smear
must be observed manually for the presence of band cells in increased numbers, toxic
granulation or vacuolization of neutrophils.

A typical WBC scattergram shows the counts of the subpopulations of leukocytes

(lymphocytes, monocytes, neutrophils, eosinophils and basophils) on a blood specimen. An
abnormal WBC scattergram indicates uncharacteristic cell distribution of any of the leukocytes.
Various changes like graying of WBC clusters, merging of clusters, multiple clustering and
abnormal blue coded events may also be observed in a scattergram (Mubeen ​et al.​ , 2014).

Indications of PBS
According to Rodak (2012), a manual microscopic review of a peripheral blood smear is
essential whenever instrument analysis indicates specimen abnormalities. Similar to the case,
the presence of an abnormal WBC scattergram, increased, WBC count, possible blast or
abnormal lymphocyte, left shift, and increased platelet count from the hematology analyzer
warrants the preparation and examination of the patient's peripheral blood smear.

12
Interpretation of PBS
Examination of peripheral blood smears must be done in low-power magnification to
assess overall film quality, color, and distribution of cells can be assessed, such as rouleaux
formation or clumps. High-power magnification enables the selection of a correct area for
differential count and morphology evaluation, and performance of the WBC estimate. The
microscopic examination of the peripheral blood smear under the 100x oil immersion
magnification is mainly for the WBC differential count, with the RBC, WBC, and platelet
morphology evaluation and platelet estimate. (Rodak, 2012)

In the examination of the patient’s peripheral blood smear under oil immersion
magnification, the RBCs appear hyperchromic with anisocytosis, with the presence of
Howell-Jolly bodies. Platelets appear normal in size and show no morphologic abnormalities.
The WBC differential count showed relative and absolute neutrophilia. Both immature cells,
such as myeloblasts, promyelocytes, myelocytes, metamyelocytes, and band neutrophils; and
mature cells, such as segmented neutrophils, can be seen in the smears. In addition,
hypersegmentation may also be appreciated in the smear as an abnormal morphology of the
neutrophil.

Table 1.​ Relative WBC differential count on the patient’s peripheral blood smear
Frequency Relative count

Segmented neutrophils 19 44%

Band neutrophils 7 16%

Metamyelocyte 10 23%

Myelocyte 4 9%

Promyelocyte 1 2%

Myeloblast 3 7%

The WHO classification classifies neoplasm primarily according to lineage - myeloid,

lymphoid, or histiocytic/dendritic. (Swerdlow ​et al., 2017) With the presentation in the peripheral
blood smear, we can infer that the cells are from a myeloid lineage. In addition, according to
Swerdlow (2017), precursor myeloid neoplasms such as Acute Myeloid Leukemia (AML) is
discussed separately from more-mature myeloid neoplasms, which include myeloproliferative
neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and
myelodysplastic Syndromes. The diagnosis of AML has a requisite blast percentage of greater
than or equal to 20% myeloblasts, monoblasts/promonocytes, and/or megaloblasts. In the
peripheral smear of our patient, only 7% in the relative WBC differential count were made up of
blasts. The mature myeloid neoplasms are classified according to their biological features, such
as myeloproliferative neoplasms with effective haematopoiesis compared to myelodysplastic
neoplasms with ineffective haematopoiesis, as well as by their genetic features. (Swerdlow ​et
al.,​ 2017)

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Indications of Bone Marrow Aspirate
Obtaining a bone marrow aspirate is an invasive procedure. It is usually obtained from
the posterior superior iliac crest, anterior superior iliac crest, sternum, anterior medial surface of
the tibia, or spinous process of the vertebrae. The aspirate is examined to identify the types and
proportions of hematologic cells and to look for morphologic variance. Bone marrow
examinations may be used to diagnose and stage hematologic and non-hematologic neoplasia,
to determine the cause of cytopenia, and to confirm or exclude metabolic and infectious
conditions suspected in correlation to clinical symptoms and peripheral blood findings.
Indications for requesting a bone marrow aspirate are summarized in Table 2 (Rodak, Fritsma,
Keohane, 2012).

Table 2. ​Indications for requesting a bone marrow aspirate (Rodak, Fritsma, Keohane, 2012).

In this case, the bone marrow aspirate was done for the investigation of abnormal
peripheral blood smear morphology suggestive of bone marrow pathology. In reading a bone
marrow aspirate, at low power magnification (100x), the following are assessed: peripheral
blood dilution, bony spicules and areas of clear cell morphology, fat-to-marrow ratio, estimate of
cellularity, tumor cells in clusters, and estimate of megakaryocytes. In the higher power
magnification (500x) myelocytic and erythrocytic maturation, and cell maturation stages are
observed. Moreover, a differential count on 300 to 1000 cells is also done, and the
myeloid-to-erythroid ratio is computed (Rodak, Fritsma, Keohane, 2012).

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Low Power Examination (100x)

Initially, bony spicules or aggregations of bone and hematopoietic marrow, which stain
dark blue are located. They may be sparse or absent, so hematopoietic cells may be searched
instead. Within these areas, intact and nearly contiguous nucleated cells are selected for
examination, whereas areas showing distorted morphology or dilution with sinusoidal blood are
avoided. Near the spicules, cellularity is estimated by observing the proportion of hematopoietic
cells to adipocytes. Normally, the cellularity is 50% for patients aged 30 to 70 years. Based from
this, the area is classified as hypocellular, normocellular, or hypercellular. Moreover, tumor cell
nuclei often stain darkly (hyperchromatic), and vacuoles are seen in the cytoplasm. Tumor cell
clusters are often found near the edges of the smear. At this magnification, erythrocytic
maturation stages stain more intensely, and their margins are more sharply defined.
Megakaryocytes are the largest cells, with multilobed nuclei, usually 2 to 10 per low-power field
(Rodak, Fritsma, Keohane, 2012).

High Power Examination (500x)

Cells are identified based on morphology and maturation. Megakaryocytes, cells of

myelocytic and erythrocytic series are seen, along with eosinophils, basophils, lymphocytes,
plasma cells, monocytes, and histiocytes. Expected percentages are summarized in Table 3.

Table 3. ​Anticipated distribution of cells in bone marrow aspirate (Rodak, Fritsma, Keohane, 2012).

In the case slides, the bone marrow aspirate is hypercellular with absence of monotony,
and with myeloid predominance. Megakaryocytes are seen around adipose cells. Eosinophils
and basophils are increased in number. Scattered sea blue histiocytes are also seen. Accurate
percentage of the distribution of specific cells cannot be determined due to the resolution of the
images, and the M:E ratio cannot be computed. There is high M:E ratio only upon estimation.

Correlating the data from the history, physical examination, peripheral blood smear, and
bone marrow aspirate, possible diagnoses include: Chronic Myeloid Leukemia (CML), Chronic
Neutrophilic Leukemia (CNL), and Atypical Chronic Myeloid Leukemia (aCML). Although a
leukemoid reaction appears almost the same as CML in the PBS and bone marrow aspirate,
this diagnosis is ruled out because of the absence of an underlying condition such as infection,
stress, allergy, intoxication, or non-hematologic malignancy. Moreover, clinical history presents
that the patient is asymptomatic.

For the final diagnosis, there should be integration of peripheral blood, bone marrow
aspirate and trephine biopsy findings, together with the results of supplementary tests such as

15
 immunophenotyping, cytogenetic analysis and molecular genetic studies as appropriate, in the
context of clinical and other diagnostic findings (Lee ​et al​., 2008).

DIFFERENTIAL DIAGNOSIS

ATYPICAL CHRONIC MYELOID LEUKEMIA

CASE SPECIMEN REFERENCE SPECIMEN

PBS reveals marked granulocytosis with frequent

Green - metamyelocyte immature forms. Dysgranulopoiesis is demonstrated
Blue - band by hyposegmented nuclei, abnormal chromatin
Red - neutrophil pattern, and abnormal cytoplasmic granules.
Yellow - RBC with Howell-Jolly bodies

Reference: Faramarz, N., Nagesh, R., Sophin, S., & Wayne, W.

Peripheral blood smear shows leukocytosis and (2013). Myelodysplastic syndrome/neoplasms. Atlas of
left-shift myeloid and neutrophil maturation. RBC’s hematopathology: Morphology, Immunophenotype,
are hyperchromic with anisocytosis and dimorphic Cytogenetics, and Molecular approaches.​
population. Platelets have normal morphology.

16
 Atypical chronic myeloid leukemia, BCR-ABL1
Green - metamyelocyte negative with peculiar abnormal neutrophils.
Blue - band
Red - neutrophil
Peripheral blood smear shows leukocytosis, with
Yellow -promyelocyte neutrophils showing abnormally twisted and
Black arrow - myeloblast branched nuclei that are hyperchromatic.
White arrow - myelocyte
Reference: Wang, S. A., & Hasserjian, R. P. (Eds.). (2018).
Peripheral blood smear shows leukocytosis and Diagnosis of Blood and Bone Marrow Disorders. Springer​.
left-shift myeloid and neutrophil maturation.
Myeloblasts have prominent nucleoli.

17
 A: PBS shows presence of leukocytosis
characterized by dysplastic neutrophils and
left-shifted granulocytes with very few platelets.
B: Marked dysplasia is present with hypolobation
and hypercondensed nuclear chromatin ccompanied
by decreased cytoplasmic granulation

Reference: ​Pereira, I., George, T. I., & Arber, D. A. (2011). Atlas

of peripheral blood: the primary diagnostic tool. Lippincott
Williams & Wilkins.

Bone marrow aspirate is hypercellular with

absence of monotony. There is presence of large
cells around fat cells. Bone marrow biopsy shows marked hypercellularity
and myeloid preponderance.

Reference:Faramarz, N., Nagesh, R., Sophin, S., & Wayne, W.

(2013). Myelodysplastic syndrome/neoplasms. Atlas of
hematopathology: Morphology, Immunophenotype,
Cytogenetics, and Molecular approaches.​

18
 Bone marrow aspirate is hypercellular with
Bone marrow aspirate smear shows myeloid
marked myeloid predominance. There is an
hyperplasia with left-shift maturation and
increased megakaryocytes in clusters with some
dysgranulopoiesis, with cytoplasmic hypogranulation,
micromegakaryocytes (“dwarf” forms) and large,
nuclear hypolobation.
multilobulated forms. Eosinophils and basophils
are increased in number. Scattered sea blue Reference: Wang, S. A., & Hasserjian, R. P. (Eds.). (2018).
histiocytes and adipose cells are also seen. Diagnosis of Blood and Bone Marrow Disorders. Springer​.

Brief Description
Atypical chronic myeloid leukemia, BCR-ABL1-negative (aCML) is arare leukemic disorder characterized by the
simultaneous myelodysplastic as well as myeloproliferative features present at the time of initial diagnosis. The
hallmark of the disease is dysplastic neutrophilic leukocytosis with left-shifted neutrophilic precursors (>10%) in the
blood. However, multilineage dysplasia is common, and reflects the stem cell origin of this entity. The neoplastic
cells do not have BCR-ABL1 fusion; rearrangement of PDGFRA, PDGFRB or FGFR1, or PCM1-JAK2. (Wang &
Hasserjian, 2018)

Rule in Rule out

Epidemiology ● Typically occurs in elderly (median ● Female (Male predominance 2:1)

age is 72 years old)

Clinical ● May be asymptomatic ● Thrombocytosis (usually presents

● Leukocytosis with thrombocytopenia)
● Anemia ● No hepatosplenomegaly

Histologic Peripheral Blood Smear Peripheral Blood Smear

● Leukocytosis ● Absence of dysgranulopoiesis
● Increased neutrophils and (cytoplasmic hypogranulation,
neutrophil precursors nuclear hypolobation,
● No to minimal monocytosis pseudo-Pelger-Huet neutrophils,

19
 hypersegmented neutrophils with
Bone marrow aspirate abnormal branching nuclei)
● Hypercellular bone marrow ● Presence of basophilia
● Predominance of myeloid
precursors/Elevated M:E ratio Bone marrow aspirate
● Dysmegakaryopoiesis ● Granulocytic dysplasia (same
megakaryocytes characteristic as PBS)
● Blasts

DECISION: ​RULED OUT

Perform Cytogenetic Testing including fluorescence in situ hybridization (FISH) or PCR
for (-) BCR/ABL to confirm the diagnosis of aCML.

CHRONIC NEUTROPHILIC LEUKEMIA

CASE SPECIMEN REFERENCE SPECIMEN

Peripheral Blood Smear 1

Green - metamyelocyte The peripheral blood smear shows increased neutrophils,

Blue - band without a significant number of circulating immature
Red - neutrophil granulocytic forms.
Yellow - RBC with Howell-Jolly bodies

Peripheral blood smear shows leukocytosis and Reference: Wang, S. A., & Hasserjian, R. P. (2018). Diagnosis of
left-shift myeloid and neutrophil maturation. RBC’s Blood and Bone Marrow Disorders. Basingstoke, England:
Springer.
are hyperchromic with anisocytosis and dimorphic
population. Platelets have normal morphology.

20
Peripheral Blood Smear 3

Green - metamyelocyte The neutrophils lack morphologic dysplasia, with normal

Blue - band
nuclear lobation and normal granulation.Toxic granulation
Red - neutrophil
Yellow - promyelocyte is commonly observed.
Black arrow - myeloblast
White arrow - myelocyte Reference: Wang, S. A., & Hasserjian, R. P. (2018). Diagnosis of
Blood and Bone Marrow Disorders. Basingstoke, England:
Springer.
Peripheral blood smear shows leukocytosis and
left-shift myeloid and neutrophil maturation.
Myeloblasts have prominent nucleoli.

Bone Marrow Aspirate 2

The bone marrow aspirate shows a myeloid predominance

with complete maturation and without granulocytic
dysplasia.

Reference: Wang, S. A., & Hasserjian, R. P. (2018). Diagnosis of

21
 Blood and Bone Marrow Disorders. Basingstoke, England:
Springer.

Bone marrow aspirate smear demonstrates neutrophil

proliferation from myelocytes to segmented forms with
toxic granulation, but no other significant abnormalities.

Reference: Swerdlow, S.H., Campo, E., Harris, N.L., Jaffe, E.S.,

Pileri, S.A., Stein, H., Thiele, J., Arber, D.A., Hasserjian, R.P., Le
Beau, M.M., Orazi, A., and Siebert, R. (2017). World Health
Organization Classification of Tumours, Revised 4th Edition.
International Agency for Research on Cancer (IARC).

Brief Description
Chronic neutrophilic leukemia (CNL) is a rare and distinct BCR–ABL-negative chronic myeloproliferative neoplasm
characterized by sustained (mature) neutrophilia, affecting mostly elderly patients. For the diagnosis of CNL, the
following diagnostic criteria must be fulfilled, which includes (1) peripheral blood neutrophilic leukocytosis (≥25 ×
10^9/L) with segmented neutrophils and bands (≥80%), myeloblasts rarely observed, and immature granulocytes
(promyelocytes, myelocytes, and metamyelocytes) (<10%), monocyte count (< 1 x 10^9/L), no dysgranulopoiesis ;
(2) hypercellular bone marrow with a normal neutrophil maturation pattern, myeloblasts (<5%), and neutrophil
granulocytes increased in percentage and number; (3) not meeting WHO criteria for BCR-ABL1-positive chronic
myeloid leukaemia, polycythaemia vera, essential thrombocythemia, or primary myelofibrosis; (4) No
rearrangement of PDGFRA, PDGFRB, or FGFR1, and no PCM1-JAK2 fusion; (5) CSF3R T6181 or another
activating CSF3R mutation or persistent neutrophilia (≥3 months), splenomegaly, and no identifiable cause of
reactive neutrophilia including absence of a plasma cell neoplasm or, if a plasma cell neoplasm is present,
demonstration of clonality of myeloid cells by cytogenetic or molecular studies (Swerdlow ​et al.​ , 2017).

The most common clinical features of CNL include splenomegaly, bleeding from mucocutaneous surfaces, pruritus,
and hepatomegaly. This condition has a generally poor prognosis attributed to its heterogeneous clinical course
with a definite risk of death from either blastic transformation, brain hemorrhage, or progressive neutrophilic
leukocytosis. The neutrophilia seen in CNL is usually slowly progressive followed by anemia and thrombocytopenia
(Gorczyca, 2014).

22
 Rule in Rule out

Epidemiology ● Generally affects older adults ● Slight male predominance,

● Median age at CNL diagnosis is 66 male―to―female ratio is 1.6:1
years

Clinical ● Splenomegaly is present in almost all

cases at diagnosis
● Common clinical features include
hepatomegaly, mucocutaneous surface
bleeding and pruritus

Histologic Histologic Features (Peripheral Blood

Smear)
● Presence of Promyelocyte ● Presence of Myeloblasts
● Presence of Myelocytes
● Presence of Metamyelocytes
● Presence of Band Neutrophils
● Presence of Neutrophils
● Presence of hypersegmented
Neutrophils

Histologic Features (Bone Marrow

Aspirate)
● Hypercellular bone marrow
● Presence of megakaryocytes
(Should be less prominent)
● High myeloid to erythroid ratio
● Presence of myeloblast ​(<5%)

DECISION: ​RULED OUT

*Cytogenetics including fluorescence in situ hybridization (FISH) or PCR for BCR/ABL-1 for the exclusion
of underlying BCR–ABL1-driven oncogenesis, such as in chronic myeloid leukemia (CML)

23
 CHRONIC MYELOID LEUKEMIA

CASE SPECIMEN REFERENCE SPECIMEN

Patients usually present with leukocytosis with

left-shifted neutrophilic maturation, eosinophilia, and
Green - metamyelocyte basophilia in the peripheral blood (Sangle, 2018)
Blue - band
Red - neutrophil
Yellow - RBC with Howell-Jolly bodies

Peripheral blood smear shows leukocytosis and

left-shift myeloid and neutrophil maturation. RBC’s
are hyperchromic with anisocytosis and dimorphic
population. Platelets have normal morphology.

24
 Green - metamyelocyte
Blue - band
Red - neutrophil
Yellow -promyelocyte A Peripheral blood smear showing leukocytosis and
Black arrow - myeloblast neutrophilic cells at various stages of maturation;
White arrow - myelocyte basophilia is prominent; no dysplasia is present.

Peripheral blood smear shows leukocytosis and

left-shift myeloid and neutrophil maturation.
Myeloblasts have prominent nucleoli.

Bone marrow aspirate is hypercellular with absence There is a decrease in adipose tissue and marked
of monotony. There is presence of large cells hypercellularity in the bone marrow aspirate. The
around fat cells. myeloid:erythroid ratio is elevated, basophils are
increased, and small megakaryocytes with rounded
nuclei are evident. Small “dwarf” megakaryocytes

25
 form clusters amid numerous maturing myeloid
elements (Wang & Hasserjian, 2018).

Bone marrow aspirate is hypercellular with marked

myeloid predominance. There is an increased The myeloid:erythroid ratio is elevated, basophils are
megakaryocytes in clusters with some increased, and small megakaryocytes with rounded
micromegakaryocytes (“dwarf” forms) and large, nuclei are evident. Small “dwarf” megakaryocytes
multilobulated forms. Eosinophils and basophils are form clusters amid numerous maturing myeloid
scattered. Also, scattered sea blue histiocytes and elements (Wang & Hasserjian, 2018).
adipose cells are also seen. No fibrosis can be
appreciated in the case slide due to the resolution
of the image. M:E ratio cannot be determined with
the slide’s given magnification

Brief Description

Chronic myeloid leukemia (CML) is a stem cell disorder arising from fusion of the ​ABL1 ​gene on
chromosome 9 with the ​BCR g ​ ene on chromosome 22 which is referred to as the Philadelphia
chromosome. CML is a distinct being ​BCR/ABL1 ​positive compared to other myeloproliferative disorders
which are ​BCR/ABL1 ​negative. It has a mean age of diagnosis of 65 years old and has a slightly male
predominance. CML has three phases, respectively the chronic phase where majority of the patients are
diagnosed, and the accelerated and blast phase which may occur without the recognition of the chronic
phase. Initially, chronic phase is characterized by marked leukocytosis, thrombocytosis, and basophilia,
and splenomegaly. Also, the number of myeloblasts in the blood and marrow smears usually does not
exceed 5%. Macrophages resembling Gaucher cells which usually occurs singly and are more prominent
in perivascular locations may also be present is bone marrow smears and sections (Rosai et al., 2018;
WHO, 2017).

26
Clinically, nearly 50% of the patients are asymptomatic, but if symptomatic, fatigue, malaise, weight loss,
night sweats, and anemia, and up to 50% patients with splenomegaly may be seen (WHO,2017).

Rule in Rule out

Epidemiology ● Median age of diagnosis 65

years old with slight male
predominance

Clinical ● Patient is asymptomatic

● No hepatosplenomegaly &
lymphadenopathy.
● Normocytic normochromic
anemia (Low hematocrit,
hemoglobin, RBC count)
● Leukocytosis
● Thrombocytosis

Histologic PBS PBS

● Hyperchromic erythrocytes ● No monocytosis
with anisocytosis ● No eosinophilia
● Presence of Platelets ● No basophilia
● Leukocytosis
● Myeloid & Neutrophil
maturation

Bone Marrow Aspirate

Bone Marrow Aspirate
● No eosinophilia
● hypercellular
● Increase in granulocytes ● No basophilia
● Myeloid & Neutrophil
maturation
● Presence of dwarf
megakaryocytes

DECISION: ​CANNOT BE TOTALLY RULED OUT

Submit for immunophenotyping and molecular studies using FISH analysis/karyotype to confirm the
diagnosis (Philadelphia chromosome positive t(9,22)) and/or qRT-PCR to establish the BCR-ABL fusion​.

27
 WORKING IMPRESSION

Chronic myeloid leukemia (CML) is a stem cell disorder arising from fusion of the
ABL1 g ​ ene on chromosome 9 with the ​BCR ​gene on chromosome 22 which is referred to as the
Philadelphia chromosome. CML is a distinct being ​BCR/ABL1 ​positive compared to other
myeloproliferative disorders which are ​BCR/ABL1 ​negative. Among three phases of CML,
chronic, accelerated, and blast phase, majority of patients are diagnosed in the chronic phase.
Initially, chronic phase is characterized by marked leukocytosis, thrombocytosis, and basophilia,
and splenomegaly. Also, the number of myeloblasts in the blood and marrow smears usually
does not exceed 5%. Macrophages resembling Gaucher cells which usually occurs singly and
are more prominent in perivascular locations may also be present is bone marrow smears and
sections. Usually after 3-4 years, chronic phase is followed by accelerated phase of shorter
duration which is characterized by increasing blasts in the blood and marrow, progressive
basophilia, increasing myelofibrosis and additional cytogenetic changes. Abruptly, blast phase
may occur and even without the intermediate accelerated phase wherein the blasts exceed 20%
in the blood or marrow. Extensive myelofibrosis may develop early or in late disease in CML and
is associated with more aggressive clinical course. This is characterized by an increase in
reticulin fibers correlated with an increase in angiogenesis (Rosai et al., 2018).The diagnosis
requires detection of the Ph chromosome and/or BCR-ABL 1 in the appropriate clinical and
laboratory settings (WHO, 2017).

BCR-ABL1 ​translocation is found in all cells of the myeloid lineage including erythroid
and granulocyte precursors and megakaryocytes. The unregulated proliferation of
BCR-ABL1​-positive stem cells is responsible for massive expansion, primarily in granulocyte
production leading to leukocytosis. Also, ​BRL-ABL1 oncoprotein is constitutively active as
tyrosine kinase that phosphorylates intermediate molecules in different pathways that affects
proliferation, maturation, resistance to apoptosis, and cell adhesion which ultimately result in the
typical leukemic phenotype (Rodgers ​et al.​ , 2013).

In epidemiology, CML is a rare disease with its general incidence of 1.5 in 100,000. It
represents 10% -15% of all leukemias. Its incidence increases with age (<0.1 cases per 100,000
children to >or = 2.5 cases per 100,000 in elderly individuals) with a median age of diagnosis of
65 years old and a slight male predominance (1.5:1). Worldwide, CML has no socio geographic
preponderance, however earlier onset has been reported in areas with lower socioeconomic
status. Ionizing radiation is the only known causative factor, developing usually within 6-8 years
of exposure. Also, there are no known genetic factors that could determine susceptibility to CML
(Rodgers ​et al​., 2013; WHO, 2017).

About 85% of patients with CML present in the chronic phase. Patients are often
asymptomatic early on (20-50%), with insidious onset of nonspecific symptoms (eg, fatigue,
weakness, anorexia, weight loss, night sweats, a sense of abdominal fullness particularly in left
upper quadrant, gouty arthritis, symptoms of leukostasis such as tinnitus, stupor, and urticaria),
which may prompt evaluation (Etten et al., 2017). Initially, pallor, bleeding, easy bruising, and
lymphadenopathy are unusual, but moderate or occasionally extreme splenomegaly is common
(60 to 70% of cases). Anemia (45 and 62 %), white blood cell count above 100,000/microL (52
and 72 %), and platelet count above 600,000 to 700,000/microL (15 and 34 %) are also seen.

28
With disease progression, splenomegaly may increase, and pallor and bleeding occur. Fever,
marked lymphadenopathy, and maculopapular skin involvement are ominous developments.

Although the patient in the case presented none of the aforementioned nonspecific
symptoms, the patient still fits the clinical diagnosis of CML, as she is asymptomatic, has no
lymphadenopathy and hepatosplenomegaly which is typically seen initially in CML before the
disease progresses.

Histologically, in the chronic phase of CML, the blood shows prominent leukocytosis
(with predominance of neutrophils), absolute basophilia, and often eosinophilia. Granulocytic
cells show a leftward shift with a “non symmetrical” distribution (myelocytes > metamyelocytes).
Although myelocytes predominate among immature cells, promyelocytes and blasts are
increased (blasts usually do not exceed 2%). The blast count between 10% and 19% defines
AP and the blast. Platelets are usually increased, although platelet count may be normal or
even reduced. Platelets display atypia with anisocytosis and an occasional giant form.
Monocytes are also increased (absolute monocytosis), but disproportionally to the number of
granulocytes.

Bone marrow biopsy is not required to diagnose CML in most cases, but should be done
if the findings in the peripheral blood are atypical or if a cellular aspirate cannot be obtained.

In the chronic phase (CP), bone marrow specimens are hypercellular, with marked
granulocytic proliferation and a maturation pattern similar to that in the blood, including
expansion at the myelocyte stage.There is no significant dysplasia. Blasts usually account for
<5% of the marrow cells; 10% suggests advanced disease. The proportion of erythroid
precursors is usually significantly decreased. Megakaryocytes may be normal or slightly
decreased in number, but 40-50% of cases exhibit moderate to marked megakaryocytic
proliferation . In CP, the megakaryocytes are smaller than normal and have hyposegmented
nuclei; they are referred to as 'dwarf' megakaryocytes.

Eosinophils and basophils are usually increased in number, and pseudo-Gaucher cells
are common. These features are mirrored in marrow biopsy sections, in which a layer of
immature granules (5-10 cells in thickness) is common around the bone trabeculae, in contrast
to the normal thickness of 2-3 cells. Moderate to marked reticulin fibrosis, which correlates with
increased numbers of megakaryocytes and may be associated with an enlarged spleen.
(Campo, 2017)

The diagnosis of CML is first suspected by identifying the typical findings in the blood
and bone marrow, and then confirmed by the demonstration of the Philadelphia chromosome,
the BCR-ABL1 fusion gene or the BCR-ABL1 fusion mRNA by conventional cytogenetics,
fluorescence in situ hybridization (FISH) analysis, or reverse transcription polymerase chain
reaction (RT-PCR).

First- and second-generation oral TKIs target the constitutively active tyrosine kinase
implicated in the pathogenesis of CML. Although they do not cure the disease, these agents are
able to achieve long-term control of CML in the majority of patients; thus, they have become the
initial treatment of choice for almost all newly diagnosed patients with CML. (Negrin & Schiffer,
2018)

29
 Overall, the complete cytogenetic response rate to first-line TKI(Tyrosine Kinase
Inhibitor) is 70- 90%, with 5-year progression-free and overall survival rates of 80- 95%. Today,
few patients die from leukaemia, and the overall survival is similar to that of the non-leukaemic
population. (Campo, 2017)

CLINICAL CORRELATION

Most patients with CML are diagnosed during the chronic phase which is usually
insidious in onset. Patients may be asymptomatic (nearly 50%) during the time of their WBC
count is discovered to be abnormal. However if symptomatic, fatigue, malaise, weight loss, night
sweats, and anemia, and up to 50% patients with splenomegaly may be seen. There are also
atypical presentations which include marked thrombocytosis without leukocytosis that mimics
essential thrombocythemia. About 5% of the patients are diagnosed in accelerated or blast
phase without a recognized chronic phase. Usually, chronic phase progresses to blast phase
within 3-5 years after diagnosis if without effective therapy. Clinically, this is characterized by
declining performance status, constitutional signs such as fever and weight loss, and symptoms
such as chloroma, petechiae, and bruising related to severe anemia, thrombocytopenia,
increased WBC count, splenic enlargement, and in blast phase, a poor outcome (WHO, 2017;
Rodgers, 2013).

Hematologic findings reflect elevated WBC counts with increases in both immature and
mature granulocytes. Usually <5% circulating blasts and <10% blasts and promyelocytes are
noted, with the majority of cells being myelocytes, metamyelocytes, and band forms. Platelet
counts are almost always elevated at diagnosis, and a mild degree of normocytic normochromic
anemia is present. Leukocyte alkaline phosphatase is low in CML cells. Also, phagocytic
functions are usually normal at diagnosis and remain normal during the chronic phase.
Histamine production secondary to basophilia is increased in later stages, causing pruritus,
diarrhea, and flushing. Bone marrow cellularity is increased, with an increased
myeloid-to-erythroid ratio at diagnosis. Marrow or blood basophilia, eosinophilia, and
monocytosis may also be present. On the other hand, collagen fibrosis in the marrow is unusual
at presentation but significant degrees of reticulin stain–measured fibrosis are noted in about
half of the patients (Longo, 2013).

In chromosomal studies, these must be evidence of the cytogenetic hallmark of CML

which is t(9;22)(q34;q11.2), found in 90–95% of patients by molecularly or by cytogenetics or
FISH to make a diagnosis of CML (Longo, 2013).

EPIDEMIOLOGY

According to the Philippine Council for Health Research and Development, leukemia
ranks 4th in the leading causes of cancer deaths in the Philippines, and the World Health
Organization (2017) reported that worldwide statistics of Chronic Myelogenous Leukemia (CML)
has an annual incidence of 1-2 cases per 100, 000 population, with a slight male predominance,

30
and increases with age, from < 0.1 cases per 100, 000 children to 2.5 cases per 100,000
elderly individuals. Most patients with CML are diagnosed during the chronic phase which is
usually insidious in onset. Patients may be asymptomatic (nearly 50%) during the time of their
WBC count is discovered to be abnormal. However if symptomatic, fatigue, malaise, weight
loss, night sweats, and anemia, and up to 50% patients with splenomegaly may be seen. There
are also atypical presentations which include marked thrombocytosis without leukocytosis that
mimics essential thrombocythemia. About 5% of the patients are diagnosed in accelerated or
blast phase without a recognized chronic phase. Usually, chronic phase progresses to blast
phase within 3-5 years after diagnosis if without effective therapy. Clinically, this is characterized
by declining performance status, constitutional signs such as fever and weight loss, and
symptoms such as chloroma, petechiae, and bruising related to severe anemia,
thrombocytopenia, increased WBC count, splenic enlargement, and in blast phase, a poor
outcome (WHO, 2017; Rodgers, 2013).

No association with race or ethnicity and geographical variations have been reported.
CML is reported to be most common in older populations, with a median age at diagnosis of
around 65 years. Though CML is more common in men, it is reported that women tend to have
a higher survival rate than men. However, an earlier patient age at onset has been reported in
areas with lower socioeconomic status (Swerdlow ​et al.​, 2017 and WHO, 2014).

MICROSCOPIC FEATURES

Chronic Phase
In CP, the peripheral blood shows leukocytosis (12-1000 x 10​9​/L, median: ~80 x 10​9​/L)
due to neutrophils in various stages of maturation, with peaks in the proportions of myelocytes
and segmented neutrophils. Significant granulocytic dysplasia is absent. Blasts typically account
for <2% of the WBCs. Absolute basophilia and eosinophilia are common. Absolute monocytosis
may be present, but the proportion of monocytes is usually <3%, except in rare cases. Platelet
counts are normal or increased to >1000 x 109/L; marked thrombocytopenia is uncommon in
CP.
Bone marrow specimens are hypercellular, with marked granulocytic proliferation and a
maturation pattern similar to that in the blood. There is no significant dysplasia. Blasts usually
account for <5% of the marrow cells; >10% suggests advanced disease. The proportion of
erythroid precursors is usually significantly decreased. Megakaryocytes may be normal or
slightly decreased in number, but 40-50% of cases exhibit moderate to marked megakaryocytic
proliferation. The megakaryocytes are smaller than normal and have hyposegmented nuclei;
they are referred to as 'dwarf' megakaryocytes, but are not true micromegakaryocytes.
Eosinophils and basophils are usually increased in number, and pseudo-Gaucher cells are
common.
In bone marrow biopsy, a layer of immature granulocytes (5-10 cells in thickness) is
found around the bone trabeculae. Moderate to marked reticulin fibrosis, which correlates with
increased numbers of megakaryocytes and may be associated with an enlarged spleen, has
been reported in 30- 40% of biopsies at diagnosis.

31
Accelerated Phase
i​t was recommended that the diagnosis of AP be made if any of the following parameters
were present: a persistent or increasing high WBC count (> 10 x 109/L) and/or persistent or
increasing splenomegaly, unresponsive to therapy; persistent thrombocytosis (> 1000 x 109/L),
unresponsive to therapy; persistent thrombocytopenia (< 100 x 109/L), unrelated to therapy;
evidence of clonal cytogenetic evolution, defined by cells harbouring the Ph chromosome and
additional cytogenetic changes; >20% basophils in the peripheral blood; and 10-19% blasts in
the peripheral blood and/or bone marrow.
Large clusters or sheets of small, abnormal megakaryocytes associated with marked
reticulin or collagen fibrosis were considered to be presumptive evidence of AP, particularly if
accompanied by any of the haematological parameters listed above. Although other defining
criteria for AP have been suggested
Bone marrow specimens are often hypercellular, and dysplastic changes may be seen.
Clusters of small megakaryocytes may be present and may be associated with significant
reticulin and/or collagen fibrosis. The increased proportion of blasts in AP (10-19%) may be
highlighted with immunohistochemical staining for CD34. In most cases, the blasts have a
myeloid phenotype. Lymphoid blasts may be seen, but some data suggest that the finding of
any bona fide lymphoblasts in the blood and/or bone marrow in CP or AP should raise concern
that a lymphoblastic crisis may be imminent, because lymphoblastic BP is reported to
sometimes have an abrupt onset, without preceding AP.
Blast Phase
The criteria for BP include >20% blasts in the blood or bone marrow or the presence of
an extramedullary proliferation of blasts. Some investigators and clinical trials have instead used
a threshold of >30% blasts in the blood and/or bone marrow to define BP, but most patients in
BP have a very poor prognosis regardless of which cut-off point is used.
The blast lineage is myeloid in most cases, and may include neutrophilic, monocytic,
megakaryocytic, basophilic, eosinophilic, or erythroid blasts. In 20-30% of BP cases, the blasts
are lymphoblasts. Sequential lymphoblastic and myeloblastic crises have also been reported. In
BP, the blast lineage may be morphologically obvious, but the blasts are often primitive and/or
heterogeneous, and expression of antigens of more than one lineage is common; therefore,
cytochemical and immunophenotypic analysis of the blasts is recommended.
Extramedullary blast proliferations are most common in the skin, lymph nodes, bone,
and CNS. They may be of myeloid, lymphoid, or mixed-lineage phenotype. In bone marrow
biopsy specimens, sheets of blasts that occupy focal but substantial areas of the bone marrow
(e.g. an entire intertrabecular space or more) can be considered presumptive evidence of BP
even if the rest of the marrow shows CP.

PATHOPHYSIOLOGY

Chronic myeloid leukemia is a clonal hematopoietic stem cell disorder characterized by a

reciprocal translocation between the long arms of chromosomes 9 (ch9) and 22 (ch22). The
abnormal ch22 was first observed in Philadelphia, USA—hence the common terminology,

32
Philadelphia (Ph) chromosome—but the reciprocal translocation of ch9 was not recognized until
1973.2 t(9;22) results in the juxtaposition of the human analogue of the v-ABL oncogene from
ch9 with the BCR housekeeping gene on ch22 to produce the fusion BCR-ABL1 gene. This
fusion gene is transcribed into BCR-ABL1 mRNA and translated into the Bcr-Abl1 protein. The
identification of the fusion gene was facilitated by the close proximity (5 kb) of the breakpoints
on ch22, hence the term breakpoint cluster region (BCR). Sequencing of the BCR showed five
exons (b1–b5), with the most frequent breakpoints being between b2 and b3 or b3 and b4. The
breakpoints within the ABL1 gene on ch9 were distributed along a much wider region, but
always resulted in fusion upstream of the second ABL1 exon, so the fusions were originally
known as b2a2 or b3a2. Later, b1–b5 exons were shown to be exons 12–16 of a much larger
gene, also named BCR, and b2a2 and b3a2 became e13a2 and e14a2. ABL1 encodes a
non-receptor tyrosine kinase that phosphorylates substrate proteins via its SH1 domain, and
affects crucial cellular activities, such as increased proliferation, loss of stromal adhesion, and
resistance to apoptosis.3 Through the loss of upstream control elements in the creation of the
fusion gene, Bcr-Abl1 is capable of autophosphorylation and uncontrolled signaling to a plethora
of downstream proteins, activating these effector pathways (Swerdlow ​et al., 2 ​ 017; Apperley,
2015; Granatowicz ​et. Al, 2​ 015).

Fig 2​. The Philadelphia chromosome. ABL, Abelson murine leukemia; BCR, breakpoint cluster
region. (Granatowicz, A., Piatek, C. I., Moschiano, E., El-Hemaidi, I., Armitage, J. D., & Akhtari,
M. (2015). An Overview and Update of Chronic Myeloid Leukemia for Primary Care Physicians.
Korean journal of family medicine,​ ​36​(5), 197-202.)

Several mechanisms for the transforming activity of BCR/ABL-p210 have been proposed
such as an altered adhesion of myeloid progenitor cells to surrounding stromal cells, constitutive
mitogenic signaling, and molecular changes leading to a reduced rate of apoptosis. These
mechanisms apparently involve a number of different (BCR/ABL-dependent) effector molecules
such as adhesion receptors or anti-apoptotic molecules of the bcl-2 family. More recent data
suggest that BCR/ABL also contributes to an upregulated expression of angiogenic growth
factors. BCR/ABL exerts its stimulating effects through activation of a number of signaling
molecules. In BCR/ABL-dependent signaling networks, three major pathways can be

33
distinguished: the p21Ras, the PI-3- kinase-Akt and STAT5-pathway. These pathways are
complex and exhibit several cross interactions with numerous substrates and associated
molecules. BCR/ABL exists in a complex with Grb2 and SOS, the latter being a nucleotide
exchange factor for Ras. BCR/ABL-Grb2 interaction activates Ras which in turn leads to
phosphorylation and activation of MAP-kinases thus activating mitogenic signaling in BCR/ABL
transformed cells. Signaling through PI3-kinase leads to activation of a number of downstream
effector molecules including Akt and the mammalian target of rapamycin (mTOR) which are
important regulators of cell viability and cell cycle progression. In addition, the PI3-
kinase-Akt-mTOR pathway is involved in BCR/ABL-dependent expression of several critical
effector molecules including angiogenic cytokines. Finally, it has been described that BCR/ABL
phosphorylates STAT5, and that activation of STAT5 contributes to growth and viability of
BCR/ABL-transformed cells. Together, BCR/ABL is a well characterized oncogene that activates
a number of critical signaling pathways pivotal to proliferation and inhibition of apoptosis as well
as expression of critical effector molecules in CML cells. In CML-CP, BCR/ABL appears to be a
primary and selective molecular trigger of growth and survival of leukemic cells (Swerdlow ​et al.,
​ 003).
2017; Sillaber ​et al. 2

Fig 3​. BCR-ABL1–dependent pathways to blastic transformation. Schematic representation of

the potential BCR-ABL1–dependent molecular mechanisms leading to CML disease
progression. (Perrotti D, Jamieson C, Goldman J, Skorski T. (2010). Chronic myeloid leukemia:
mechanisms of blastic transformation. J Clin Invest. 2010;120(7):2254-2264.
https://doi.org/10.1172/JCI41246

34
 BCR-ABL1 fusion gene is present in all cases of chronic myeloid leukemia and results in
two critical events in the disease. First, the gene provides a unique biomarker for diagnosis and
monitoring response to treatment, and second, the fusion tyrosine kinase is susceptible to drug
targeting. TKIs block the ATP binding pocket of Abl1 kinase domain, inhibiting phosphorylation,
and resulting in cell death (Apperley, 2015).

DIAGNOSTICS

The workup for chronic myelogenous leukemia (CML) consists of a complete blood
count with differential, peripheral blood smear, and bone marrow analysis. Although typical
hepatomegaly and splenomegaly may be imaged by using a liver/spleen scan, these
abnormalities are often so obvious clinically that radiologic imaging is not necessary. The
diagnosis of CML is based on the histopathologic findings in the peripheral blood and the
Philadelphia (Ph1) chromosome in bone marrow cells.

Other laboratory abnormalities include hyperuricemia, which is a reflection of high bone

marrow cellular turnover, and markedly elevated serum vitamin B-12–binding protein (TC-I). The
latter is synthesized by the granulocytes and reflects the degree of leukocytosis.

(Source: Anderson Young & Poulsen (2014). Anderson’s Atlas of Hematology, 2nd Edition. PA: Wolters Kluwer Health: Lippincott
Williams & Wilkins)

35
 DIAGNOSTIC CRITERIA OF CML PHASES​ (WHO)

CHRONIC ACCELERATED BLASTIC

PBS ● Leukocytosis due to One or more of the following: ● ≥20% blasts in blood or bone
neutrophils in various stages ● 10–19% myeloblasts marrow
of maturation ● ≤20% basophils ● Extramedullary proliferation of
● Usually <2% blasts ● Persistent platelet count blasts
● Basophilia >1000 × 109/L or <100 × ● Presence of large clusters of
● <3% monocytes 109/L blasts in bone marrow
● Normal or increased platelet ● Increasing numbers of white
count blood cells
● Mild anemia

BONE ● Hypercellular One or more of the following:

MARROW ● <5% blasts ● 10–19% myeloblasts
● Smaller than normal ● Dysgranulopoiesis
megakaryocytes with ● Dysmegakaryopoiesis
hypolobulated nuclei ● Marked collagen or reticulin
● Reticulin fibers increased fibrosis
● Eosinophils may be increased
● Pseudo-Gaucher cells and
sea-blue histiocytes may be
seen

A. IMMUNOPHENOTYPE

Immunohistochemistry
Immunohistochemical stains are helpful for the estimation of blast counts and identification
of their lineage, particularly when there is no access to flow cytometry or there is inadequate
marrow aspirate (dry tap). Blasts are positive for CD34, CD117, CD33, myeloperoxidase (MPO),
and HLA-DR. Promyelocytes are positive for CD117, CD33, and MPO; they do not express
CD34 and HLA-DR. The staining with MPO, CD71, and glycophorin A (GPHA) shows marked
predominance of myeloid cells. Megakaryocytes are positive for CD61. In cases with increased
blasts, immunostaining with terminal deoxynucleotide transferase (TdT), CD34, CD117, myeloid
markers, and B- and T-cell markers is helpful to establish the lineage of immature cells and their
proportion (e.g., to differentiate between myeloid and lymphoid blast crises).

The following are commonly used markers:

CD34 Blasts

CD117 Myeloblasts

TdT Lymphoblasts

CD31, CD61, & factor VIII Megakaryoblasts

Glycophorin A & hemoglobin A Erythroid precursors

Lysozyme & CD68 Granulocytic and monocytic lineages

36
Flow Cytometry (FC)
The analysis of the peripheral blood by flow cytometry (FC) shows characteristic features in
the CP CML, which allow its distinction from reactive neutrophilia or eosinophilia.
Multiparametric immunophenotyping by flow cytometry (MIFC) can be useful in diagnosis and
monitoring of CML in the following areas:
● Blast enumeration and lineage assignment
● Identification of abnormal blasts with various aberrancies including expression of
cross-lineage markers
● Detection of dysmaturation patterns of myeloids, similar to those seen in MDS/MPN
● Enumeration of basophils. Basophils are located at a unique position by CD45 gating,
which is similar to that of monocytes but with slightly lower side scatter. Basophils
express CD13, CD33, and dim CD22. Immature and abnormal basophils may also
express CD117 and/or cross lineage aberrancies

B. CYTOGENETICS AND MOLECULAR STUDIES

Detection of the BCR-ABL1 fusion gene is the hallmark of CML in diagnosis, monitoring,
and targeted therapy. Conventional cytogenetics has been the gold standard for the diagnosis
of CML and has been effectively used for monitoring the response to therapy. However,
cytogenetic analysis requires fresh sample (BM aspirate), depends on the presence of actively
dividing cells, and may miss submicroscopic BCR–ABL1 translocations, and its sensitivity is too
low for detection of minimal residual disease (MRD). Fluorescence in situ hybridization (FISH)
and polymerase chain reaction (PCR) methods offer more sensitive way to diagnose and
monitor CML and can be performed on the blood sample. FISH analysis can detect large DNA
deletions involving chromosome 9. which can be seen in ∼10%–20% of CML patients.

The sensitivity of classical cytogenetics is limited by the number of cells cultured to the
number of metaphases counted. Moreover, about 5% of CML cases have t(9;22) translocations
that may not be visible under the light microscope and will thus be missed by this approach.
Molecular testing should be able to detect such “cryptic” translocations and will therefore
approach 100% sensitivity for initial diagnosis.

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Fig 4.​ (A) G-banded karyotype with a classic t(9;22) showing the “Philadelphia” chromosome. (B) An
ideogram of chromosomes 9 and 22 showing the FISH probes for the ABL1 and BCR loci. The FISH
panel shows cells with red ABL1; green BCR, and yellow (fusion) signals. (​Anderson Young & Poulsen
(2014). Anderson’s Atlas of Hematology, 2nd Edition. PA: Wolters Kluwer Health: Lippincott Williams & Wilkins)

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C. DIFFERENTIAL DIAGNOSTICS

CONDITION DIFFERENTIATING TESTS

Chronic Neutrophilic ● Cytochemistry:​ Neutrophilic alkaline phosphatase is normal

Leukemia (CNL) or increased
● Genetics:​ Philadelphia chromosome or BCR-ABL1 fusion
genes are absent (defect in CSF3R)
● FISH:​ negative for BCR-ABL fusion gene.

Atypical CML ● Cytochemistry: ​Leukocyte alkaline phosphatase level

variable
● Immunophenotype: N ​ o specific characteristic is seen
● Genetics​ : most common abnormalities include +8 and del
(20q). There is no BCR-ABL 1 fusion gene and no
rearrangement of PDGFRA or PDGFRB genes
● FISH:​ negative for BCR-ABL fusion gene

Chronic ● Cytochemistry: ​Nonspecific esterase positive,

myelomonocytic Myeloperoxidase and Sudan black B negative or weakly
Leukemia (CMML) positive and Periodic acid-Schiff negative
● Immunophenotype:
○ Express the myelomonocytic antigen such as CD33
and CD13
○ Variable expression of CD14, CD68, CD64
● Genetics:​ Frequent abnormalities include +8, −7/del (7q)
and structural abnormalities of 12p
● FISH: ​may rarely reveal a PDGFR gene rearrangement

D. DIAGNOSTIC TEST TAILORED TO THE PATIENT

The previously done tests to the patient was CBC, PBS and bone marrow aspirate which
highly considers/suspects CML. To further strengthen the diagnosis, immunohistochemistry
and/or flow cytometry, if available, can be performed for the estimation of blast counts and
identification of their lineage which is useful in identifying the phase of CML that is important
since it alters the treatment of the patient. To confirm the diagnosis and to check the baseline
before treatment, cytogenetic studies and molecular analysis is needed. Fluorescent in situ
hybridisation (FISH), and/or quantitative reverse transcription polymerase chain reaction
(qRT-PCR) of bone marrow are required to confirm the diagnosis and establish the BCR-ABL
breakpoint. A complete metabolic profile is also recommended, since potassium, LDH, and uric
acid may be elevated due to extensive cell turnover.

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 TREATMENT AND MANAGEMENT

The two main treatment options for patients with newly diagnosed chronic phase (CP)
CML are BCR-ABL1 tyrosine kinase inhibitors (TKIs) (imatinib, dasatinib, nilotinib, bosutinib, and
radotinib) and Allogeneic hematopoietic cell transplantation (HCT). Allogeneic HCT remains the
only treatment option with proven ability to cure CML and had been the mainstay for younger
patients in CP before the development of TKIs. However, TKIs have shown long-term disease
control and good tolerability, thus few patients choose to pursue allogeneic HCT as initial
therapy. However, allogeneic HCT remains a principal therapeutic option for patients who
develop resistance or intolerance to TKIs.

Imatinib was the first TKI available for the treatment of patients with CP CML. Studies
have shown that second generation TKIs (eg, dasatinib or nilotinib) are more potent and
produce faster and deeper responses than imatinib. However, the clinical significance of these
endpoints is unclear, as patients on imatinib could catch up in subsequent years, and there is no
difference in survival rate. It is also noted that second generation TKIs have an increased rate of
progression to accelerated phase or blast crisis in the imatinib-treated patients in the first year.
However, progression was seen in only 3 to 4 percent of imatinib-treated patients and there is
no information available on the fate of these patients or the possibility of salvage treatment with
a second generation TKI. Further follow-up is needed to confirm whether the short-term
improvements seen with second generation TKIs will result in longer term benefits, such as
improved survival. For patients with low- or intermediate-risk CP CML, treatment with any of the
first or second generation TKIs (eg, imatinib, dasatinib, nilotinib, bosutinib, radotinib) is the
treatment of choice. With this, side effect profiles, comorbid illnesses, and cost are important
factors to the choice of TKI. For patients with high-risk CP CML, use of one of the second
generation TKIs are preferred because early molecular responses are more frequent with
second generation TKIs, and this is associated with improved survival in this setting.

Allogeneic HCT is another treatment option with curative ability but comes at the cost of
increased toxicity. With HCT, the probability of survival can be predicted with reasonable
accuracy using a scoring system devised by the European Group for Blood and Marrow
Transplantation (EBMT). The five-year overall survival rates for patients in the best risk groups
ranged from 60 to 80 percent. However, with the advent of newer approaches to HCT, current
survival rates are likely to be higher. Among patients with newly diagnosed CML in chronic
phase, we recommend initial treatment with a BCR-ABL1 tyrosine kinase inhibitor rather than
HCT due to its toxicity. HCT may be considered in rare circumstances like that of a younger
patient with an HLA-matched sibling donor (EBMT score of 0). In this setting, HCT may be
preferred since it offers the possibility of cure.

40
 Source: Gratwohl, A., Stern, M., Brand, R., Apperley, J., Baldomero, H., de Witte, T., et al (2009). Risk
score for outcome after allogeneic hematopoietic stem cell transplantation. Cancer, 115(20), 4715–4726.
doi:10.1002/cncr.24531

Other agents were much more commonly used in CML prior to the advent of the TKIs.
These include hydroxyurea, interferon alfa with or without cytarabine, and busulfan. In addition,
omacetaxine mepesuccinate (previously known as homoharringtonine) is a protein synthesis
inhibitor that has demonstrated activity in patients with CML and is approved for patients with
disease refractory to TKIs. Hydroxyurea can be used to reduce white blood cell counts while
awaiting confirmation of a suspected diagnosis of CML in a patient with significant leukocytosis
or in patients with systemic symptoms or with symptomatic splenomegaly. most patients will
have a hematologic remission, amelioration of symptoms, and reduction or elimination of
splenomegaly with these agents. However, these are considered palliative therapy since they
are not curative, do not prolong overall survival, and only rarely result in attainment of a
cytogenetic response.

The aim of initial therapy with TKI is to achieve the following milestones. Long-term
survival is significantly better for patients who achieve rapid molecular responses regardless of
which TKI is used. At three months, BCR-ABL1 ≤10 percent on the International Scale and/or
Ph+ metaphase cells ≤35 percent, at six months, BCR-ABL1 <1 percent and/or Ph+ 0, and at 12

41
months, BCR-ABL1 ≤0.1 percent. Failure to achieve these milestones should be confirmed with
repeat studies before changes in therapy are initiated. A decision to change therapy must also
take into consideration the trends in these values over time

PROGNOSIS AND STAGING

The 3 phases of chronic myelogenous leukemia (CML), as defined by the World Health
Organization (WHO), are listed below.

CML Phase WHO Definition

Chronic stable phase Peripheral blood blasts fewer than 10% in

the blood and bone marrow

Accelerated phase Blasts 10-19% of white blood cells in

peripheral and/or nucleated bone marrow
cells ; persistent thrombocytopenia (< 100
× 10​9​/L) unrelated to therapy or persistent
thrombocytosis (> 1000 × 10​9​/L)
unresponsive to therapy; increasing white
blood cells and spleen size unresponsive to
therapy; cytogenetic evidence of clonal
evolution

Blast crisis Peripheral blood blasts ≥ 20% of peripheral

blood white blood cells or nucleated bone
marrow cells; extramedullary blast
proliferation; and large foci or clusters of
blasts on bone marrow biopsy

Chronic stable phase is usually asymptomatic. It is the phase where the patient is more
responsive to treatment. Accelerated phase is usually where the symptoms begin to appear
such as hepatosplenomegaly, easy fatigability, etc. Untreated accelerated phase will eventually
progress to blast crisis, which has the worse prognosis of the three.

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 REFERENCES

Anderson, S. C., & Poulsen, K. (2013). Atlas of hematology. Lippincott Williams & Wilkins.
Campo, E., Harris, N. L., Pileri, S. A., Jaffe, E. S., Stein, H., & Thiele, J. (2017). WHO
Classification of Tumours of Haematopoietic and Lymphoid Tissues (4th ed.). IARC Who
Classification of Tum.
Eroschenko, V. P., & Di Fiore, M. S. (2013). DiFiore's atlas of histology with functional
correlations. Lippincott Williams & Wilkins.
Gorczyca, W. (2014). Differential Diagnosis in Neoplastic Hematopathology, 3rd Edition. CRC
Press. Taylor & Francis Group, Boca Raton, FL 33487-2742.
Gratwohl, A., Stern, M., Brand, R., Apperley, J., Baldomero, H., de Witte, T., et al (2009). Risk
score for outcome after allogeneic hematopoietic stem cell transplantation. Cancer,
115(20), 4715–4726. doi:10.1002/cncr.24531
Lee, S., Erber, W., Porwit, A., Tomonaga, M., Peterson, L. (2008). ICSH guidelines for the
standardization of bone marrow specimens and reports. ​Int. Jnl. Lab. Hem, 3​ 0: 349–364.
doi:10.1111/j.1751-553X.2008.01100.x.
Luca, D.C. 2012. Bone marrow - nonneoplastic - Normal - General . Retrieved from
http://www.pathologyoutlines.com/topic/bonemarrownormalbonemarrow.html
Negrin, R. S., & Schiffer, C. A. (2018, March 30). UpToDate: Overview of the treatment of
chronic myeloid leukemia. Retrieved January 16, 2019, from
https://www.uptodate.com/contents/overview-of-the-treatment-of-chronic-myeloid-leukem
ia?search=chronic%20myeloid%20leukemia&source=search_result&selectedTitle=2~15
0&usage_type=default&display_rank=2
Pathpedia (n.d.). Histology images of bone marrow. Retrieved from
https://www.pathpedia.com/education/eatlas/histology/bone_marrow/Images.aspx?5
Philippine Council for Health Research and Development. (2014). Leukemia. Philippine Council
for Health Research and Development.
Rodak, B., Fritsma, G., Keohane, E. (2012). ​Hematology Clinical Principles and Applications, 4th
​
ed.​ Missouri: Elsevier Saunders.
Sangle, N. (2018, December 30). Chronic myelogenous leukemia (CML). Retrieved January 16,
2019, from http://www.pathologyoutlines.com/topic/myeloproliferativecml.html
Swerdlow, S.H., Campo, E., Harris, N.L., Jaffe, E.S., Pileri, S.A., Stein, H., Thiele, J., Arber,
D.A., Hasserjian, R.P., Le Beau, M.M., Orazi, A., and Siebert, R. (2017). World Health
Organization Classification of Tumours, Revised 4th Edition. International Agency for
Research on Cancer (IARC).
Wang, S. A., & Hasserjian, R. P. (2018). Diagnosis of Blood and Bone Marrow Disorders.
Basingstoke, England: Springer.
World Health Organization. (2014). Chronic Myelogenous Leukemia. World Health Organization
Union for International Cancer Control

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