Clinicopathological Evaluation of Acute Leukemias in a Tertiary Care Hospital: A Cross-Sectional Study
Gayatri N. PATEL1, Rashmi GUDUR2, Anand GUDUR2 , R. M. OSWAL1, Sujatha KANETHKAR2
1Department of Pathology, Krishna Institute of Medical Sciences (Deemed to University), MAHARASHTRA, INDIA
2Department of Oncology, Krishna Institute of Medical Sciences (Deemed to University), MAHARASHTRA, INDIA
Keywords: Acute myeloid leukemia, WHO classification 2016, Flow cytometry, Cytogenetics, Molecular study
Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) are clinically and biologically diverse phenotypic
diseases amongst hematological malignancies. The current study objectives were to diagnose and classify cases of AL as per revised 4th edition
of WHO 2016 classification of AL’s and study their clinicopathological profiles.
Material and Method: This cross-sectional, observational study included 68 patients, diagnosed with AL were recruited. Diagnosis was based
on peripheral blood smear examination, bone marrow aspiration, flowcytometry, and cytogenetic and molecular studies.
Results: Sixty-eight cases of AL were diagnosed in a period of 2 years, where 25 cases were of ALL and 43 cases were of AML. In the subclassification
of AML as per WHO 2016, 20 cases were of AML, RGA, 21 cases were of AML, NOS, and 2 cases were of AML, MRC. In AML, RGA, APL
with PML-RARA positive cases were 10 out of 20 cases, AML with (8;21) RUNX1-RUNX1T1 were 7/20 cases; there were two cases of AML
with mutated NPM1 gene and one case of AML with biallelic mutation of CEBPA. In AML, NOS subcategory AML with maturation was more
common with 9/21cases. In subcategory of ALL, B-ALL was more common than T-ALL. B-ALL, NOS was more common than B-ALL, RGA
and we had 1 case of NK cell Leukemia.
Conclusion: The application of revised 4th edition WHO 2016 classification confers uniformity in reporting acute leukemia cases that aids in
the treatment by using targeted therapies and helps in the prediction of prognosis. The WHO classification for acute leukemias is very objective,
therapy oriented and the need of the hour.
Acute leukemias (AL) constitute a heterogeneous group
of hematological malignancies, characterized by an
uncontrolled proliferation of hematopoietic cells that
infiltrate into the bone marrow and blood 1
Acute myeloid leukemia (AML) and acute lymphoblastic
leukemia (ALL) are the two main types of AL, with a
global prevalence of 3-4 and 0.4-2 per 100,000 individuals,
respectively (2-4). The annual incidence in India is
estimated to be 4.3 and 1.4 per 100,000 individuals for
AML and ALL, respectively 5,6. AML is more prevalent
in adults and ALL in children 7.
The FAB classification system was proposed in 1976 for
classifying these diseases in order to treat them as per
their biologic behavior. However, it had its own set of
drawbacks as it was based on morphology alone and led
to subjective errors 8. This subjectivity in diagnosis was
eliminated by the WHO classification system, proposed
in 1999 and revised in 2001, 2008, and 2016 (published
in 2017). The WHO (2016) classification system was evolved to improve the objectivity and reproducibility
by additionally utilizing the cytogenetic, molecular,
cytochemical, and immunologic characteristics for an
integrated diagnosis 9,10. This has greatly contributed to
the determination of differential diagnosis and prognosis
of leukemic proliferations, facilitating targeted treatment as
per their pathologic behavior. This includes management
of recognizable genetic lesions, stem cell transplantation
as well as immunotherapy such as treatment directed at
specific cluster differentiation (CD) markers 11. The
regular revisions of AL classifications have given rise to
a constant need to study these pathologies in order to
upgrade the management strategies.
The present study aims to diagnose and classify AL cases
according to the WHO Classification of Tumors of
Hematopoietic and Lymphoid Tissues, 2016, revised 4th
edition and study their clinicopathological profiles 9.
This cross-sectional, observational study was conducted at
the Department of Pathology, in a tertiary teaching hospital in India, from June 2017 to May 2019, after obtaining
ethical clearance from the Institutional Review Board
(protocol number: 031/2017-18, Date: 23.11.2017).
In all, 68 patients diagnosed with AL (AML and ALL) as
per the WHO Classification of Tumors of Hematopoietic
and Lymphoid Tissues, 2016, revised 4th edition criteria,
irrespective of age and gender, were recruited into the
study after obtaining written informed consent from
them (or their legal guardians in case of minor patients)
6. AL cases without cytogenetic studies or advanced
ancillary techniques were excluded from the study.
Patients were examined for pallor, fever, generalized
weakness, bony tenderness, petechiae, ecchymosis, and
gum bleeding. Presence of hepatomegaly, splenomegaly,
and lymphadenopathy was also recorded. Ultrasonography
(USG) was performed where necessary. Morphologic
dysplasia, cytogenetic abnormalities, and discontinuation
of chemotherapy due to low blood cell count were
considered as unfavorable prognostic factors.
This included estimation of hemoglobin (Hb) levels, total
leukocyte count (TLC), differential leukocyte count (DLC),
and platelet count, using the Sysmex XT-1800i (XT-1800i,
Sysmex Corporation, Kobe, Japan). Peripheral venous
blood samples were collected in ethylenediaminetetraacetic
acid (EDTA)-anticoagulated vacutainers. Peripheral
smears were made, stained with the Romanowsky Leishman
stain, and studied in detail for morphology. In cases of
leukopenia, buffy-coat smears were prepared and stained
with the Leishman stain and examined for the presence of
blast cells. A provisional diagnosis was made and ancillary
All cases underwent flow-cytometric analyses using BD
FACSCanto™II/FACSDiva™ 6-color flowcytometry (BD
Biosciences, Becton, Dickinson, and Company, New Jersey,
USA), molecular studies using nested/reverse transcriptase
polymerase chain reaction (PCR), and cytogenetic studies
using fluorescence in-situ hybridization (FISH).
Bone Marrow Aspiration Study
This was performed on all patients under aseptic conditions
and local infiltration anesthesia with 3 cc of 2% lignocaine.
Bone marrow aspiration was performed from the posterior
superior iliac spine using Salah’s aspiration needle. Smears
were prepared, air dried, and stained with the Giemsa
stain. If the marrow particles were not visibly present in
the aspirate, it was centrifuged and the smears prepared accordingly. The smears were meticulously examined for
cellularity, predominant series, myeloid:erythroid ratio,
erythroid series cells, myeloid series cells, lymphocytes,
plasma cells, megakaryocytes, blast cells, atypical cells,
mitosis, iron stores, and parasites. At least 500 cells were
counted to obtain the myelogram.
Bone Marrow Trephine Biopsy
Indicated only when a dry tap was obtained or the aspirate
was inadequate for diagnosis (6 out of 68 cases). Bone
marrow was obtained from the posterior superior iliac
spine using a Jamshidi™ needle (BD Biosciences, Becton,
Dickinson, and Company, New Jersey, USA) under
aseptic conditions and local infiltration anesthesia with 2%
lignocaine. The biopsy needle was introduced in the bone
cortex with a rotatory movement and gently advanced
after removing the stylet. An optimal 1-2 cm long biopsy
sample was obtained in its maximum diameter, placed in
10% formalin fixative, decalcified in 14% EDTA solution,
submitted for routine paraffin processing, and stained with
hematoxylin and eosin for further examination.
The data was collected, compiled, and analyzed using SPSS
version 20 software (IBM Corp., released 2011, IBM SPSS
Statistics for Windows, Version 20.0. Armonk, NY: IBM
Corp.). Categorical variables were expressed in terms of
frequencies and percentages.
The present study included 68 cases of AL, of which 43
(63.24%) were diagnosed with AML and 25 (36.76%) with
ALL. Covering an age range of 10-70 years, the mean age
of the included participants was 37.07±21.15 years, with a
slight male predominance (M:F = 1.7:1). The average age
was 48.16±16.18 years for the AML cases and 8.28±13.85
years for the ALL cases. Both were more common in
males (M:F = 1.5:1 in AML; 2.1:1 in ALL). The frequency
distribution of different types of AL is described in Table
. The frequency distribution of the clinical features of
AL is shown in Table II
. Fever was the most common
presenting symptom (91.81%) followed by generalized
weakness (80.88%). The most common presenting sign
was pallor (94.19%) followed by splenomegaly (80.88%).
summarizes the hematological characteristics
of these subjects. Anemia (Hb 7.2 g%), leukocytosis
(>100,000/mm3), and thrombocytopenia (<40,000mm3)
were seen in all the cases, with a low neutrophil count
(7.6%) and an increased peripheral blast cell count of
>20% (reaching an average of 63%). Granulocyte precursor
cells like myelocytes, metamyelocytes, promyelocytes, and promonocytes were increased in cases of AML. The
immunophenotyping findings in these AL cases are detailed
in Table IV
. Table V
presents the distribution of AL cases
according to their prognosis. The comparative results
revealed that the prognosis was more on the unfavorable side for all the leukemia types, and all the patients having
unfavorable prognosis succumbed to their illness within 6-9
months after diagnosis. The histological findings from the
peripheral smear and bone marrow aspirate are presented
in Figure 1A-J
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|Table II: Frequency distribution of clinical features of acute leukemias
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|Table III: Mean values of hematological parameters in cases of acute leukemia
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|Figure 1: A) Hypercellularity in BMA with blast cells, Arrow shows blast cell with Auer rod in AML. B) PBS Showing large blasts with
variable N:C ratio and scant-to-moderate amount of basophilic cytoplasm, absence of maturing granulocytes indicate AML-NOS. C) PBS
showing large blasts features with matured nuclei favoring AML-NOS with maturation. D) PBS Showing severe leukocytosis with blasts
features favoring AML-NOS (Black arrow showing Monoblasts ; Red arrow showing Promonocytes). E) BMA showing case of AMLNOS.
F) BMA of AML-NOS showing neutrophilic precursors (Black arrow showing myelocytes ; Red arrow showing Promonocytes;
green arrow shows auer rod myeloblast). G) PBS of APL showing bilobed or buttock-shaped nuclei with moderate cytoplasm. H) PBS of
A case of APL with PML-RARA. I) PBS showing large sized lymphocytes and azurophilic granules indicating PBP-NKLL. J) PBS showing
Multiple Auer rods cells in criss-cross pattern indicating AML. (All the images shown are x400; inserts are x1000 ; BMA and PBS samples
were stained by Giemsa and Leishman stains, respectively).
AML: Acute myeloid leukemia, APL: Acute promyelocytic leukemia, BMA: Bone marrow aspirate, NOS: Not otherwise specified, PBS: Peripheral blood smear,
s/o: Suggestive of
The present study was conducted to diagnose and classify
AL cases according to the WHO (2016) Classification and
study their clinicopathological profiles 9
. Sixty-eight AL
cases were studied for a period of 2 years. AML was more
commonly diagnosed than ALL, similar to previous studies
. ALL was found to be more common in children (aged 1-14 years) and AML in adults (aged >30 years) in
concordance with the findings of Dores et al. and others
. The presenting signs and symptoms also found
resonance with these studies 12-17
. Fever was the most
common complaint in both the leukemias, similar to
Kumar et al., Ghosh et al., and others (72-89%) 12-16
This was most likely associated with granulocytopenia and
concomitant infections 17,18
. Generalized weakness and bleeding manifestations were also similar to these studies
. Gingival bleeding was not seen in any of the ALL
cases but was present in AML cases, akin to the studies
by Ghosh et al. (23%), Preethi CR (25%), and Sultan et al.
(22%). This is suggested to be due to thrombocytopenia
and neutropenia 14-16
. The most common presenting
sign in both ALL and AML was pallor followed by
hepatosplenomegaly, similar to the above-mentioned
studies, likely related to anemia and organ infiltration,
. The increased peripheral smear blast
cell % (68% in ALL and 61% in AML) also mirrors that
seen by Kumar et al. (85% in ALL) and Ghosh et al. (57.6%
in AML). This correlates to overproduction and blood
infiltration of immature white blood cells 12-14
Acute Lymphoblastic Leukemia (ALL)
B-cell ALL (B-ALL) was found to be more common than
T-cell ALL (T-ALL), in agreement with the prevalence
noted by Kumar et al. (63%) 12. Most B-ALL blasts showed
CD10 and CD19 positivity. B-ALL not otherwise specified
(NOS) revealed CD56 aberrancy, a normal karyotype, and
posterior reversible encephalopathy syndrome, with death
reported within 5 days of admission. This is in agreement
with the findings of Swerdlow et al. and Arber et al. who
stated that cranial nervous system (CNS) involvement
leads to an adverse prognosis in ALL patients 9,19.
B-ALL cases with recurrent genetic abnormality (RGA)
t(12;21)(p13;q22) TEL-AML1 (ETV6-RUNX1) revealed
blast cell positivity for CD19, CD10, CD34, and CD13
but were typically negative for CD20, CD9, CD66c. These
patients achieved molecular remission with a cure rate of
>90% 9,19,20. B-ALL RGA (9;22) BCR-ABL1 showed
a higher incidence in adults with the blasts cells typically
showing high-frequency expression of CD25. Despite
imatinib therapy, mortality was recorded within 3 months
from diagnosis. They are usually associated with the worst
prognosis among all B-ALL RGA cases 9,19. T-ALL cases
revealed CD2 and CD7 positivity. T-ALL natural killer
cell leukemia (T-ALL NK), diagnosed in a 50-year old
male, had an immunophenotypic picture showing CD45
bright+ as well as cytoplasmic myeloperoxidase (cMPO)-
negative blasts and marked leukocytosis with 85-90%
large-sized lymphoid cells (having high N:C ratio, clumped
chromatin and cytoplasmic granules with 1-2 prominent
gated nucleoli). Death was recorded within a month
from diagnosis. T-LL NOS also presented with mostly
an unfavorable prognosis. Other studies have also found
T-ALL NK to have an extremely poor prognosis 9,20,21.
Acute Myeloid Leukemia (AML)
The present study found AML-NOS to be the most common
type of AML, unlike Nunes et al. who found AML-RGA to
be the most prevalent. However, the distribution of AMLRGA
subtypes mirrored the works of Nunes et al. 22.
AML-NOS was a diagnosis of exclusion, made for cases
that cytogenetically did not fulfill the criteria for other
AML subtypes, as per the WHO classification (9). However,
cytogenetic studies were not performed by Ghosh et al. and
Preethi CR, and instead the FAB classification (1976) was
employed 8. They found that a small percentage of cases
remained, and these were categorized under AML M3 as
per FAB classification, which is now categorized as acute
promyelocytic leukemia (APL) with PML/RARA under
AML-RGA 8,14,15. It was noted that the WHO (2017) 9
AML-NOS subcategory closely matched the FAB (1976)
AML with maturation (FAB M2) subcategory 8,14,15.
The presence of APL with PML-RARA, with concomitant
disseminated intravascular coagulation (DIC) and CNS
involvement, typically led to a very poor prognosis
9,19,23. An increase in promyelocytes on peripheral
smear and bone marrow aspirate myelogram was seen,
along with the absence of megakaryocytes. Hypergranular
variants had multiple Auer rods in the cytoplasm of
myeloblasts and/or progranulocytes. Cases of AML-RGA
t(8;21)(q22;q22)RUNx1-RUNX1T1 with tuberculosis (TB)
required stopping anthracylines and cyclophosphamide for
3 weeks due to a risk of flaring up of TB, but with chances
of increase in mortality. Hence, careful anti-leukemic
therapy was continued after starting anti-tubercular
therapy as early as possible 9,19,23. AML with mutated
NPM1 was diagnosed in older individuals with the blast
cells typically negative for CD34. Nucleophosmin (NPM)
is a surrogate marker for this gene mutation, seen in about
33% of AML cases, with the morphology resembling
acute myelomonocytic/monocytic leukemia and has a
good prognosis. AML with biallelic mutations of CCAAT
enhancer binding protein alpha (CEBPA) was typically
associated with a normal Hb level, normal karyotype, and
higher blast cell count with a good response to induction
chemotherapy 9,19,24,25. According to Walter et al.,
AML-NOS patients corresponded to the FAB M0 category,
known to have significantly worse outcomes than non-M0
cases, similar to the present study 26.
The present study establishes the WHO (2016) classification
as a practically useful system for diagnosing the various
types of AL, primarily with a focus of providing targeted therapy. Cytogenetics is one of the most important
diagnostic parameters as recurrent genetic aberrations
have provided insights into the molecular mechanisms
of leukemogenesis. The uniformity in categorizing these
diseases, afforded by this classification system, permits
the use of immunotherapy such as specific targeting of
CD expression especially in patients failing induction
therapies. Lately, monoclonal antibody therapy has
become a significant component of the treatment protocol,
for example the use of Gemtuzumab for CD33-positive
AML. Stem cell transplantation is also required in many
cases receiving chemotherapy and radiotherapy as it helps
to replenish their bone marrow reservoir with normal
hematopoietic stem cells 27.
This study has its limitations in being a single-center, crosssectional
study with a limited sample size. Multicentric,
prospective studies with larger sample size and longer
follow-up periods are encouraged to validate the results.
In conclusion, the application of revised 4th edition WHO
2016 classification of acute leukemias confers uniformity in
reporting of acute leukemia cases, which aids in treatment
by using targeted therapies and helps in prediction of
prognosis. The categorization of acute leukemia cases in
favorable and unfavorable prognosis groups tells us about
the future outcome of cases, and findings of this study
show that the unfavorable prognosis group has a dismal
prognosis. Overall, the revised 4th edition WHO 2016
classification is very objective, therapy oriented and the
need of the hour.
The authors are thankful to Dr. Garima Agrawal, Dr. Nikita
Vohra, Dr. Dhiraj Shukla, Dr. Sourish Hota, Dr. Chintan
Sangoi, Nitin H Patel, Aruna Sangoi, and Mayuri Patel for
extending their help and support in this research work.
CONFLICT of INTEREST
The authors declare no conflict of interest.
The study was not funded by any government or private
Concept: GNP, RG, SK, Design: SK, Data collection or
processing: GNP, RG, Analysis or Interpretation: GNP, RG,
AG, RMO, Literature search: AG, RMO, SK, Writing: GNP,
AG, RMO, Approval: GNP, RG, AG, RMO, SK.
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