| |

Received: 26 July 2018    Revised: 10 September 2018    Accepted: 20 September 2018

DOI: 10.1111/ijlh.12939

ORIGINAL ARTICLE

Apoptosis: A biomarker of high-­risk phenotype in pediatric

acute myeloid leukemia?

Anudishi Tyagi1 | Raja Pramanik1 | Radhika Bakhshi2 | Sreenivas Vishnubhatla3 | 

Sameer Bakhshi1

1
Department of Medical Oncology, Dr. B. R.
A. Institute Rotary Cancer Hospital, All India
Institute of Medical Sciences, New Delhi,
India
2
Shaheed Rajguru College of Applied
Sciences, University of Delhi, New Delhi,
India
3
Department of Biostatistics, All India
Institute of Medical Sciences, New Delhi,
India
Correspondence
Dr. Sameer Bakhshi, Department of Medical
Oncology, Dr. B. R.A. Institute Rotary
Cancer Hospital, All India Institute of
Medical Sciences, New Delhi, India.
Email: sambakh@hotmail.com
Funding information
Department of Biotechnology, Ministry
of Science and Technology, Grant/Award
Number: BT/PR7197/MED/30/899/2012
Abstract
Introduction: Dysregulation of apoptosis has been explored in acute myeloid leuke-
mia (AML); yet, its correlation with clinical outcomes in pediatric AML is unknown.
This study was aimed to analyze percentage of apoptosis and apoptosis mediated
through the intrinsic pathway with clinical outcomes in patients with pediatric
AML.
Methods: This prospective study included pediatric AML patients enrolled from July
2013 to August 2016. Annexin-­V (marker of total apoptosis) and caspase-­9 expres-
sion (marker of intrinsic pathway) was determined in baseline bone marrow (BM)
samples by flow cytometery and compared with controls (unaffected BM of solid
tumors and peripheral blood [PB] of unaffected siblings). Overall survival (OS) and
event-­free survival (EFS) were compared using log-­rank test.
Results: A total of 151 AML patients were enrolled, median age 10 (range: 0.7-­
18 years). Annexin-­V expression in blast cells was significantly high in AML patients
as compared to BM of subjects with solid tumors (P = 0.01) and PB of healthy sub-
jects (P = 0.04). Caspase-­9 expression in blast cells was not significantly different.
Median annexin-­V expression was significantly higher in patients with WBC count
≥11 000/mm3 (P = 0.02), poor-­risk cytogenetics (P = 0.02), the absence of RUNX1-­
RUNX1T1 translocation (P = 0.004), and the absence of NPM1 mutation (P = 0.05).
Patients with high annexin-­V expression had significantly inferior OS (P = 0.05) in
univariate analysis but not in multivariate analysis (P = 0.32).
Conclusion: Apoptosis as a whole was found to be activated in baseline BM samples
of AML patients. High apoptosis may be associated with high-­risk phenotype in this
disease.

KEYWORDS
acute myeloid leukemia, annexin-V, apoptosis, caspase-9

1 |  I NTRO D U C TI O N hematopoiesis. AML develops through a multistep process of ge-

nomic and epigenetic alterations which results in disease progres-
Acute myeloid leukemia (AML) is a genetic heterogeneous dis- sion.1 Dismal survival and poor outcomes in this disease result in a
ease characterized by the rapid proliferation of immature myeloid large unmet need to discover newer biomarkers and potential ther-
progenitor cells, ultimately resulting in suppression of normal apeutic targets. 2,3

Int J Lab Hematol. 2018;1–7. © 2018 John Wiley & Sons Ltd |  1
wileyonlinelibrary.com/journal/ijlh  
 |
2       TYAGI et al.

Abnormalities in apoptotic pathways lead to the development
of hematological malignancies and reduce the efficacy of chemo-
therapy.4 There are two interconnected pathways of apoptotic cell
death; the intrinsic pathway involves the mitochondria and cyto-
chrome C, activation of the apaf-­1 complex finally culminating into
caspase-­9, and the extrinsic pathway, on the other hand, comprises
the death receptor (DR) DR4, DR5, FAS leading to the proteolytic ac-
tivation of caspase-­8, 3 and 7 finally leading to cell death. Decreased
susceptibility to apoptosis is an important biological feature of leu-
kemic cells and reflects their survival potential and resistance to
chemotherapeutic drugs. As a genuine hallmark of cancer, “evasion
of apoptosis” has been the focus of research for many tumors in
recent years. 5 Several preclinical studies have evaluated the role of
6
extrinsic apoptotic pathway in the pathogenesis of AML. However,
the role of intrinsic apoptotic pathway in AML appears less clear.
Annexin-­V is widely used as a marker for total apoptosis in
scientific research.7 Some preclinical studies have used caspase-­9
as a marker of the intrinsic apoptotic pathway. 8-10 To date, there
are no published reports correlating total apoptosis and intrinsic
apoptosis with clinical outcome in pediatric AML patients. The
present study was designed to analyze total apoptosis and apop-
tosis induced due to intrinsic pathway, and correlate these data
with clinic-­p athological features and outcome in pediatric AML
subjects.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Patient’s sample
This was a single-­centre, prospective cohort study conducted on de
novo pediatric (≤18 year) AML subjects at the tertiary cancer centre
at our institute, who were registered between July 2013 and June
2016. Study was approved by the institute ethics committee.Assent
form from parents was taken from subject’s ≤8 years of age and
informed consent was taken from rest. We excluded patients with

(A) (B) (C) (D)

Necrotic Late apoptosis

Caspase-9
PI

Viable cell Early apoptosis

CD45 Annexin-V Annexin-V

(E) ** (F) **
80

60

P* =0.76
* P* =0.01 * P**=0.78
P**=0.04
60

Caspase -9 Expression
40
Annexin-V (%)
40

20
20
0

BM Patients BM Controls PB Controls BM Patients BM Controls PB Controls

(n = 151) (n = 30) (n = 50) (n = 151) (n = 30) (n = 50)

FIGURE 1 Analysis of annexin-­V (total apoptosis) and caspase-­9 expression (intrinsic pathway of apoptosis) by flow cytometery: (A) Side
scattered vs Forward scattered dot plot showing normal distribution of different cells population, (B) Blasts were gated based on low
side scatter and dim CD45 (C) Gated blasts showing percentage of annexin-­V expression (total apoptosis) and (D) Gated blasts showing
percentage of caspase-­9 expression (intrinsic pathway of apoptosis) (E) Comparison of Annexin-­V expression and (F) Caspase-­9 expression
between AML patients, PB controls and BM controls. Y axis of each graph shows the percentage of annexin-­V expression and caspase-­9
expression. P* represents the comparison of patients with PB controls while P** represents their comparison with BM controls. BM, bone
marrow; PB, peripheral blood [Colour figure can be viewed at wileyonlinelibrary.com]
TYAGI et al. |
      3

TA B L E   1   Association of baseline
Variable Annexin-­V Caspase-­9
parameter with caspase-­9 and annexin-­V
(N = 151) (Median IQ Range) P (Median IQ Range) P

Age (y)
<10 (77) 46.7 (34.6-­56.4) 0.97 20.4 (10.5-­32.1) 0.65
≥10 (74) 47.1 (35.4-­55.6) 19.3 (9.9-­33.6)
Sex
Male (108) 46.8 (35.5-­56.7) 0.70 18.6 (10.1-­3 0.3) 0.20
Female (43) 46.7 (32.6-­55.1) 27.3 (11.2-­3 4.5)
Hb (g/dL)
<8 (82) 46.2 (34.6-­53.3) 0.73 19.9 (9.0-­3 0.2) 0.49
≥8 (69) 47.5 (35.1-­57.3) 20.3 (12.4-­36.3)
Platelet (/mm3)
<50 000(48) 47.1 (35.1-­56.7) 0.94 18.9 (12.4-­31.6) 0.90
≥50 000(103) 46.7 (34.6-­55.6) 20.4 (9.9-­33.6)
WBC (/mm3)
<11 000 (55) 42.6 (30.7-­51.5) 0.02 18.8 (12-­27.7) 0.63
≥11 000 (96) 48.3 (42.3-­57.4) 20.6 (6.6-­33.8)
LDH (n = 93)
<280 (12) 42.6 (30.6-­49.2) 0.56 17.5 (10.1-­38.3) 0.82
≥280 (81) 45.5 (32-­52.9) 18.2 (5.9-­31.6)
Karyotype (n = 125)
Good (61) 42.5 (29.8-­51.3) 0.02 22.4 (12.4-­36.3) 0.09
Intermediate (49) 46.7 (38.5-­51.5) 18.1 (7.2-­27.6)
Poor (15) 52.5 (42.6-­62.7) 20.3 (13.7-­39.4)
NPM1 (n = 122)
Negative (113) 48.3 (42.5-­56.7) 0.05 20.1 (9-­3 0.6) 0.17
Positive (9) 31.7 (23.7-­52.9) 35.2 (17.8-­36.4)
*RUNX1-­RUNX1T1 (n = 125)
Negative (76) 45.7 (42.3-­55.6) 0.004 15.2 (9.4-­3 0.1) 0.43
Positive (49) 38.0 (28.2-­51.3) 15.0 (12-­36.3)
FlT3ITD (n = 127)
Negative (117) 47.3 (36.5-­55.7) 0.16 20.1 (9-­32.1) 0.53
Positive (10) 55.9 (47.5-­57.4) 23.9 (14.5-­37.7)

FLT-3ITD, FMS like tyrosine kinase 3 internal tandem duplication; Hb, hemoglobin; IQ, inter-­quartile;
LDH, lactate dehydrogenase; NPM1, nucleophosmin; WBC, white blood cells.
*
Done by cytogenetics.

AML-­M3 and MDS-­related AML. Peripheral blood (PB) samples from
50 age and sex-­matched normal individuals (siblings accompanying
the patients) were taken along with bone marrow (BM) samples from
30 age-­and sex-­matched patients with solid tumors without any mi-
croscopic bone marrow involvement.
and FLT3-­ITD was done using PCR.12,13 Based on this information,
the European leukemia network (ELN) classification was used to
categorize the patients into three different prognostic risk groups;
good, intermediate, and poor risk.14

2.2 | Baseline cytogenetic and molecular analysis
Pretreatment samples from all patients underwent cytogenetic
analysis at NABL-­accredited laboratory, and chromosomal abnor-
malities were described according to the International System for
Human Cytogenetic Nomenclature.11 Molecular analysis for NPM1
2.3 | Treatment
All patients were treated on a uniform protocol. They were induced
with 3 + 7 regimen (daunorubicin 60 mg/m2 for 3 days and cyto-
sine arabinoside 100 mg/m2 as a 24-­hour continuous infusion for
7 days). Patients, who did not achieve CR after 1st induction, were
given ADE as a 2nd induction.15 After achieving remission, the
4       |
(A) Apoptosis (Overall survival)
1.00
Log rank; P = 0.05
Cumulative survival
0.75
Apoptosis Low (n = 74)
0.50
Apoptosis High (n = 77)
0.25
0.00
0 10 20 30 40
Time (Mo)
F I G U R E   2   Kaplan-­Meier curves showing (A) Overall survival and (B) Event-­free survival for apoptosis [Colour figure can be viewed at
wileyonlinelibrary.com]
patients received three cycles of high-­dose cytosine arabinoside at
18 g/m2. Twenty-­eight patients received consolidation at 12 g/m2
as a part of a randomized controlled trial, which was underway dur-
ing this period at our institute. Patients who relapsed received al-
logeneic transplantation in CR2, if a matched sibling was available.
2.4 | Measurement of apoptosis using
Annexin-­V and caspase-­9
Five milliliters BM samples was collected in EDTA vials and pro-
6
cessed within 5 hour after collection. The whole blood (10 cells)
were mixed with the BD FACS lysing solution and kept in dark for
10 minutes. Followed centrifugation, supernatant was discarded
and white blood cell (WBC) pellets were washed with phosphate-­
buffered saline. One microliter of Caspase-­9 (FITC) was added
into the tube and incubated for 30-­6 0 minutes in 37°C incubator
with 5% CO2. Cells were again washed with phosphate-­buffered
saline wash buffer twice and suspended in 200 μL Annexin-­V bind-
ing buffer. Cells were then incubated with 5 μL Annexin-­V (APC)
and 5 μL CD45 (Percp-­c y5.5) (e-­bioscience, Thermo fisher, San
Diego, CA, USA) for 15 minutes in dark at room temperature. The
cells were then washed and resuspended in 200 μL binding buffer.
Finally, the cells were mixed with 5 μL propidium iodide (PE) (e-­
bioscience, Thermo fisher). Cells were immediately acquired into
FACS Calibur (BD Bioscience, Becton Dickinson, San Jose, CA,
USA) and analyzed using the Cell Quest Pro software program (BD).
2.5 | Statistical analysis
Baseline characteristics for all the patients were summarized using
median and range. Primary overall survival (OS) was measured as the
duration from the date of enrollment to death from any cause. Event-­
free survival (EFS) was measured as the duration from date of enroll-
ment to date of relapse or death due to any cause. These data were
censored at the date of last follow-­up (22-­December-­2017) for alive
patients. Annexin-­V and caspase-­9 expression were categorized into
high or low based on their mean value. Kaplan-­Meier curves were
TYAGI et al.
(B) Apoptosis (Event-free survival)
1.00
Log rank; P = 0.07
Cumulative survival
0.75
0.50
Apoptosis Low (n = 74)
Apoptosis High (n = 77)
0.25
0.00
50 0 10 20 30 40 50
Time (Mo)
used to estimate OS and EFS and log-­rank test was used to compare
differences between survival curves. Comparison of Annexin-­V and
caspase-­9 expression between controls and AML patients was done
using Mann-­Whitney test. Comparison between Annexin-­V and cas-
pase-­9 expression with baseline patient’s characteristics was done
using Dunn test or Ranksum test. For multivariate analysis, a Cox
proportional hazards model was constructed for outcome. Stepwise
multivariate Cox regression method was employed to evaluate the
independent prognostic factors. P value ≤0.05 was considered sig-
nificant. All statistical analysis was done using STATA 11.2 (Lakeway,
TX, USA).
3 | R E S U LT S
3.1 | Baseline parameters
A total of 200 patients were registered at the centre during the
study period. Out of these, 49 were excluded from the study (five
patients were acute promyelocytic leukemia, 15 patients had only
one visit and 29 patients had technical failure during processing).
Therefore, 151 patients were eligible for this study. Median age
was 10 years (0.7-­18 years); male: female ratio was 2.5:1. Median
blast percentage of AML patients and BM controls was 75 (range:
10-­95) and 0.8 (range: 0.2-­2 .3), respectively. Cytogenetics was
evaluable in 125 patients (82.8%), 48.8% of the patients had
good-­r isk cytogenetics while intermediate and poor-­r isk cytoge-
netics were present in 39.2% and 12.0% subjects, respectively.
French American British (FAB) subclassification of AML was avail-
able in 59 cases. Out of these, 23/59 (38.9%) cases were of M2,
11/59 (18.6%) cases of M5, 9/59 (15.2%) cases of M4, 6/59 (10.2%)
cases of M0 and M1 while 2/59 (3.4%) cases were of M6 and M7.
Recurrent RUNX1-­RUNX1T1 translocation was seen in 49/125
(39.2%) cases. FLT3-­I TD and NPM1 mutations were analyzed
in 127 and 122 cases, respectively. Out of these, 10/127 (7.9%)
had mutated FLT3-­I TD while 9/122 (7.4%) had mutated NPM1.
Complete remission (CR) was achieved in 123 patients with induc-
tion chemotherapy. Median follow-­u p was 33.8 months.
TYAGI et al.       5|
TA B L E   2   Association of baseline
Variable Event-­free survival Overall survival
parameters and apoptosis with clinical
(n = 151) Hazard Ratio (95%CI) P Hazard Ratio (95%CI) P
outcomes
Age (y)
≤10 (n = 77) 1.00 0.46 1.00 0.62
≥10 (n = 74) 0.8 (0.6-­1.3) 0.9 (0.6-­1.4)
Sex
Male (n = 108) 1.00 0.16 1.00 0.55
Female (n = 43) 1.3 (0.9-­2.1) 1.2 (0.7-­1.8)
Hemoglobin (g/dL)
≤8 (n = 82) 1.00 0.14 1.00 0.18
≥8 (n = 69) 1.4 (0.9-­2.1) (1.3 (0.8-­2.1)
WBC (/mm3)
≤11 000 (=55) 1.00 0.46 1.00 0.57
≥11 000 (=96) 1.2 (0.8-­1.8) 1.1 (0.7-­1.8)
Platelets (/mm3)
≤50 000 (n = 48) 1.00 0.87 1.00 0.84
≥50 000 (n = 103) 1.0 (0.7-­1.6) 1.0 (0.7-­1.7)
LDH (n = 93)
≤280 (n = 12) 1.00 0.51 1.00 0.29
≥280 (n = 81) 0.8 (0.4-­1.6) 0.6 (0.3-­1.4)
CSF (n = 105)
Negative (n = 92) 1.00 0.20 1.00 0.20
Positive (n = 13) 1.5 (0.8-­2.9) 1.5 (0.7-­2.9)
Karyotype (n = 125)
Good (n = 61) 1.00 0.05 1.00 0.002
Intermediate (n = 49) 1.2 (0.7-­1.9) 1.3 (0.8-­2.3)
Poor (n = 15) 2.2 (1.2-­4.3) 3.1 (1.6-­6.2)
FLT3-­ITD (n = 127)
Negative (n = 117) 1.00 0.39 1.00 0.16
Positive (n = 10) 1.4 (0.6-­3.0) 1.8 (0.8-­3.9)
NPM1 (n = 122)
Negative (n = 113) 1.00 0.41 1.00 0.43
Positive (n = 9) 1.4 (0.6-­3.0) 1.4 (0.6-­3.3)
*
RUNX1-­RUNX1T1 (n = 125)
Negative (n = 76) 1.00 0.68 1.00 0.48
Positive (n = 49) 0.9 (0.6-­1.4) 0.8 (0.5-­1.4)
Total apoptosis
Apoptosis (Median=46.7)
≤Median (n = 74) 1.00 0.07 1.00 0.05
≥Median (n = 77) 1.4 (0.9-­2.1) 1.5 (0.9-­2.4)
Caspase-­9 (Median=20.1)
≤Median (n = 74) 1.00 0.28 1.00 0.15
≥Median (n = 77) 1.2 (0.8-­1.8) 1.4 (0.9-­2.1)

CI, confidence interval; CSF, cerebrospinal fluid; FLT-3ITD, FMS like tyrosine kinase 3 internal tan-
dem duplication; LDH, lactate dehydrogenase; NPM1, nucleophosmin; WBC, white blood cells.
*
Done by cytogenetics.
 |
6       TYAGI et al.
TA B L E   3   Factors associated with
–event-­free survival and -­overall survival,
based on multivariate analysis Variables
Apoptosis
*
Karyotype (others vs
poor)
CI, confidence interval; HR, hazard ratio.
*
Others include good and intermediate risk group of cytogenetics.
3.2 | Comparison of apoptosis between AML
patients and controls
Annexin-­V expression in blast cells (Figure 1A-­C) of AML patients
was significantly higher than the lymphocytes of PB (P = 0.04) and
BM (P = 0.01) controls at diagnosis (Figure 1E). However, intrinsic
pathway of apoptosis as determined by caspase-­
9 expression in
blast cells (Figure 1D) was not significantly different between AML
patients, PB and BM controls (Figure 1F).
3.3 | Association of baseline patient’s
characteristics with annexin-­V and caspase-­9
Annexin-­V was found to be significantly higher in patients with high
WBC count (P = 0.02), poor-­risk cytogenetics (P = 0.02), the absence
of NPM1 mutation (P = 0.05), and the absence of RUNX1-­RUNX1T1
translocation (P < 0.01). However, no association was observed
between caspase-­9 and any of the baseline patient characteristics
(Table 1).
3.4 | Association of baseline patient’s
characteristics, annexin-­V and caspase-­9 with
survival outcomes
In univariate analysis, poor-­r isk cytogenetics adversely affected
EFS and OS. Higher Annexin-­V expression was significantly as-
sociated with inferior overall survival (P = 0.05) (Figure 2A).
However, no association was found between Annexin-­V and EFS
(Table 2; Figure 2B). No association could be demonstrated be-
tween caspase-­9 with either EFS or OS (Table 2). In multivariate
analysis, karyotype was the only significant factor (P = 0.004)
(Table 3).
4 |  D I S CU S S I O N
Recent studies have demonstrated that the true “Achilles heel” of
leukemia may be exploited by targeting both signal transduction
and apoptotic programs simultaneously (synthetic lethality).16,17
Indeed, apoptosis has been established as a mechanism of antican-
cer defense and several components of the apoptotic machinery
are being currently targeted in clinical trials of leukemia.18 Although
alterations in the apoptotic cascade have been revealed by studies
Event-­free survival Overall survival
HR (95% CI) P value HR (95% CI) P value
1.2 (0.8-­1.8) 0.43 1.3 (0.8-­2.1) 0.32
1.9 (1.0-­3.6) 0.04 2.6 (1.3-­4.9) 0.004
on the genomic landscape of AML, several questions remain unan-
swered; which apoptotic circuitry is activated (intrinsic or extrinsic)?
Can there be some predictive biomarker for targeting apoptosis in
AML? Can some reliable prognostic biomarker be identified related
to these cascades?
We envisaged to explore these points and got some key find-
ings. Firstly, Annexin-­V (a marker of total apoptosis) was signifi-
cantly overexpressed in baseline BM of AML patients as compared
to BM or PB of the matched controls. Increased apoptosis defi-
nitely gives a survival advantage to leukemic cells; it generates
vacant niches that potentially become repopulated by more ag-
gressive subclones. Thus, apoptosis increases proliferative pres-
sure, promotes clonal selection, and drives tumor evolution.18
However, caspase-­9, the key component of intrinsic pathway, was
not overexpressed in the blast of AML patients. This indirect clue
suggests that the extrinsic pathway might be more active in AML,
a less clear fact that needs validation in large studies. Secondly,
Annexin-­V was overexpressed significantly in patients with poor
prognostic factors such as poor cytogenetics, high WBC count,
lower expression of RUNX1-­RUNX1T1 and NPM1, which appears
quite logical. Thirdly, Annexin-­V over expression was significantly
associated with inferior OS in univariate analysis although not in
multivariate analysis.
Hess et al6 in their study using reverse transcriptase multiplex
ligation-­dependent probe amplification (RT-­MLPA) of several pro
and antiapoptotic genes, validated a three gene expression signature
(BIRC3, BAX-­(1) and BMF) which predicted OS. In another study,
Kohler et al19 concluded that high levels of Bax and Bad correlate
with poor outcome, particularly in patients who do not enter CR and
may serve as prognostic markers in AML.19 Another study by our
group also suggested that high BAX/BCL2 RMFI ratio was positively
associated with CR rates in AML. 20 It is interesting to note that com-
ponents of the intrinsic cascade upstream of the mitochondria seem
to be upregulated while caspase-­9 (the final components of the in-
trinsic cascade) is neither overexpressed nor prognostic for survival.
In a recent study by Singh et al, 21 the apoptotic index (AI) at base-
line was reported to be significantly associated with poor prognostic
features of pediatric ALL. We also observed a similar association of
total apoptosis at baseline with features of a high-­risk phenotype for
pediatric AML.
Our study is unique because for the first time, we have seen the
association of apoptosis at baseline directly with survival. Although
apoptosis had a significant association with OS in univariate analysis
TYAGI et al.
it could not retain its effect in multivariate model. We hypothesized
that the interaction of karyotype (a strong prognostic factor in AML)
is the reason for this effect. Larger studies would be beneficial in
elucidating the exact relationship.
As more and more newer molecules targeting different compo-
nents of the apoptotic circuitry are making moves into early phase
clinical trials of AML, a disease which is molecularly highly heteroge-
neous, it becomes all the more essential to select out patients who
have specific defects in the apoptotic machinery. It is also import-
ant to identify which component (intrinsic or extrinsic) is activated
in a particular patient, so that we may have appropriately enriched
dose expansion cohorts during these clinical trials. Markers such as
Annexin-­V can thus be simple enrichments strategies.
To conclude, apoptosis as a whole was found to be activated in
baseline BM samples of AML patients. High apoptosis may be as-
sociated with high-­risk phenotype in this disease and the extrinsic
pathway may actually be overexpressed in these clones, which is
hypothesis generating.
AC K N OW L E D G E M E N T S
We thank our nursing staff, data entry operator, patients, and their par-
ents who participated in the study. We also acknowledge the follow-
ing funding sources: (a) Department of Biotechnology, Government
of India under grant (Number: BT/PR7197/MED/30/899/2012
Dated: 26 September 2013) (b) AIIMS, Institutional Junior Research
Fellowship grant.
C O N FL I C T O F I N T E R E S T
All authors declare no conflict of interests.
AU T H O R S C O N T R I B U T I O N
AT, RP, RB, and SB: contribution to design the research study, ac-
quisition of data, interpretation of data, drafting the manuscript and
critical review the intellectual content of the manuscript; SV: was the
statistician. The final draft was approved by all.
ORCID
Sameer Bakhshi  http://orcid.org/0000-0001-9367-4407
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How to cite this article: Tyagi A, Pramanik R, Bakhshi R,
Vishnubhatla S, Bakhshi S. Apoptosis: A biomarker of
high-­risk phenotype in pediatric acute myeloid leukemia? Int
J Lab Hematol. 2018;00:1–7. https://doi.org/10.1111/
ijlh.12939