Molecular characteristics and chromatin texture
features in acute promyelocytic leukemia
Mariana R. B. De Mello1, Dulcineia M. Albuquerque2, Fernanda Gonçalves Pereira-Cunha2, Krizzia B Albanez3,
Katia B. B. Pagnano2, Fernando F. Costa1, Konradin Metze3and Irene Lorand-Metze2*
Background: Acute promyelocytic leukemia is a cytogenetically well defined entity. Nevertheless, some features
observed at diagnosis are related to a worse outcome of the patients.
Methods: In a prospective study, we analyzed peripheral (PB) leukocyte count, immunophenotype, methylation
status of CDKN2B, CDKN2A and TP73; FLT3 and NPM1 mutations besides nuclear chromatin texture characteristics
of the leukemic cells. We also examined the relation of these features with patient’s outcome.
Results: Among 19 cases, 4 had a microgranular morphology, 7 presented PB leukocytes >10x109/l, 2 had FLT3-ITD
and 3 had FLT3-TKD (all three presenting a methylated CDKN2B). NPM1 mutation was not observed. PB leukocyte
count showed an inverse relation with standard deviation of gray levels, contrast, cluster prominence, and
chromatin fractal dimension (FD). Cases with FLT3-ITD presented a microgranular morphology, PB leukocytosis and
expression of HLA-DR, CD34 and CD11b. Concerning nuclear chromatin texture variables, these cases had a lower
entropy, contrast, cluster prominence and FD, but higher local homogeneity, and R245, in keeping with more
homogeneously distributed chromatin. In the univariate Cox analysis, a higher leukocyte count, FLT3-ITD mutation,
microgranular morphology, methylation of CDKN2B, besides a higher local homogeneity of nuclear chromatin, a
lower chromatin entropy and FD were associated to a worse outcome. All these features lost significance when the
cases were stratified for FLT3-ITD mutation. Methylation status of CDNK2A and TP73 showed no relation to patient’s
Conclusion: in APL, patients with FLT3-ITD mutation show different clinical characteristics and have blasts with a
more homogeneous chromatin texture. Texture analysis demonstrated that FLTD-ITD was accompanied not only by
different cytoplasmic features, but also by a change in chromatin structure in routine cytologic preparations. Yet we
were not able to detect chromatin changes by nuclear texture analysis of patients with the FTLD-TKD or
methylation of specific genes.
Keywords: Promyelocytic leukemia, Prognosis, FLT3-ITD, Chromatin texture
Acute promyelocytic leukemia (APL) is a well character-
ized subtype of acute myeloid leukemia (AML) defined
by a specific cytogenetic alteration of the tyrosine kinase
3 gene [1-5]. In Brazil, APL accounts for about 20% of
the adult patients with de novo AML, which is a higher
proportion than what is found in USA or Europe [1-7].
APL promyelocytes express regularly CD33, CD13 and
CD117, and infrequently HLA-DR and CD34 antigen
. Although the disease is a cytogenetically clearly
defined entity, several clinical and biological features
have shown to be of prognostic importance, such as
presence of the so-called variant (microgranular) morph-
ology of the leukemic cells, high peripheral leukocyte
counts at diagnosis or different RARα fusion partners
[1-3]. A prognostic index based on peripheral leukocyte
and platelet counts has been established by PETHEMA
and GIMEMA Groups and validated also in brazilian
patients . Whereas in other AML subtypes, cytogenetic
* Correspondence: email@example.com
2Hematology/Hemotherapy Center, State University of Campinas, Rua Carlos
Chagas 480, 13083-878 Campinas, SP, Brazil
Full list of author information is available at the end of the article
© 2012 Mello et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Mello et al. Diagnostic Pathology 2012, 7:75
alterations and specific gene mutations are relevant for
patients’ outcome, the prognostic relevance of additional
karyotype abnormalities or gene mutations in APL
patients are still controversial [1-3,6,7].
In APL, two mutations of the fms-related tyrosine kin-
ase 3 gene (FLT3) are more frequent: the internal tan-
dem duplications (FLT3-ITD) of the justamembrane
region is found in 12-38% of the cases and the missense
point mutations involving mainly the D835/I836 resi-
dues of the second tyrosine kinase domain (FLT3-TKD)
are observed in 2-20% of the patients  .
FLT3-ITD has been associated with high peripheral
leukocyte counts at diagnosis and a lower disease-free
survival [2,3,9,10]. The association of the FLT3-TKD
mutation with the outcome of APL patients has been
reported in only few studies, but with controversial
Epigenetic mechanisms play an important role in the
pathogenesis of acute leukemias [11-19]. They are im-
portant for DNA stability as well as for the activation of
several pathways of cell cycle regulation and apoptosis.
Recent studies indicate that DNA methylation status
and post-translational modifications of histones may be
at least as important as gene mutations and deletions for
the activation of oncogenes and silencing of tumor sup-
pressor genes [12-14]. Methylation of specific genes
involved in cell cycle control such as CDKN2B (p15),
TP73 (p73) and ESR1 (ER) have been studied in AML
and MDS [13,15,19]. Hypermethylation of CDKN2B has
been associated with a poorer prognosis of the patients.
There are only few investigations about the interaction
between molecular alterations and DNA methylation
profile in APL.
The interaction of genetic and epigenetic mechanisms
leads to chromatin remodelling which may be measured
in an objetive way by analysis of the nuclear chromatin
texture in routinely stained slides. It has been demon-
strated that in Giemsa-stained cells, the deeply stained
heterochromatin domains correspond to the methyl-rich
regions of CpG islands . Therefore, the chromatin
methylation pattern may be evaluated by computer-
assisted analysis of the nuclear texture in cytological pre-
parations. This principle has been applied to routine
histological and cytological material of several solid
tumors and hematologic neoplasias including AML, dis-
closing the prognostic importance of a variety of features
of quantitative analysis of the nuclear chromatin pattern
Special attention has always been drawn to cytoplasmic
features of the APL blasts. To our knowledge, however, a
nuclear texture analysis has never been performed in this
type of AML. Thus, the aim of our study was to examine
the relation among clinical and molecular features, more
precisely, the relation between alterations in the FLT3
gene, methylation of specific genes, nuclear chromatin
texture characteristics and outcome in APL patients.
The study included all consecutive new cases of de novo
APL diagnosed at the Hematology and Hemotherapy
Center of Campinas between 2007 and 2009. Peripheral
blood (PB) counts, bone marrow (BM) examination, cyto-
genetics, immunophenotyping, texture analysis of nuclear
chromatin, methylation of CDKN2B, CDKN2A and TP73
genes as well as mutations in FLT3 were performed at
diagnosis. According to morphology, cases were divided
into those with the classical, hypergranular morphology
(Figure 1A) and cases that showed predominantly a
Figure 1 Bone marrow smears of cases of APL. A - classical
morphology: the leukemic cells present a folded nucleus and a
broad and hypergranular cytoplasm. Several cells present Auer rods
(upper left and lower middle). B – variant morphology: the
neoplastic cells present an oval or bilobated nucleus and few small
granula in the less abundant cytoplasm. May-Grünwald-Giemsa.
Mello et al. Diagnostic Pathology 2012, 7:75
Page 2 of 8
bilobated nuclear shape (Figure 1B) and few small granula
in a less abundant cytoplasm (microgranular or variant
All the patients were treated by the modified AIDA
protocol [30,31]. Overall survival (OS) of the patients
was calculated from the date of diagnosis to the date of
death or last follow-up.
This study was approved by the Ethics Committee of
Faculty of Medical Sciences of the University of Campinas
(proc nr 389/2007).
A two-step tree color platform as described by Pereira et al
 was used. The screening panel comprised three anti-
body combinations: CD3/CD19/CD45; CD7/CD13/CD45
and HLA-DR/CD33/CD45. If leukemic blasts expressed
CD13 and/or CD33, the study was complemented with the
combinations CD11b/CD14/CD45; CD15/CD34/CD45 and
cMPO/CD117/CD45. For each sample at least 10,000
events were acquired on a FACs CaliburTMequipment
(Becton-Dickinson, San Jose – California -USA) with the
Cell-QuestTM(BD) software. Quantitative analysis was per-
formed using the Paint-a-GateTMsoftware (BD).
Bone marrow slides at diagnosis, stained with May-
Grünwald-Giemsa were retrieved from the files. Nuclear
chromatin texture analysis was performed on at least
100 randomly choosen, non-overlapping tumor nuclei
per patient using the Leica DC 500 system (ocular lens
10x and objective 100x, oil immersion). Neoplastic cells
were acquired in 24-bit color bitmap format (12 mega-
pixels per image) The nuclei were interactively segmen-
ted and then converted to 8 bit gray scale with levels of
luminance ranging between 0 and 255.
We analyzed features of geometric morphometry (nu-
clear area, form factor 8, gray level and its standard devi-
ation) and variables of the gray level co-occurrence
matrix (entropy, contrast, local homogeneity and cluster
prominence) [28,29]. We also determined the fractal di-
mension (FD) of the nuclear chromatin according to the
Minkowski-Bouligand method extended to pseudo-3D
images [25-28]. We performed a pseudo-3D transform-
ation of the images, considering the x and y coordinates
as the position of the pixel and the z axis was its grey
level . The FD of this surface was calculated using an
in-house developed software. The goodness of fit of the
FD (the adaptation of the real FD to the ideal one)
was also determined, using a linear regression. The
point distribution of the slope was rotated to 45
degrees as previously described . The coefficient
of the regression between the real and the estimated
values was calculated. The R2value obtained is an es-
timate of the “quality of fractality”. An ideal fractal
has a R2=1.0. The real fractals have R2values <1.0
For methylation studies, DNA samples were treated with
sodium bisulfite . Reactions for CDKN2A and TP73
genes were performed by methylation specific PCR
(MSP), and combined bisulfate restriction analysis
(COBRA) was used to study the methylation status of
CDKN2B gene. Primer sequences and temperature
anneling are shown in Table 1. After amplification, the
PCR products were digested with specific enzymes
(Table 1) and the products were separated on 3% agarose
gel stained with ethidium bromide.
Dicionário - Ver dicionário detalhado
The genotyping technique was used to study muta-
tions in FLT3-ITD. MegaBACE 1000 equipment (GE
Healthcare - Amersham) was used and analysis was
made with the Fragment Profiler v1.2 software. In brief,
0.5 μl DNA was amplified in a volume of 50 μl contain-
ing 120 mm FLT3 sense primer labeled with HEX fluor-
ophore (Table 2) and 120 mm FLT3 antisense primer
unmarked; 10X buffer, 3 mM MgCl2, 0.2 mM dNTP's,
0.05 U Taq polymerase and water. It consisted of an ini-
tial denaturation step at 94°C for 10 minutes followed by
35 cycles at 94°C for 30 seconds, 56°C for 1 minute, and
72°C for 2 minutes and a final extension step at 72°C for
7 minutes. PCR products were diluted 40x in water.
Then, 2 μl of each dilution was distributed in plates with
Table 1 Sequences of primers and conditions used
TP73 U AGGGGATGTAGTGAAATTGGGGTTT60-
Mello et al. Diagnostic Pathology 2012, 7:75
Page 3 of 8
96 wells previously prepared with a mixture containing
7.75 μl Genotyping Loading Solution (0.1% Tween 20 in
H2O)+0.25 μl of MegaBACE ET550-R Size Standards,
making a final volume of 10 μl. Samples were denatured
at 95°C for 5 minutes before being placed on the
PCR for the detection of FLT3-TKD mutation was per-
formed using 0.025U Taq DNA polymerase, 200 nM of
each primer (Table 2), 0.2 mM dNTP's, 2 mM MgCl2,
10x buffer and water. Cycling conditions: 94°C for 5 for
minutes, 40 cycles of 94°C for for 1 minute, 55°C for 40
seconds, 72°C for 30 seconds and final extension at 72°C
for 5 minutes. After amplification PCR products were
digested with enzyme EcoRV and products were sepa-
rated by electrophoresis on 3% agarose gel stained with
The differences between groups were examined by the
Mann–Whitney test. Correlations were examined by the
Spearman rank order correlation. The differences were
considered significant when p<0.05. The relations be-
tween clinical, phenotypic, morphometric and molecular
features with OS of the patients were analysed using the
Cox proportional hazard regression. Because of the small
number of events we did not perform a multivariate
analysis. Instead, we compared the univariate Cox-
regressions for each of the variables studied before
and after stratification for the presence of FLT3-ITD.
Winstat 3.1. and SPSS 15 programs were used. The
exact confidence intervals were calculated with the
free graph pad software (t http://www.graphpad.com/
We detected 19 cases of APL in a cohort of 106 con-
secutive cases of de novo AMLs (17.9%), diagnosed at
our Institution during the period of the study. Among
them, 10 patients were diagnosed by classical cytogenet-
ics: 8 cases had only t(15;17); 1 had t(4;6), t (15;17); 1
had del(7), t(15;17). Six cases were diagnosed only by
molecular diagnostics (PML/RARα) and in 3 cases the
diagnosis was made only by morphologic criteria, since
metaphases could not be achieved for cytogenetic
Clinical data and PB counts are shown in Table 3.
Patients had a median age of 44 years. PB leukocyte
counts were >10x109/l in 7 cases. Concerning the
immunophenotypic features, 2 cases showed an expres-
sion of HLA-DR (mean fluorescence intensity – MFI -
of 100 and 141) and four had a dim expression of CD34
(mean MFI of 47). Four cases presented microgranular
morphology; all of them presented a high PB leukocyte
count (Table 4). Concerning nuclear texture features,
these cases had higher local homogeneity, contrast and
R2values, but a lower standard deviation of gray levels,
entropy, cluster prominence, and fractal dimension.
Peripheral leukocyte count at diagnosis had no relation
with phenotypic features, but showed an inverse correlation
with standard deviation of gray levels (r=−0.49; p=0.03),
contrast (r=−0.48; p=0.03), cluster prominence (r=−0.57;
p=0.01) and fractal dimension (r=−0.46; p=0.04).
FLT3-TKD mutation was found in 3 cases (15.8%; 95%
confidence interval from 0.0478 to 0.384) and FLT3-ITD
was present in 2 cases (10.5%; 95% confidence interval
0.013 - 0.331). All patients presenting FLT3-TKD had
also a methylated CDKN2B gene. This gene was methy-
lated in only one patient with no abnormalities in FLT3.
CDKN2A was methylated in 21.0% (95% confidence
interval 0.0605 - 0.4557) and TP73 in 10.5% of the
patients (95% confidence interval 0.0130 - 0.3314).
The leukemic cells of patients with FLT3-ITD expressed
HLA-DR, CD34 and CD11b (Table 5). They also had
higher PB leukocyte counts. Both presented a variant
morphology. Concerning nuclear texture features (Table 6),
these patients presented a lower standard deviation of grey
levels, entropy, contrast, cluster prominence and the fractal
dimension (FD), but higher values for local homogeneity,
and the goodness-of-fit of the FD.
During the period of the study (median 23 months;
1 – 41 months) 5 patients had died, four of them within
the first month of diagnosis. All these four had a high
leukocyte count, and presented microgranular morph-
ology or had FLT3-ITD. Using the Cox model, in the uni-
variate analysis a higher leukocyte count (B=+0.032;
p=0.038), presence of FLT3-ITD mutation (B=+2.55;
p=0.01), a microgranular morphology (B=+2.39; p=0.01),
methylation of CDKN2B gene (B=+1.70; p=0.066),
expression of HLA-DR (B=+0.017; p=0.025) and CD34
(B=+0.028; p=0.077) as well as a higher local homogeneity
of nuclear chromatin of the leukemic cells (B=+54.24;
Table 3 Clinical and hematological data of the patients
Age (years) 44 (20–82)
Table 2 Sequence of primers and enzymes used in FLT3
Mello et al. Diagnostic Pathology 2012, 7:75
Page 4 of 8
p=0.035) were associated with a shorter overall survival.
However, higher values of standard deviation of gray level
(B=−1.28; p=0.025), of entropy (B=−3.63; p=0.031) and
chromatin fractal dimension (B=−85.7; p=0.036) were
associated to a longer survival. All these values lost
significance when the univariate Cox regressions were
stratified by presence or absence of the FLT3-ITD muta-
tion. Methylation status of CDNK2A and TP73 showed no
relation with the survival of the patients.
The outcome of patients with APL has improved over
the last 2 decades as a consequence of a precise molecu-
lar diagnosis, better supportive care as well as the use of
trans-retinoic acid (ATRA) and arsenic trioxide in
chemotherapy protocols [30,34]. Nevertheless, 20% of
the patients still die during the first month of treatment,
mainly due to bleeding. So, it would be interesting to de-
tect the clinical and laboratory features that could pre-
dict a shorter overall as well as disease-free survival.
Peripheral leukocyte counts above 10x109/l and a micro-
granular morphology have been recognized as such
variables [2-5]. Additional chromosomal abnormalities,
however, do not seem to influence the outcome of the
patients [6,7]. Therefore, it would be interesting to study
the relation of these features with molecular characetris-
tics of this disease. Nuclear texture features of neoplastic
cells have shown to be morphological correlates of chro-
matin remodelling with genetic and epigenetic altera-
tions in acute leukemias [12-17], and have shown to be
related to patients’ survival. Recently, computerized
image analysis has been used to examine chromatin re-
modeling in several hematological neoplasms and tex-
ture variables of the nuclear architecture have shown to
be independent prognostic factors in acute leukemias
In the present study, in a rather small cohort of
patients, diagnosed and treated according to the inter-
national standard of care, we could observe a mortality
rate of 21% during the first month of treatment which
was similar to that observed in other countries [4,30,34].
Table 5 Hematological and phenotypic features of the
patients according to presence of FLT3-ITD
8.8 (8.0-9.7) 8.7 (3.0-14.1)0.78
63.0 (61.0-65.0)2.0 (0.69-160.0)0.09
27.0 (20.0-34.0) 14.5 (5.0-85.0)0.32
MFI HLA-DR 121.0 (100.8-141.2)0.0 (0.0-25.9)0.0004
MFI CD11b 41.1 (39.8-42.5) 0.0 (0.0-20.1)0.01
MFI CD34 42.9 (35.4-50.4) 0.0 (0.0-63.5)0.009
Table 6 Morphometric features of patients with APL
according to FLT3-ITD
Leukocyte count x109/l 63.0 (61.0 – 65.0)
Nuclear area (μ2) 100.9 (86.6-115.3)
2.0 (0.7 – 160.0) 0.04
105.4 (83.4-126.9) 0.63
Form factor1.07 (1.02 - 1.12)0.97 (0.80 - 1.12) 0.07
Mean gray level108.2 (106 – 109) 129.7 (102 – 158) 0.028
Standard deviation of
6.3 (6.2-6.5) 8.7 (7.4-10.4)0.026
Local homogeneity 0.60 (0.59-0.61)0.55 (0.53-0.58)0.026
Entropy 6.95 (6.90-7.01)7.74 (7.29-8.21)0.026
Cluster Prominence (x105)
2.59 (2.29-2.89) 3.69 (2.87-4.83) 0.04
2.09 (1.97-2.2.20) 5.04 (2.46-11.94)0.026
2.10 (2.09-2.10)2.12 (2.10-2.14) 0.05
Table 4 Peripheral leukocyte counts and morphometric features of patients with APL according to the morphological
type (median and range)
Leukocyte count x109/l 63.0 (24.0 – 160.0)
Nuclear area (μ2) 86.5 (83.4 – 115.3)
1.75 (0.69 – 68.0) 0.007
113.4 (87.1 – 115.3) 0.15
Form factor 1.11 (1.02 - 1.12)0.97 (0.80 - 1.12)0.03
Mean gray level109.7 (106 – 110)129.7 (102 – 158)0.012
SD gray level * 6.4 (6.2 - 7.6)8.7 (7.3 - 10.4) 0.018
Local homogeneity0.59 (0.58 - 0.61) 0.55 (0.53 - 0.58)0.008
Entropy7.01 (6.89 – 7.32) 7.76 (7.29 – 8.21)0.01
Cluster Prominence (x105)
2.88 (2.29 - 3.15)2.59 (2.29 - 2.89)0.02
2.20 (1.97 - 4.48) 5.29 (2.26 – 11.94) 0.05
2.09 (2.08 - 2.10) 2.11 (2.10 - 2.13) 0.01
0.99910 (0.99900 - 0.99940)0.99885 (0.99802 - 0.99910)0.02
* Standard deviation of gray level.
Mello et al. Diagnostic Pathology 2012, 7:75
Page 5 of 8
We could also confirm the prognostic value of periph-
eral leukocyte counts and microgranular morphology.
More recently, several investigations have focused on the
impact of mutations of the FLT3 gene in APL [2,3,5,7,9-
11,35] that has been a well recognized adverse prognostic
factor in AML with a normal karyotype. In APL, the find-
ing of FLT3-ITD has been associated with increased periph-
eral leukocyte counts, higher early mortality and more
frequent recurrences. The presence of FLT3-ITD in APL is
also associated with a different expression of 147 genes
involved in cytoskeleton organization, cell proliferation and
migration, adhesion, as well as the coagulation and inflama-
tion pathways . Thus, it is linked to a more aggressive
clinical behaviour with higher peripheral leukocytosis and a
more pronounced coagulopathy, provoking a higher rate of
early death, mainly due to bleeding. The higher leukocytosis
is also indicative of a more pronounced proliferative activ-
ity. Our study also corroborated these findings. FLT3-ITD
was a significant adverse prognostic factor for survival in
the univariate Cox-regression. Furthermore, other variables,
such as peripheral leukocyte count, microgranular morph-
ology and texture features of nuclear chromatin were sig-
nificant prognostic factors only when examined isolately,
but lost their importance when the regressions were strati-
fied for the presence of FLT3-ITD. From these findings we
may hypothesize that the presence of the FLT3-ITD muta-
tion might be more important for a dismal outcome than
leukocytosis alone or a microgranular morphology. Add-
itional studies with larger number of patients, however, are
necessary to clarify this question.
Neither the FLT3-TKD mutation nor the methylation
of CDNK2B, were unfavourable prognostic features in
our investigation. Yet, a possible role of CDNK2B for the
outcome of patients with other types of acute leukemias
has been postulated . Bit this could not be confirmed
for APL in the present study, perhaps due to the reduced
test power caused by the small cohort of patients.
Recently, the importance of epigenetic changes for
normal hemopoietic maturation as well as in the
pathogenesis of AML has been emphasized [14,17-
19]. Hypermethylation of CpG islands within the pro-
moter regions together with deacetylation of certain
histones is an important mechanism of gene silencing in
hematologic neoplasias. Several studies have shown that
APL is generally associated with a specific methylation
pattern, but some genes, such as CDKN2B, may be
methylated isolately in only a part of the patients [15,19].
Patients with the FLT3-ITD mutation show a gene expres-
sion profile where various genes relevant for cytoskeleton
organization, cell adhesion and migration, proliferation and
coagulation pathways are expressed .
MGG staining of cytologic smears permits to evalu-
ate the topographic localization of methylated regions
in the nucleus, since deeply Giemsa-stained compact
heterochromatin domains are co-localized with methyl-
rich CPG islands . Computer-assisted image ana-
lysis is able to document discrete changes in the
chromatin structure, which may not be visible for the
trained observer [38-43]. Discrete morphologic altera-
tions of the nucleus and its substructures accompany
functional and molecular changes of the cell. This
is also true for benign and malignant hemopoesis
High order chromatin architecture alterations may par-
allel chromosomal alterations in cancer [44,45]. Moreover,
disrcrete morphologic changes of the chromatin texture
may be equivalent to alterations of the methylation pat-
tern and therefore of the gene expression. Probably for
these reasons computerized texture analysis of chromatin
has shown to be helpful for diagnosis as well as for prog-
nosis of several neoplasias including AML [16-18,23-
28,38-43]. In particular, a fractal model of nuclear
chromatin has been associated with cellular activity, the
organization of nuclear chromatin and the surrounding
nucleoplasmic space, especially the distribution of hetero-
cromatin, and with prognosis [16,25-29,46].
In our study several variables of geometric morphom-
etry, the gray-level co-ocurrence matrix and also R2
showed different values in leukemic cells of cases with and
without the FLT3-ITD mutation. In patients presenting
FLT3-ITD, cells had a smoother and more homogeneously
distributed chromatin which would more likely corres-
pond to profound alterations of the epigenome. The pres-
ence of FLT3-ITD has shown to be associated with a
higher proliferative activity and a more undifferentiated
phenotype . This is in keeping with the different chro-
matin texture features found in our patients as well as the
higher PB leukocytosis.
Several clinical, phenotypical and nuclear chromatin
texture features were associated to the survival of our
patients. However, when stratified for the presence of
FLT3-ITD, all of them lost their prognostic value. In that
way, our explorative study indicated that this mutation
is a very important prognostic factor in APL. This is in
keeping with the concept of a previous study that APL
with FLT3-ITD mutation represents a distinct subtype of
APL with a worse prognosis .
In conclusion, our work underlines the necessity of
the detection of the FLT3-ITD mutation in APL, that
constitutes a separate entity of a worse prognosis and
where new treatment strategies are necessary in order to
decrease early mortality in an otherwise highly curable
form of acute leukemia. Texture analysis demonstrated
that the FLTD-ITD alterations were accompanied not
only by different cytoplasmic features, but also by a
change in the chromatin structure in routine cytologic
preparations. Yet we were not able to detect chromatin
changes by nuclear texture analysis of patients with the
Mello et al. Diagnostic Pathology 2012, 7:75
Page 6 of 8
FLTD-TKD mutation or methylation of specific cell
The authors have no conflicts of interests to declare.
MRBM collected the clinical data, contributed to the study design, performed
all the molecular studies and wrote the manuscript draft. The results are a
part of her PhD thesis, supervised by Irene Lorand-Metze (Postgraduate
Course in Medical Pathophysiology, University of Campinas). DMA trained
and supervised MRBM in the molecular techniques, and helped in the data
interpretation. FGPC performed the diagnostic immunophenotyping of the
patients and helped in the interpretantion of the data. KBA performed the
acquisition of all nuclear images and made their segmentation. KBBP made
the diagnostic molecular study and treated the patients. FFC supervised the
molecular studies. KM contributed to essential parts of the the study design,
performed the statistical analysis, interprettion of the results, wrote essential
parts of the manuscript and made a critical review of the final version. ILM:
conception and study design. Supervision of the collection of the data,
microscopic analysis, interpretation of the data, manuscript draft and final
approval. All authors have read and approved the manuscript.
This work was supported by a grant from FAPESP (São Paulo Research
Foundation - project 2007/54686-0), Irene Lorand-Metze, Konradin Metze and
Fernando F Costa are researchers of the National Council of Technological
and Scientific Development (CNPq - projects 302277/2009-9, 307270/2010-6
and 402022/2010-6 respectively). We thank Randall L Adam for kind support
in the morphometry analysis.
1Department of Internal Medicine, Faculty of Medical Sciences, State
University of Campinas, Rua Tessalia Vieira de Camargo 126, 13083-887
Campinas, Brazil.2Hematology/Hemotherapy Center, State University of
Campinas, Rua Carlos Chagas 480, 13083-878 Campinas, SP, Brazil.
3Department of Pathology, Faculty of Medical Sciences, State University of
Campinas, Rua Tessalia Vieira de Camargo 126, 13083-887 Campinas, Brazil.
Received: 4 April 2012 Accepted: 21 May 2012
Published: 28 June 2012
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Cite this article as: Mello et al.: Molecular characteristics and chromatin
texture features in acute promyelocytic leukemia. Diagnostic Pathology
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