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Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant relapses in patients with acute myeloid leukemias

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  • First Pavlov Saint-Petersburg Medical University
Article

Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant relapses in patients with acute myeloid leukemias

Abstract and Figures

The presented data are showing the crucial role and great prognostic significance of BAALC-expressing leukemic precursors in origin and development of posttransplant relapses (PTR) in acute myeloid leukemia patients with different cytological variants. For evidence simultaneous serial measurements of BAALC and WT1 transcript copy numbers by means of quantitative real time polymerase chain reaction were used at diagnosis, before conditioning regimen as well as at PTR in 50 patients treated with allogeneic hematopoietic stem cell transplantation (alloHSCT). Thirty-eight of them were adults and twelve pediatric patients aged 1-60 years (median – 25.8 years). It was shown, that BAALC gene overexpression to be presented in all studied cytological and being combined with increased level of WT1 expression at PTR in most of them. This combination was prognostically poor since it is largely associated with increased cumulative incidence of PTR (p<0.0001), and shortened event free (p<0.0001) and overall survival (p=0.002). Keywords: acute myeloid leukemia, hematopoietic stem cell transplantation, posttransplant relapse, BAALC, WT1, combined overexpression
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Abbreviations: allo HSCT, allogeneic hematopoietic stem cell
transplantation; AML, acute myeloid leukemia; PTR, posttransplant
relapses; PCR, polymerase chain reaction; qRT-PCR, quantitative real
time PCR; BAALC, Brain and Acute Leukemia, Cytoplasmic gene;
WT1, Wilm’s 1 tumor gene; FAB, French-American-British; M1-
M7, FAB variants of AML; OS, overall survival; RFS, relapse-free-
survival; CIR, cumulative incidence relapses
Introduction
Despite the obvious advances in understanding of acute myeloid
leukemia (AML) pathogenesis and successful treatment of some AML
variants (e. g. M3), this problem has not been resolved even with the
support of hematopoietic stem cell transplantation (HSCT). The main
reason of HSCT failure is often posttransplant relapse (PTR) which
can be provided by some leukemic precursors.1
The rst step toward the elucidation of basic mechanisms of PTR
origin was made by Italian investigators, who developed a method
of highly sensitive quantitative evaluation of WT1-expressing blast
elements burdens based on real-time PCR2,3 and successfully tested it at
clinics.4,5 The second step has been done in this direction after opening
and clinical investigation of Brain and Acute Leukemia, Cytoplasmic
gene, e.g. BAALC. This gene was discovered to be expressed in the
cytoplasm of neuroectoderm-derived tissues, especially in neurons6 as
well as in bone marrow CD34+ cells.7 It is localized on the long arm
of chromosome 8 and is overexpressed in 40-50% of AML patients.8,9
First evidence has recently been obtained that specic leukemic
precursors with immunophenotype CD34+CD38- are responsible
for the high BAALC mRNA production in patient’s bone marrow.
10 The number of such cells as well as the BAALC gene expression
level were the highest in patients with M0, M1 and M2 AML FAB
variants,11–13 but they were the lowest in M3 FAB variant,14,15 that may
be explained by different types of leukemia initiating cells (LICs).
The latter were discovered after elegant experiments with successful
engrafting previously sorted leukemic precursors in immunodecient
mice.16,17 As a result, at least two kind of LICs immature and mature
were recognized. The rst were more characteristic for M0, M1,
M2 and M4 FAB variants of AML,10 whereas the second one was
responsible for hematopoiesis in APL.18 Meanwhile, immature LICs
reveal immunophenotype CD34+/CD38- being capable to selective
expression of pan-specic molecular marker BAALC.10 As for mature
LICs, they are responsible for leukemic hematopoiesis in APL and
along with blasts can express another pan-specic molecular marker
WT1. First of all, this assumption is based on many cases of AML
with higher levels of WT1 gene expression and lower number of
blasts in tested samples of bone marrow as before, so after HSCT
performing.2,4,19-21 Secondly, it was recently shown that levels of WT1
expression in some patients with APL could be highest in AML group,
whereas where as the number of blasts in the tested bone marrow
samples could be lower 30 %.19,22 Since levels of BAALC and WT1
expressions may be successfully determined by means of standard
quantitative PCR in real time, a possibility appears to measure in such
a way relative burden of BAALC-expression and less correctly (due to
blasts) WT1–expressing relatively mature LICs.16–18
Developing this idea, we have recently showed the crucial role
of BAALC-expressing leukemic precursors in origin and development
of PTR.19,22 The basic evidence was obtained on the group of AML
patients (n=12), who had so called false remission just prior to
conditioning and HSCT. As a result, BAALC gene overexpression was
found in 3/12 (25%) patients in this group, that was associated with
an increased incidence of PTR in all of them (p=0.002) as well as
shortened event free survival (EFS) (p=0.019). When this molecular
indicator of paired overexpression of BAALC and WT1 genes was
Hematol Transfus Int J. 2020;8(6):127131. 127
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Crucial role of BAALC-expressing leukemic
precursors in origin and development of
posttransplant relapses in patients with acute
myeloid leukemias
Volume 8 Issue 6 - 2020
Nikolay N Mamaev, Alyena I Shakirova, Ildar
M Barkhatov, Yana V Gudozhnikova, Tatiana L
Gindina, Mikhail M Kanunnikov, Valentina M
Kravtsova, Zhamal Z Rakhmanova, Olesya V
Paina, Lyudmila S Zubarovskaya
Raisa Gorbacheva Memorial Research Institute for Pediatric
Oncology, Hematology and Transplantation at the Pavlov First
Saint Petersburg State Medical University, Russia
Correspondence: Nikolay N Mamaev, Raisa Gorbacheva
Memorial Research Institute for Pediatric Oncology,
Hematology and Transplantation at the Pavlov First Saint
Petersburg State Medical University, 197022, Roentgena St., 12.
St. Petersburg, Russian Federation, Russia,
Email
Received: December 05, 2020 | Published: December 31,
2020
Abstract
The presented data are showing the crucial role and great prognostic signicance of BAALC-
expressing leukemic precursors in origin and development of posttransplant relapses
(PTR) in acute myeloid leukemia patients with different cytological variants. For evidence
simultaneous serial measurements of BAALC and WT1 transcript copy numbers by means of
quantitative real time polymerase chain reaction were used at diagnosis, before conditioning
regimen as well as at PTR in 50 patients treated with allogeneic hematopoietic stem cell
transplantation (alloHSCT). Thirty-eight of them were adults and twelve pediatric patients
aged 1-60 years (median – 25.8 years). It was shown, that BAALC gene overexpression to
be presented in all studied cytological and being combined with increased level of WT1
expression at PTR in most of them. This combination was prognostically poor since it is
largely associated with increased cumulative incidence of PTR (p<0.0001), and shortened
event free (p<0.0001) and overall survival (p=0.002).
Keywords: acute myeloid leukemia, hematopoietic stem cell transplantation,
posttransplant relapse, BAALC, WT1, combined overexpression
Hematology & Transfusion International Journal
Research Article Open Access
Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant
relapses in patients with acute myeloid leukemias 128
Copyright:
©2020 Mamaev et al.
Citation: Mamaev NN, Shakirova AI, Barkhatov IM et al. Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant
relapses in patients with acute myeloid leukemias. Hematol Transfus Int J. 2020;8(6):127131. DOI: 10.15406/htij.2020.08.00240
tested in a larger AML patient cohort (n=110) with various cytological
and cytogenetic variants, we revealed the important role of BAALC-
producing precursors in origin and further development of PTR in
patients with M1, M2, M4, and M5 FAB variants.18,19,22 Additionally,
similar data have been obtained also in patients APL with PTR which
will be discussed here in details too.
Patients and methods
Patient cohort
Our retrospective study presents the data of BAALC and WT1gene
expression levels measured in parallel during serial sampling of bone
marrow taken from 50 patients diagnosed with AML. All the patients
were treated with alloHSCT at R. Gorbacheva Memorial Research
Institute of Children Oncology, Hematology and Transplantation (St.
Petersburg) from 2010 to 2016 years. The study group included 25
females and 25 males at the age of 1 to 60 years (median age of 25.8
years old). Among them were nine pediatric patients under the age of
17 year. Of notice, 7/50 included AML were EVI1-positive, although
they did not contained in karyotypes any specic aberrations of loci
3q26. In all patients with different AML FAB-variants, serial BAALC
and WT1 gene expression changes were evaluated individually in
combination with counting of blast cell numbers in the same bone
marrow samples. Written informed consent for all individuals was
obtained in accordance with the Declaration of Helsinki.
Analysis of BAALC and WT1 gene expression levels
Total mRNA extraction from fresh bone marrow samples, its
reverse transcription and quantitative estimation of the BAALC gene
expression level by quantitative real-time PCR (qRT-PCR) were
performed as previously described.20 BAALC transcript copy numbers
(CN) were determined using appropriate BAALC RQ-Kit (Inogene,
Russia), including plasmid standards for constructing of calibration
curves for the BAALC gene and reference gene ABL1. Basic control
time points for bone marrow sampling were as follows: at diagnosis
(e.g., D-80), just before alloHSCT, prior to the conditioning (D0) and
after alloHSCT, i.e. on D+30, D+60, D+90, D+150 and later post-
transplant. The bone marrow sampling was obligatory in cases of PTR
occurrence. A median follow-up time after HSCT was 7 months (range
from 0.6 to 52.5). The analysis of BAALC gene expression included
93 samples tested at clinical cytologically proven relapse, and 299
bone marrow aspirates that were taken during clinical remission. The
relative BAALC gene expression level was calculated as a ratio of
CNBAALC to CNABL1 and expressed in percents. The value of 31% was
chosen as a common cut-off value to study clinical signicance of
BAALC gene overexpression before and after HSCT and to perform
molecular monitoring of the interaction between leukemic precursors
and blast cell populations. This value was higher than the maximum
of BAALC gene expression level in patients with pre-transplant
cytological remission, who did not show any clinical signs of the
disease progression.
In parallel, the WT1 gene expression levels were determined in
each sample at the same time points. The copy numbers of WT1
transcripts were determined by the same qRT-PCR method according
to recommendations of European Leukemia Net group.4 The basal
WT1gene expression level of 250 copies per 104 copies of ABL1
reference gene was applied to designate low and high WT1expressors.
Statistical analysis
Descriptive statistic methods for data with asymmetric distribution
were used, with evaluation of the sample ranges and median values.
Two-year relapse-free survival (RFS) and cumulative incidence
of relapse (CIR) were calculated using Kaplan-Meier method, and
the log-rank test was used to compare differences between survival
curves. RFS and CIR were measured from D0 until the date of
death, regardless of cause, or until terms of documented relapse, or
last contact date. P < 0.05 was considered as statistically signicant
difference level. SPSS software version 22.0 (IBM corporation,
Armonk, NY, USA) was used for statistical analysis.
Results
The basic ndings of our study are presented in Table 1. These
data show higher expression BAALC gene closely related to increase
burden of immature LICs to be presented in leukemic population of
all tested FAB-cytological, cytogenetic and molecular variants of
AML In most of them there was combined with higher expression
of both tested genes indicated in the Table 1 by bold/ Therefore,
many of them were transplanted at state of relapse which in turn
could provide both shorter survival of these patients and death. It
should be also noticed the combined BAALC and WT1 genes higher
expression to be presented in cases of APL and AML with inv(16)
which were characterized by aggressive clinical course (#25 and #40,
respectively), whose detail clinical and laboratory details will be
published additionally (Figure 1).
Figure 1 The results of serial measurements of BAALC . WT1, EVI1 and PML/RARa expression along to number blasts counting in the tested bone marrow
samples from treated by HSCT APL patient (#25) at the stage of posttransplant relapse which evidence a participation in relapse formation of BAALC-expressing
immature LICs.
Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant
relapses in patients with acute myeloid leukemias 129
Copyright:
©2020 Mamaev et al.
Citation: Mamaev NN, Shakirova AI, Barkhatov IM et al. Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant
relapses in patients with acute myeloid leukemias. Hematol Transfus Int J. 2020;8(6):127131. DOI: 10.15406/htij.2020.08.00240
Table 1 BAALC and WT1 gene expression levels and other clinical and hematological characteristics of patients at different stages before and after allo HSCT
(only samples with maximal blast cells burden are pointed)
Patient,
FAB-
variant Gender Ag e,
years
Cytogenetic
group
Number
of
HSCT
Disease stage
Post
HSCT
OS
days
Diagnosis D0 PTR
BAALC,
%
WT1,
CN
Blasts,
%
BAALC,
%
WT1,
CN
Blasts,
%
BAALC,
%
WT1,
CN
Blasts,
%
1М1m 18 CK/del(7q) 1 242 13542 93,2 n/d n/d n/d - - - 730*
2М1f 19 t(8;21) + OA 1 370 402 32 n/d n/d n/d - - - 95
3М1m 21 t(8;21) + OA 1 n/d n/d n/d 0,5 379 2.5 329 7858 70 306
4М1f 25 NK 1 n/d n/d n/d 40 1764 13.2 - - - 613
5М1m 26 NK 1 n/d n/d n/d 2 47 2.6 2 35 48 179
6М1 f 26 CK, EVI1+
1130 10493 84,2 n/d n/d n/d 15 488 11,6
692
2125 6500 51,2 n/d n/d n/d
125 6500 51,2
154 2930 22
7М1f 32 NK 1 n/d n/d n/d 21 40 2 37.1 102 7,8 672+
8М1 f 44 CK/del(7q),
del(5q) 1n/d n/d n/d 29 2355 8,8 n/d n/d n/d 45
9М1f 48 n/d 1 17 386 60 n/d n/d n/d 117 281 40 160?
10 М1f 54 NK 1 n/d n/d n/d 32 18439 79,2 77 32981 35,4 140?
11 M1 f 60 NK 1 2619 1728 96,6 3 29 0.6 378 197 37 382
12 М2m 8 t(8;21) + OA 1 321 1236 69,6 22 7 4,2 - - - 469+
13 М2m 9 NK 1 99 10789 21,2 27 4 1,6 - - - 730*
14 М2f 15 t(8;21) + OA 2 89 37 71,2 34 322 9
67 2033 45
730*
133 9929 75,6
15 М2m 15 NK 1 0,6 37 8,8 n/d n/d n/d - - - 730*
16 М2m 28 OA 1n/d n/d n/d n/d n/d n/d 23 5518 24 162
17 М2m 30 t(8;21) + OA 2 4 725 26,8 n/d n/d n/d
153 2363 23
730*
47 2649 28
18 М2m 35 NK 1 n/d n/d n/d 34 4238 5 - - - 20
19 М2f 38 CK/del(7q) 1 n/d n/d n/d 787 8756 59,4 - - - 407
20 М2m 39 NK, EVI1+ 1 n/d n/d n/d n/d n/d n/d 83 18872 26 350
21 М2 f 49 CK/del (5q) 1 n/d n/d n/d 14 4 6,5 n/d n/d n/d 214
22 М2 m 58 t(8;21) + OA 1 n/d n/d n/d 366 26100 45,8 n/d n/d n/d 763
23 M3 f 17 t(15;17), EVI1+ 1 n/d n/d n/d 0.8 2957 1.8 3 32684 22.4 160
24 M3 m 18 t(15;17) 1 0.05 4375 33.6 0.2 421 10 - - - 730*
25 M3 f 45 t(15;17) 1 n/d n/d n/d n/d n/d n/d 90.4 10693 60 730*
26 М4m 5 t(8;21)/ del(7q)
+ OA 195 999 65,8 n/d n/d n/d 35 411 12,6 730*
27 М4m 6 CK/del(5q) 1 982,1 183 54,6 n/d n/d n/d 7118 296 41 779
28 М4f 16 NK 1 34 1479 22,5 n/d n/d n/d - - - 128
29 М4m 17 NK 1 n/d n/d n/d 5 51 3 6 362 7 197
30 М4m 17 NK 1 n/d n/d n/d 2 2258 12 - - - 730*
31 М4f 17 OA 2
52 8043 76 41 3078 0,6 61 7809 51
454
61 7809 51 31 8407 2.4 69 2424 50,8
32 М4m 18 NK 1 n/d n/d n/d 10 836 7,2 - - - 61
Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant
relapses in patients with acute myeloid leukemias 130
Copyright:
©2020 Mamaev et al.
Citation: Mamaev NN, Shakirova AI, Barkhatov IM et al. Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant
relapses in patients with acute myeloid leukemias. Hematol Transfus Int J. 2020;8(6):127131. DOI: 10.15406/htij.2020.08.00240
Patient,
FAB-
variant Gender Ag e,
years
Cytogenetic
group
Number
of
HSCT
Disease stage
Post
HSCT
OS
days
Diagnosis D0 PTR
BAALC,
%
WT1,
CN
Blasts,
%
BAALC,
%
WT1,
CN
Blasts,
%
BAALC,
%
WT1,
CN
Blasts,
%
33 М4m 19 NK 1 27 14929 95 9 1319 25.4 - - - 311
34 М4f 19 inv(16) + OA 1 n/d n/d n/d 25 96 10.6 - - - 370*
35 М4f 21 CK/del(7) EVI1+ 2 107 867 17 n/d n/d n/d
389 529 12
393
400 26036 24
36 М4m 21 NK 1 n/d n/d n/d 39 41 5,4 - - - 518+
37 М4f 25 NK 1 107 1790 82 n/d n/d n/d - - - 730*
38 М4m 27 inv(16) 1 n/d n/d n/d 20 1696 10 - - - 459
39 М4 f 28 OA 1n/d n/d n/d 34 7367 7,6 n/d n/d n/d 730*
40 М4f 34 inv(16) 1 n/d n/d n/d 549 10819 11,6 0,5 7223 10.4 560
41 М4m 39 NK 1 n/d n/d n/d 503 3548 62 - - - 614
42 М4f 45 NK 1 n/d n/d n/d n/d 13095 48,8 31 3381 n/d 700?
43 М4f 55 NK 1 4 11686 68 n/d n/d n/d 0,06 1020 20 101
44 М5m 11 del(7) +OA 1 45 11753 88 n/d n/d n/d - - - 730*
45 М5m 22 t(9;11) 1 17 25 94,4 0,6 6 1,4 10 25 9,6 730*
46 М5f 32 CK/del(7q) 1 n/d n/d n/d 272 3779 31,6 n/d n/d n/d 142
47 М5m 37 CK/del(7), EVI1+ 1 2 177 82 77 3,8 24 4542 21 423
48 М5m 55 NK 1 1,3 9631 40 n/d n/d n/d - - - 730*
49 М7f 1 CK, EVI1+
(chromothripsis) 1n/d n/d n/d n/d n/d n/d 9 3315 60 148
50 М7f 3 EVI1+ 1 0.25 282 12.8 3 3693 13.4 0.3 1049 20.8 258
Conventional symbols: PTR, posttransplant relapse; m, male; f, female; CN, copy number per 104 copies of ABL1; CK, complex karyotype (>3 cytogenetic
aberrations per metaphase); NK, normal karyotype; OA, other cytogenetic aberrations (<2); - – no PTR during two years after allo HSCT; n/d, no data; relapse.
Bone marrow samples with combined BAALC and WT1 gene overexpression are shown in bold; * - censored data; ? – OS in these cases was incorrect due to
absent of their correct death date
Table Continued...
Discussion
The data presented here show for the rst time the crucial role of
BAALC gene overexpression at pre-conditioning and posttransplant
stages in the origin and further development of post-transplant
relapses. According to our ndings even single BAALC gene
overexpression at the day D0 as well as at PTR is associated with an
unfavorable prognosis. The latter was especially bad in the cases with
combined overexpression of BAALC and WT1 genes in the studied
patients with different cytological and cytogenetic AML variants. In
the cohort of patients involved in this investigation, such phenomenon
was represented 33 times, including 13 of them at diagnosis, 8 on the
day D0 and 12 at PTR. Importantly for us, that overexpression of gene
BALLC was provided by immature CD34+CD38- precursors only,10,11
which allows recognize subtle changes of BAALC-expressing
burdens without great problems.
. As for WT1 gene overexpression concerns this mechanism is
not elucidated yet. In our opinion, another issue of WT1 expression
along with traditionally appreciated blasts must be presented. Indirect
evidence of it is seen in parallel measurements of BAAALC and WT1
along with blast number counting in the testes bone marrow samples
from patients with APL. As known, the levels of WT1 expression in
APL are highest among other subtypes of AML24 whereas APL cases
with aggressive course reveal higher BAALC expression.25 Since, some
of APL patents with higher WT1 expression can reveal relatively low
number of blasts, it allows to draw assumption concerning another
issue of WT1 expression in these APL patients than blasts. In our
opinion, the main candidate for this role may be recently opened in
APL by Patel et al26 so-called mature subtype of LIC which retains to
be evidenced in direct sorting experiments. Meanwhile, BAALC/WT1
molecular platform which was tested by us, will open possibilities to
obtain important data concerning mechanisms of treatment resistance
and PTR formation in treated with HSCT AML patients on top
precursor level.
So, the study hematopoiesis in patients with AML by means of
molecular platform BAALC/WT1 allows to elucidate some subtle
molecular mechanisms which could be responsible for formation both
treatment resistance and aggressive course of AML. Since BAALC
gene levels expression are closely associated with burden of its
producing LICs it is possible to study different aspects of pathogenesis
in various cytological, cytogenetic and molecular variants of AML. by
means standard enough cheap and not time-consuming qRT-PCR
Conclusion
In conclusion, it should be noted, that the involving of the BAALC
gene into molecular panel for the studying of relationships between
Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant
relapses in patients with acute myeloid leukemias 131
Copyright:
©2020 Mamaev et al.
Citation: Mamaev NN, Shakirova AI, Barkhatov IM et al. Crucial role of BAALC-expressing leukemic precursors in origin and development of posttransplant
relapses in patients with acute myeloid leukemias. Hematol Transfus Int J. 2020;8(6):127131. DOI: 10.15406/htij.2020.08.00240
immature precursor cells and blast burdens in hematopoiesis of AML
patients allows us to reach new levels in understanding of mechanisms
of PTR origin and development, and may be a basis for the further
fundamental investigations on the topic.
Conicts of interest
Authors declare no conict of interests for this article.
Authors’ contributions
NM, AS: concept and design
AS, IB, YG, TG, VK, ZR, MK, OP: submission of study materials
NM, AS: analysis and interpretation of data, drafting the manuscript
LZ: administrative support
All authors approved the nal version of the manuscript.
Gratitudes
We express gratitude to the transplantation team CIC 725 of Raisa
Gorbacheva Memorial Research Institute for Pediatric Oncology,
Hematology and Transplantation at the Pavlov First Saint Petersburg
State Medical University and especially to the Laboratory of molecular
hematology and transplantation.
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... Наш опыт изучения генов BAALC и WT1 у больных ОМЛ [6][7][8][9] говорит о наличии у них прямой связи с экспрессирующими их ранними (BAALC) и гипотетически более зрелыми (WT1) лейкозными клетками-предшественницами. Данное обстоятельство открывает реальный путь для динамической оценки содержания этих клеток с помощью стандартной количественной полимеразной цепной реакции в реальном времени (РВ-кПЦР), что для клинической практики крайне важно. ...
... Однако уровень экспрессии гена WT1 (6478 копий) по отношению к количеству бластных элементов оказался несоразмерно высоким. Отсюда правомочно допущение, что в данном наблюдении, в дополнение к ранее опубликованным нами данным [7,8], весомый вклад в экспрессию гена WT1 могли внести более зрел ые клетки-предшественницы. После выполнения неродственной аллоТГСК уровень экспрессии гена WT1 снизился до 111 копий, что в 2 раза ниже порогового уровня. ...
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The present paper provides evidence for a high detection rate of BAALC gene overexpression, also combined with WT1 gene overexpression, in patients with myelodysplastic syndromes (MDS) and FISH-verified chromosome defects. The BAALC and WT1 gene expression profiling of 16 MDS patients (6 out of them received allogeneic hematopoietic stem cell transplantation) showed an increased BAALC expression in 14 patients. The expression level in 2 patients was near the cut-off. Low expression levels were identified in a female patient with isolated 5q deletion in karyotype and also with its combination with complex karyotype. On the other hand, the highest expression levels were reported in patients with normal karyotype and 3q26 locus rearrangement, which was associated with EVI1 gene overexpression. Since the BAALC expression level, at least in patients with the major (except for М3 and М7) FAB-variants of acute myeloid leukemias (AML), was closely associated with BAALC-producing precursor cells of leukemia clone, a profound study of this phenomenon in MDS patients seems to be important for understanding the finest mechanisms underlying the pathogenesis of AML and AML relapses on the level of precursor cells.
... Moreover, this conclusion is also supported by similar discrepancy between higher WT1 gene expression and lower numbers of bone marrow blasts in 30% to 40% AML patients, both before and after HSCT [6,9,10,13,14]. In our opinion, this phenomenon may be also explained by presence of active expression of WT1 gene by more mature precursors [12,15]. Confirmation of this hypothesis should extend our opportunities for evaluation of leukemic hematopoiesis on the level of leukemic precursors, using quantitative qRT-PCR [12,14,15]. ...
... In our opinion, this phenomenon may be also explained by presence of active expression of WT1 gene by more mature precursors [12,15]. Confirmation of this hypothesis should extend our opportunities for evaluation of leukemic hematopoiesis on the level of leukemic precursors, using quantitative qRT-PCR [12,14,15]. To test this hypothesis, we performed parallel measurements of BAALC and WT1 expression levels in 14 AML patients with different cytological, cytogenetic and molecular variants of AML treated with combination of high-dose chemotherapy (ChT) and Mylotarg. ...
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Article is devoted elucidation of BAALC-expressing leukemic precursors behaving in patients with different cytogenetic and molecular variabts treated with Gentozumab-ozogamicin
... On the other hand, the data were obtained which presumed direct participation of ELP in selective expression of the BAALC gene [13,14]. On the basis of these data our hypothesis was drawn linking poor-risk BAALC overexpression directly with BAALCexpressing ELP [15][16][17] which can replace the older doubtful explanation of this phenomenon through BAALC-mRNA. If this explanation is true, then similar RT-qPCR might be used effectively in clinical setting for serial quantitative evaluation of BAALC-expressing ELP bulks. ...
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A new concept of acute myeloid leukemia (AML) relapses is proposed which is linked with direct participation of BAALC-expressing earlier leukemic progenitors (ELP). The latter may be studied effectively by means of real-time quantitative polymerase chain reaction (RT-qPCR). Recent findings in support of this conception and ongoing prospective studies in the field are shortly discussed.
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This article presents data demonstrating frequent BAALC hyperexpression, also in combination with WT1 hyperexpression, in children and adults with acute myeloid leukemia (AML). Treatment included allogeneic hematopoietic stem cell transplantation. The analysis of serial measurements of BAALC and WT1 expression level in 50 AML patients (37 adults and 13 children) showed that the increased BAALC expression is more common in patients with M1, M2, M4, and M5 FAB variants of AML with equal frequency in adults and children. Furthermore, the increased BAALC expression was rather common in combination with the increased WT1 expression, which predicted poorer prognosis. Since BAALC expression level in AML patients is closely related to AML-producing progenitor cells of leukemia hematopoiesis, a serial study of this phenomenon offers insights into the role of these cells in emergence and development of post-transplantation relapses, which is of both theoretical and practical importance.
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Article contains data showing leading role of BAALC expressing precursors in origin of post-transplant relapses in patients with different cytological and cytogenetic variants of AML
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Acute myeloid leukemia (AML) is a heterogenous clonal blood disease of a neoplastic origin. There are challeng- ing issues for the intermediate-risk AML group, which is defined as non-homogeneous due to a variety of gene mutations (FLT3, NPM1, CEBPA, etc.), prediction of differential clinical course, relapse risk, and selection of adequate therapy. In this context, a search for new mo- lecular markers with sufficient prognostic value for the relapse risk estimation in AML cases with no detecta- ble cytogenetic abnormalities represents a high-pri- ority task for clinical molecular oncohematology. We analyzed prognostic significance of BAALC (Brain And Acute Leukemia, Cytoplasmic) gene overexpression in 93 AML patients during the posttransplant period, in order to estimate feasibility of BAALC expression lev- el monitoring, to predict the relapse risk, and to eval- uate sensitivity and specificity of BAALC gene expres- sion assay, to the purpose of minimal residual disease (MRD) monitoring. BAALC expression was determined by quantitative real-time polymerase chain reaction in fresh bone marrow samples. Patients were dichotomized at BAALC's individual and general cut-off into low and high expressers. We have concluded that BAALC over- expression above both individual and common cut-off levels is recognized as a prognostically significant factor for posttransplant relapse risk estimation, overall sur- vival and relapse-free survival. A more detailed analy- sis of BAALC as a marker for estimation of therapeutic efficiency was performed. We have also compared its sensitivity to the reference techniques for minimal re- sidual disease monitoring (i.e., qPCR-based detection of chimeric gene transcripts), showing inferior sensitivity of such approach to MRD detection in post-transplant period, at least, for our study group. Serial BAALC mon- itoring may be recommended for clinical relapse predic- tion during the post-transplant period in AML patients. Keywords Acute myeloblastic leukemia, BAALC, gene expression, clinical prognosis, minimal residual disease.
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Although overexpression of the brain and acute leukemia, cytoplasmic (BAALC) gene is associated with primary resistant disease and shorter relapse-free, disease-free, and overall survival in different subsets of acute myeloid leukemia (AML), little is known about its clinical impact in acute promyelocytic leukemia (APL). Using real-time reverse transcriptase polymerase chain reaction, we showed that BAALC expression is significantly lower in APL compared with other subsets of AML (P ,<.001). We also demonstrated that BAALC overexpression was associated with shorter disease-free survival (DFS) (hazard ratio [HR], 4.43; 95% confidence interval [CI], 1.29-15.2; P = .018) in 221 consecutive patients (median age, 35 years; range, 18-82 years) with newly diagnosed APL homogeneously treated with all-trans retinoic acid and anthracycline-based chemotherapy. Cox proportional hazard modeling showed that BAALC overexpression was independently associated with shorter DFS in the total cohort (HR, 5.26; 95% CI, 1.52-18.2; P = .009) and in patients with high-risk disease (ie, those with initial leukocyte counts >10 × 10⁹/L) (HR, 5.3; 95% CI, 1.14-24.5; P = .033). We conclude that BAALC expression could be useful for refining risk stratification in APL, although this needs to be confirmed in independent cohorts.
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Acute myeloid leukemia (AML) patients show high relapse rates and some develop conventional chemotherapy resistance. Leukemia Stem Cells (LSCs) are the main player for AML relapses and drug resistance. LSCs might rely on the B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) in promoting cellular proliferation and survival. Growth of LSCs in microenvironments that are deprived of nutrients leads to up-regulation of the signaling pathways during the progression of the disease, which may illustrate the sensitivity of LSCs to inhibitors of those signaling pathways as compared to normal cells. We analyzed the expression of LSC markers (CD34, CLL-1, TIM-3 and BMI-1) using quantitative RT-PCR in bone marrow samples of 40 AML patients of different FAB types (M1, M2, M3, M4, M5, and M7). We also studied the expression of these markers in 2 AML cell lines (Kasumi-1 and KG-1a) using flow cytometry and quantitative RT-PCR. The overexpression of TIM-3, CLL-1, and BMI-1 was markedly correlated with poor prognosis in these patients. Our in vitro findings demonstrate that targeting BMI-1, which markedly increased in the leukemic cells, was associated with marked decrease in leukemic burden. This study also presents results for blocking LSCs' surface markers CD44, CLL-1, and TIM-3. These markers may play an important role in elimination of AML. Our study indicates a correlation between the expression of markers TIM-3, CLL-1, and especially of BMI-1 and the aggressiveness of AML and thus the potential impact of prognosis and therapies that target LSCs on improving the cure rates.
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Introduction: Patients with acute promyelocytic leukemia (APL) are characterized by the highest expression of Wilms' tumor 1 (WT1) gene compared with other subtypes of acute myeloid leukemia, and yet this molecular marker is almost never used for risk stratification and in therapy response monitoring. Methods: Quantitative assessment of Wilms' tumor 1 (WT1) gene transcripts was performed using real-time PCR method. The bone marrow samples were collected at the time of diagnosis for 47 APL patients, and for 31/47 patients during follow-up/relapse of the disease (129 samples in total). We examined how this molecular marker can be used for prognosis and minimal residual disease (MRD) monitoring. Results: Increased WT1 expression was found in 34% of patients. WT1high status was an independent unfavorable factor for early death occurrence and was associated with shorter overall survival (OS). Assessment of log reduction value of WT1 expression in paired diagnosis/complete remission samples did not reveal its impact on relapse rate, disease-free survival, and OS. Also, measurement of WT1 expression level at different time points during therapy was not a reliable method for MRD monitoring. Conclusion: Increased expression of WT1 gene detected in high proportion of APL patients could be considered as a marker for more precise risk stratification models in an attempt to further improve treatment and outcome of APL patients.
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Introduction: Although treatment of acute promyelocytic leukemia (APL) has evolved dramatically during the past decades, especially with the introduction of all-trans retinoic acid, risk stratification remains an important issue. To date, relapse risk can be predicted by leukocyte and platelet counts only. In the present report, we present a validation study on 3 candidate genes and a newly developed molecular risk score for APL in 2 independent patient cohorts. Patients and methods: An integrative risk score combining the expression levels of BAALC, ERG, and WT1 was calculated for 79 de novo APL patients from the original cohort and 76 de novo APL patients from a validation cohort. Gene expression analysis was executed the same for both cohorts, and the results regarding the effect on patient outcomes were compared. Results: The expression levels of BAALC, ERG, and WT1 were similar in both cohorts compared with the healthy controls. The relapse and survival rates were not different between the low- and high-risk patients according to the Sanz score. However, application of the molecular risk score on the validation cohort distinctly discriminated patients according to their risk of relapse and death just as in the original APL cohort, although single gene analyses could not reproduce the negative prognostic impact. Conclusion: The analysis clearly validated the prognostic effect of the integrative risk score on the outcome in APL patients. The value was further empowered because the single gene analyses did not show similar results. Whether the integrative risk score retains its prognostic power in the chemotherapy-free setting should be investigated further.
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Although high brain and acute leukemia, cytoplasmic (BAALC) expression is a well-characterized poor prognostic factor in acute myeloid leukemia (AML), neither the exact mechanisms by which BAALC drives leukemogenesis and drug resistance nor therapeutic approaches against BAALC-high AML have been properly elucidated. In this study, we found that BAALC induced cell cycle progression of leukemia cells by sustaining extracellular signal-regulated kinase (ERK) activity through an interaction with a scaffold protein MEK kinase-1 (MEKK1), which inhibits the interaction between ERK and MAP kinase phosphatase 3 (MKP3/DUSP6). BAALC conferred chemoresistance in AML cells by up-regulating ATP-binding cassette proteins in an ERK-dependent manner, which can be therapeutically targeted by MEK inhibitor. We also demonstrated that BAALC blocks ERK-mediated monocytic differentiation of AML cells by trapping Krüppel-like factor 4 (KLF4) in the cytoplasm and inhibiting its function in the nucleus. Consequently, MEK inhibition therapy synergizes with KLF4 induction and is highly effective against BAALC-high AML cells both in vitro and in vivo. Our data provide a molecular basis for the role of BAALC in regulating proliferation and differentiation of AML cells and highlight the unique dual function of BAALC as an attractive therapeutic target against BAALC-high AML.Leukemia accepted article preview online, 08 June 2015. doi:10.1038/leu.2015.137.
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Numerous studies have investigated the prognostic role of brain and acute leukemia, cytoplasmic (BAALC) gene expression in adult patients with acute myeloid leukemia (AML); however, the results are inconclusive. A meta-analysis was conducted to provide a comprehensive evaluation of the prognostic role of BAALC gene expression in AML. Eligible studies were searched through PubMed, Embase, the Cochrane Library, the China National Knowledge Infrastructure and the China Biology Medicine Disc. Correlations between the BAALC gene expression and clinicopathological features and prognosis were analyzed. A total of 15 studies were examined. The pooled results suggest that high BAALC expression had an unfavorable outcome in AML. The combined hazard ratio (HR) for overall survival (OS) was 1.53 and the summary HR for the disease-free survival rate was 1.64. In addition, subgroup analyses considering cytogenetic and survival analysis were also conducted. High BAALC gene expression appeared to be an adverse prognostic indicator in patients with cytogenetically normal AML (HR for OS, 1.43) and in subgroups of survival analysis with multivariate analysis (HR for OS, 2.35). These results indicate that high BAALC gene expression served as an independent poor prognostic indicator in adult patients with AML.