Leukemia & Lymphoma

ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20

The application of CD73 in minimal residual

disease monitoring using flow cytometry in B-cell
acute lymphoblastic leukemia

Wei Wang, Li Gao, Yan Li, Zhen-Ling Li, Ming Gong, Fan-Zhou Huang, Yan-
Rong Chen, Chun-Xia Zhang, Ya-Yue Gao & Yi-Gai Ma

To cite this article: Wei Wang, Li Gao, Yan Li, Zhen-Ling Li, Ming Gong, Fan-Zhou Huang,
Yan-Rong Chen, Chun-Xia Zhang, Ya-Yue Gao & Yi-Gai Ma (2015): The application of CD73
in minimal residual disease monitoring using flow cytometry in B-cell acute lymphoblastic
leukemia, Leukemia & Lymphoma, DOI: 10.3109/10428194.2015.1070153

To link to this article: http://dx.doi.org/10.3109/10428194.2015.1070153

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 LEUKEMIA & LYMPHOMA
2015; EARLY ONLINE: 1–9
http://dx.doi.org/10.3109/10428194.2015.1070153

ORIGINAL ARTICLE: CLINICAL

The application of CD73 in minimal residual disease monitoring using flow

cytometry in B-cell acute lymphoblastic leukemia
Wei Wang, Li Gao, Yan Li, Zhen-Ling Li, Ming Gong, Fan-Zhou Huang, Yan-Rong Chen, Chun-Xia Zhang,
Ya-Yue Gao & Yi-Gai Ma
Department of Hematology, China-Japan Friendship Hospital, Beijing, PR China

ABSTRACT KEYWORDS
The expression of CD73 by flow cytometry (FC) in bone marrow (BM) specimens of B-cell acute B-cell acute lymphoblastic
lymphoblastic leukemia (B-ALL) with or without minimal residual disease (MRD) was studied, and its leukemia, flow cytometry,
advantages were evaluated using the MRD assay. This study also detected the expression profile of minimal residual disease
CD73 in hematogones and mature B cells in BM specimens of 18 healthy donors. Results showed
that the mean value of CD73 expression in MRD-positive B cells was 6-fold greater than that in the HISTORY
Received 6 May 2015
Downloaded by [McMaster University] at 02:40 09 October 2015

MRD negative ones. Also, 41.82% MRD-positive B-ALL cases expressed high CD73 and the
Revised 23 June 2015
sensitivity of CD73-based MRD detection reached 10 4. Since the expression of CD73 increases with Accepted 29 June 2015
the maturation of normal B cells, it is better to mix it with CD34, CD10 and CD20 in one tube Published online
to prevent the disturbance of mature B cells. CD73 is recommended as an optional MRD marker for 1 September 2015
B-ALL patients by using FC.

Background from hematogones, because on one hand the two

The presence of minimal residual disease (MRD) after
chemotherapy in patients with B-cell acute lympho-
blastic leukemia (B-ALL) has been demonstrated to be a
crucial prognostic parameter [1–4]. For that reason, MRD
detection has been used in therapeutic protocols for risk
stratification and therapeutic regimens [5,6]. The first
method for MRD detection is quantitative real-time PCR
analysis of immunoglobulin (IG) gene rearrangements,
which can be performed in the majority of patients.
However, such an approach is labor-intensive and time-
consuming because, for MRD monitoring, the specific
rearrangement should be sequenced in each patient up
front. The second one is PCR studies of fusion genes
which can be easily used in 40% of children and adults
with B-ALL [1,7]. The third method is flow cytometry (FC),
an approach noted for its rapidity, sensitivity, wider
application and accurate qualification in MRD detection.
Recently, the development of FC make it possible to use
more markers simultaneously, which can significantly
enhance the sensitivity of MRD detection [8,9].
Generally, the detection of MRD in B-ALL by means of
FC can be made by comparing antigen profiles of
leukemic B cells with those of their normal counterparts
(hematogones) [1,10,11]. However, in some cases it
would be difficult to distinguish the leukemic B cells
different types of cells share many phenotypic features
and, on the other hand, the phenotypic switch of MRD B
cells is not uncommon during treatment. Such a dilemma
can be resolved by following up closely, making repeated
analyses or referring to the results of molecular biology.
In order to enhance the accuracy of FC in MRD
detection, new specific markers should be developed.
Recently, several new markers of MRD detection in B-ALL
have been reported [12]. Based on the report and the
result of our preliminary experiments, we evaluated
CD73 in MRD detection using multiple-parameter FC
among patients with B-ALL.
Materials and methods
Patients
Eighty-three adult patients (51 males and 32 females)
with B-ALL from China-Japan Friendship Hospital were
enrolled in the study, from July 2011 to February 2015.
Diagnosis of B-ALL was established by applying standard
WHO criteria [13]. The median age of the B-ALL group
was 46 years. B-ALLs were sub-classified immunologic-
ally according to the criteria of the European Group
for the Immunological Characterization of Leukemias
(EGIL) [14]. Twenty-one patients were progenitor

Correspondence: Yi-Gai Ma, Department of Hematology, China-Japan Friendship Hospital, No. 2 Yinghuayuan East Road, Beijing, PR China 100029. Tel: +86-
10-84205522. Fax: +86-10-64217749. dr_myg@163.com
! 2015 Taylor & Francis
 2 W. WANG ET AL.
B-ALL, 54 common B-ALL and eight precursor B-ALL.
Twenty-four patients were positive for BCR-ABL1, two
MLL-ENL and one MLL-AFF1. The other 56 B-ALL patients
had no specific fusion gene. The control group consisted
of 18 healthy donors (10 males and eight females) with a
median age of 41 years. All clinical samples were
obtained with informed consent. The study was
approved by the Ethical Committee of the China-Japan
Friendship Hospital and in full compliance with the
guideline of the Helsinki Declaration of 2008.
Sample timing
Bone marrow aspirate samples of B-ALL patients and
healthy donors were collected. The MRD detection of 62
B-ALL patients was performed at day 29 of the induction
chemotherapy. Twelve B-ALL patients were evaluated
Downloaded by [McMaster University] at 02:40 09 October 2015
after completion of the intensification chemotherapy. In
addition, nine patients were detected at different time
points before or after chemotherapies in order to
observe the stability of CD73.
Flow cytometry assessment
The following mouse anti-human monoclonal antibodies
(mAbs) were used for each patient in this study: CD10
FITC, CD10 PE-CY7, CD13 PE-CY7, CD19 PE-CY7, CD19
APC, CD20 APC-Cy7, CD22PE, CD34 PE, CD34 APC, CD38
PE-CY7, CD45 PerCP, CD58 PE, CD123 PE (all from BD
Bioscience, San Diego, CA) and CD73 PE (from BD
Pharmingen, San Diego, CA) and CD33 FITC (from
Beckman Coulter, San Diego, CA). Antibody combin-
ations used in the study for MRD detection included:
CD33/CD123/CD45/CD13/CD19/CD20, CD10/CD22/
CD45/CD19/CD34/CD20, CD10/CD34/CD45/CD38/CD19/
CD20, CD10/CD58/CD45/CD19/CD34/CD20 and CD10/
CD73/CD45/CD19/CD34/CD20. CD34, CD10, CD20, CD19
and CD45 were used as backbones, except for the
first tube.
Bone marrow aspirates were collected in heparin
anticoagulant tubes and then stained and acquired
within 12 h after procurement. Per tube, 5  105 bone
marrow cells were stained using a standard procedure.
Data were acquired and analyzed using FACS CantoII
flow cytometry and DIVA (both from BD Biosciences).
The gating strategy was as follows: alive cells were
gated on the FSC/SSC dot plot. Then distinct cellular
clusters were delineated on the SSC/CD45 dot plot.
Generally, B cells in different mature stages were
distinguished on CD19/CD45 dot plot, i.e. mature B
cells were gated as CD19+CD45high, immature B cells
were CD19+CD45dim+, while most of the leukemic B
cells were CD19+CD45 . However, in order to observe
the expression of CD73 in different development stages
of normal B cells, CD34/CD10 and CD10/CD20 dot plots
were also used, so as to delineate B cells in each
development stage clearly.
Antigen expression was quantiEed as the mean
Fuorescence intensity ratio (MFIR) (values obtained
with speciEc mAbs divided by values of the isotype
control mAb). MRD was identified on the basis of the
leukemia-associated phenotype (LAP) [1,10,15,16].
Briefly, the LAP includes CD10high, CD10 CD20 ,
CD13+, CD22high, CD33+, CD34high, CD38low/ ,
CD45low/ , CD58high and CD123high. A distinct cluster
of at least 20 cells with two or more LAPs was defined as
a leukemic population.
PCR assessment
Every patient was detected by PCR assessment before
chemotherapy in order to find out the genetic markers
for MRD detection. Six types of fusion genes were
detected by using a standardized quantitative real-time
PCR [17] in the study, including BCR-ABL1, MLL-AFF1,
MLL-AF9, MLL-ENL, ETV6-RUNX1 and TCF3-PBX1. In this
case, if a patient had no fusion gene, IG rearrangements
were detected by consensus PCR assessment before
chemotherapy. The primers were described in another
manuscript [18]. Based on the IG rearrangement results,
the corresponding primer could be selected. Then, after
chemotherapy, the specific IG rearrangement was
detected by allele specific quantitative PCR using SYBR
Green I dye (Life Technologies Corporation, Carlsbad,
CA). If either the fusion gene or the IG rearrangement
was positive, MRD would be suspected.
MRD diagnosis criteria
A diagnosis of MRD + was made if the aberrance was
consistently detected by both FC and PCR assessment.
In the meanwhile, a diagnosis of MRD was made if there
was no MRD detected by either of the two methods.
Cases were excluded from our research if the MRD was
detected by one method, but not confirmed by the other.
Statistics
The non-parametric Mann–Whitney U-test was used to
evaluate the difference of CD73 between leukemic blasts
and hematogones in B-ALL patients. The 2-tailed
Student’s t-test was applied to evaluate the difference
of CD73 between hematogones and mature B cells in
the healthy control group. The chi-squared test was used
to evaluate the characteristic parameters in CD73high
B-ALL cases. All the statistical analyses were performed
 MINIMAL RESIDUAL DISEASE DETECTION BY CD73 3

Table I. Specificity of all LAPs in MRD + B-ALLs.

LAP Case number Frequency
high
CD10 29/55 52.73%
CD10 CD20 7/55 12.73%
CD13+ 4/55 7.27%
CD22high 18/55 32.73%
CD33+ 13/55 23.64%
CD34high 4/55 7.27%
CD38low/ 45/55 81.82%
CD45low/ 19/55 34.55%
CD58high 31/55 56.36%
CD73high 23/55 41.82%
CD123high 34/55 61.82%
LAP, leukemia-associated phenotype; MRD, minimal residual disease; B-ALL,
B-cell acute lymphoblastic leukemia; Case number, the number of cases
having a certain LAP among the total 55 MRD + B-ALL cases.
Figure 1. Expression of CD73 in MRD B cells and hematogones.
The results of flow cytometry are shown. MRD B cells of B-ALL
cases (closed circle) expressed a higher level of CD73 than We also evaluated the specificity of other confirmed
hematogones from MRD B-ALL cases (triangle). The non- LAPs in these MRD + B-ALL [Table I]. We got a low
parameter Mann–Whitney U-test was used. Mean value is
specificity of CD10 CD20 , CD13+, CD22high, CD33+ and
represented by horizontal bars. The star stands for p-value low
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than 0.05. MRD, minimal residual disease; MFIR, mean fluores- CD34high. However, CD58high, CD123high, CD10 high and
cence intensity ratio; B-ALL, B-cell acute lymphoblastic leukemia. CD38low/ expressed by MRD B cells showed high
specificity.

using the SPSS software, 13.0 version (SPSS Inc., Chicago,

IL). The MFIR was presented as mean ± standard error of
mean (SEM).
Results
Fifty-five (66.27%) B-ALL patients were detected as
MRD + by using FC and PCR assessment, while 28
(33.73%) B-ALL patients were detected as MRD . The
information of MRD + B-ALL patients is listed in
Supplemental data in greater detail.
The expression of CD73 in MRD B cells and
hematogones
We compared the expression of CD73 in MRD B cells
with hematogones from MRD B-ALL patients. It was
found out that MRD B cells (20.45 ± 4.29) demonstrated
significantly higher expression of CD73 than hemato-
gones (3.14 ± 0.42, p 5 0.05) [Fig. 1]. Except for that, its
expression in hematogones showed smaller variation.
However, the variation of CD73 from MRD B cells was
considerably larger.
Specificity of CD73 in MRD detection
In order to minimize the possibility of false-positive
diagnosis, the cut-off value of CD73 MFIR was set in
accordance with the maximum value (6.56) expressed by
hematogones of MRD B-ALLs. There were 41.82%
(23/55 cases) MRD + B-ALL cases who expressed an
aberrantly high level of CD73.
Stability of CD73 in MRD B cells during
chemotherapy
In our study, nine B-ALL patients were
MRD + throughout. The bone marrow samples were
collected at different time points before or after
chemotherapies (e.g. at day 29 of induction, after
completion of the intensification and during the con-
solidation chemotherapy). This procedure allowed us to
observe the expression stability of CD73 in leukemic B
cells at different time points [Table II]. From our
observation, the expression of CD73 in five B-ALL
patients was stable at different time points, while that
of the other four B-ALL patients varied between normal
value and abnormal value.
high
Characteristic of CD73 B-ALL
We analyzed the characteristic of CD73high B-ALL. We
found out that the frequency of CD73high was 57.14%
(12/21 cases) in MRD + B-ALL cases with BCR-ABL1.
However, the frequency was only 32.35% (11/34 cases)
in other MRD + B-ALL cases. As for the sub-type of B-ALL,
the frequency of CD73high was 35% (7/20 cases), 45.45%
(15/33 cases) and 50% (1/2 cases) in progenitor B-ALL,
common B-ALL and precursor B-ALL, respectively.
Expression profile of CD73 in normal B cells
during different development stages
In order to clarify the expression profile of CD73 in
normal B cells during different development stages, we
 4 W. WANG ET AL.

Table II. The stabilities of CD73 in MRD B cells at different time points.
Sex/age Sub-type Time point Leukemic blast %* CD73 MFIR
F/74 common after the 1st induction chemotherapy 3% 25.70
after intension chemotherapy 0.49% 11.15
during consolidation therapy 65.7% 16.40
after the 2nd induction chemotherapy 15.2% 6.19
after the intension chemotherapy 4.2% 16.00
during the consolidation chemotherapy 8.5% 8.14
during the consolidation chemotherapy 2.2% 20.55
during the consolidation chemotherapy 79.6% 8.23
after the 3rd induction chemotherapy 45.2% 7.97
after the 4th induction chemotherapy 32.6% 14.20
M/28 common after 1st induction chemotherapy 1.52% 7.38
after the intension chemotherapy 0.01% 5.15
after the intension chemotherapy 0.03% 1.15
M/23 common after 1st induction chemotherapy 0.2% 1.16
after the intension chemotherapy 0.1% 1.14
M/30 common after 1st induction chemotherapy 0.03% 4.63
after the intension chemotherapy 54.44% 3.51
after the 2nd induction chemotherapy 64.40% 4.23
after the 3rd induction chemotherapy 95% 2.18
M/23 common after 1st induction chemotherapy 0.01% 1.19
after the intension chemotherapy 0.02% 1.98
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after the intension chemotherapy 0.04% 2.16

after the intension chemotherapy 0.01% 2.97
during the consolidation chemotherapy 0.69% 9.72
during the consolidation chemotherapy 0.06% 7.02
M/21 progenitor untreated 63.5% 1.74
after 1st induction chemotherapy 0.01% 2.14
after the intension chemotherapy 0.11% 1.55
after the intension chemotherapy 0.20% 2.17
after the intension chemotherapy 0.2% 2.33
during the consolidation chemotherapy 0.01% 1.30
during the consolidation chemotherapy 12.01% 2.23
F/25 common untreated 78.4% 2.60
after 1st induction chemotherapy 0.14% 55.71
after the intension chemotherapy 0.55% 1.11
F/49 progenitor untreated 81.78% 3.55
after 1st induction chemotherapy 12.10% 4.84
after the intension chemotherapy 0.97% 3.21
F/64 progenitor untreated 82.9% 1.29
after 1st induction chemotherapy 0.11% 1.20
F, female; M, male; MFIR, mean fluorescence intensity ratio.
*Leukemic blast% means the percentage of leukemic blast cells in the nucleated cells of the bone marrow sample. Italics show that the value is aberrantly high.

evaluated the expression of CD73 in hematogones and

mature B cells in healthy donors’ specimens. As shown in
Fig. 2, the expression of CD73 in mature B cells
(3.42 ± 0.23) was significantly higher than that in
hematogones (1.87 ± 0.20, p50.05). As shown in Fig. 3,
the expression of CD73 increased gradually with the
maturation of normal B cells, for the expression of CD73
in CD34+ CD10+ CD45dim CD20 B cells was the lowest,
while in CD34 CD10 CD45high CD20+ B cells it was the
highest.

Sensitivity of CD73 in MRD detection

To evaluate the sensitivity of CD73 in MRD detection,
leukemic cells from bone marrow samples of three
newly diagnosed B-ALL patients with high expression of
CD73 were serially diluted in those of three healthy
donors. The procedures were reported by another group
[19]. As shown in Fig. 4, we got a linear quantification of
Figure 2. The expression of CD73 in hematogones and mature B
cells from a healthy control. Results of flow cytometry of normal
B cells from the control group are shown. The expression level of
CD73 increased with the B cell maturation process. The 2-tailed
Student’s t-test was used. The inverted triangles stand for
hematogones and the squares stand for mature B cells. The star
stands for p value lower than 0.05.
 MINIMAL RESIDUAL DISEASE DETECTION BY CD73 5

Figure 3. The expression profile of CD73 in different development stages of normal B cells. The expression profile of CD73 from a
healthy donor’s bone marrow sample is shown. The immunophenotype of the blue cellular cluster is CD19+, CD34+, CD45+,
CD10dim+ and CD20 . The immunophenotype of the green cellular cluster is CD19+, CD45+, CD34 , CD10 and CD20 . The
immunophenotype of the yellow cellular cluster is CD19+, CD45high, CD20+, CD10 and CD34 . With the maturation of B cells, the
expression of CD73 increases gradually. As shown, the expression profile of CD73 in the mature B cells is bimodal distribution.
Downloaded by [McMaster University] at 02:40 09 October 2015

Figure 4. Sensitivity of CD73 and other LAPs in MRD detection. Leukemic B cells from the bone marrow aspirate sample of a newly
diagnosed B-ALL patient expressing high CD73 were diluted at different concentrations (10 2, 10 3 and 10 4) in the bone marrow of
a healthy donor. The black cellular cluster represents hematogones (CD19+, CD34+, CD10+, CD45+ and CD20 ). The red cellular cluster
represents leukemic B cells (CD19+, CD34+, CD10+, CD45dim and CD20 ). By using flow cytometry, the MRD detection sensitivity of
CD73high, CD38low and CD58high was compared. LAP, leukemia-associated phenotype; BM, bone marrow.

CD73-based MRD detection. The maximum sensitivity of In order to determine the rate of false positive
CD73 could reach 10 4, which was in accordance events using the same gating strategy in
with the quantification by using other LAPs [Fig. 4 and MRD samples, we add the bone marrow samples
Table III]. of three MRD B-ALL patients to those of three
 6 W. WANG ET AL.

Table III. Sensitivity of CD73 and other LAPs in MRD detection. [20,21], we set up the highest threshold of CD73 by
Sensitivity Sensitivity Sensitivity choosing hematogones in those B-ALL patients without
LAPs (dilution 10 2) (dilution 10 3) (dilution 10 4) MRD who were confirmed by both FC and PCR assess-
CD73high 2.7% 0.2% 0.03% ment. Most patients were treated with the same
CD38low 3.1% 0.1% 0.03%
CD58high 2.3% 0.1% 0.02% chemotherapeutic regimens, so the influence of che-
LAP, leukemia-associated phenotype; MRD, minimal residual disease. motherapies on CD73 expression in the two cohorts
would be the same. It turned out that the mean value of
CD73 MFIR in MRD B cells was 6-fold of that in
hematogones. Besides, the difference of CD73 between
MRD B cells from CD73high cases and hematogones from
MRD cases was so obvious that we could easily
distinguish them.
An ideal MRD marker is the one that can be frequently
detected in MRD + cases. CD73 showed high specificity,
for in our study 23/55 (41.82%) MRD + B-ALL patients
expressed high CD73, while in another study 54.5%
newly diagnosed B-ALL patients expressed high CD73
Downloaded by [McMaster University] at 02:40 09 October 2015

[12]. As shown in Table III, some LAPs such as CD38low/ ,

Figure 5. False-positive events in MRD detection by using CD73
and other LAPs. The bone marrow aspirate sample of a B-ALL
patient without MRD was diluted at a 1:1 ratio with that of a
healthy donor. The black cellular cluster represents hemato-
gones (CD19+, CD34+, CD10+, CD45+ and CD20 ) of the healthy
donor in the left column. In the right column, the black cellular
cluster represents the mixed hematogones (CD19+, CD34+,
CD10+, CD45+ and CD20 ) from both the healthy donor and the
MRD B-ALL patients. By using flow cytometry, false-positive
events of MRD were not detected based on CD73high, CD38low
and CD58high. LAP, leukemia-associated phenotype.
healthy donors. As shown in Fig. 5, MRD events were
not detected.
Discussion
In this study, we analyzed the expression of CD73 in
B-ALL patients with or without MRD. Unlike other studies
CD123high, CD58high and CD10 high had higher specificity
than CD73 did. However, it was worth noting that the
specificity of CD73high was higher than the rest of the
LAPs (i.e. CD10 CD20 , CD13+, CD22high, CD33+ and
CD34high) that have been widely used in MRD detection.
Generally speaking, an ideal immunophenotypic
marker for MRD detection should be stable over time.
In our study, CD73 expression varied over time in 44%
(4/9) cases, while it was stable in the other 56% (5/9)
cases. However, although some crucial markers have
been reported to shift significantly due to chemother-
apy, they have still been broadly used as backbones in
MRD detection strategies [22–24]. CD73 expression in
4/9 cases was detected before treatment and none of
them showed high expression. However, after chemo-
therapy, one case out of the four showed high expres-
sion of CD73. This phenomenon indicates that CD73
should not be excluded from being used as the marker
for MRD detection, even in those B-ALL patients with low
expression of CD73 before treatment. However, in the
above stability analysis, the number of cases and
detecting time points was limited, therefore more
cases would be needed in the future.
In our study, B-ALL cases with BCR-ABL1 expressed
higher CD73 than other B-ALL without the fusion gene,
but the difference had no significance (p 4 0.05). We still
need more cases to study whether CD73high is correlated
with BCR-ABL1. As for the correlation between CD73high
and the sub-type of B-ALL, it is too early to draw any
conclusion. More cases will be needed.
As indicated in the study, CD73 expression increased
with the maturation of normal B cells. Also, our
observation showed that the expression profile of
mature B cells was bimodal, for part of the mature B
cells expressed a high level of CD73, while the rest

expressed a low level. For this reason we recommend
that CD73 should be placed with CD34, CD10, CD19 and
CD20 in one tube, so that the mature B cells can be
excluded from the blasts gate.
It is obvious that the MRD marker should have high
sensitivity. By using the serial dilution experiment, we
observed the sensitivity of CD73 was just the same as
other LAPs.
In summary, based on our study of a small cohort,
we recommend that CD73 would be an optional
marker for B-ALL patients in MRD detection by using
FC. More patients should be needed for a precise
conclusion.
Potential conflict of interest: Disclosure forms provided
by the authors are available with the full text of this article at
www.informahealthcare.com/lal
Downloaded by [McMaster University] at 02:40 09 October 2015
This work was supported by grants from the ‘‘National
Natural Science Foundation of China’’ (No. 81300450 and No.
81300425) and grant from the ‘‘China-Japan Friendship
Hospital Youth Science and Technology Excellence Project’’
(No. QNYC-2014-B-07).
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