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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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2015; EARLY ONLINE: 1–9


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

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
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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

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

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

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

LAP Case number Frequency
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

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

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

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

Sensitivity of CD73 in MRD detection

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

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.
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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

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

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-
Figure 5. False-positive events in MRD detection by using CD73 sion of CD73. This phenomenon indicates that CD73
and other LAPs. The bone marrow aspirate sample of a B-ALL should not be excluded from being used as the marker
patient without MRD was diluted at a 1:1 ratio with that of a for MRD detection, even in those B-ALL patients with low
healthy donor. The black cellular cluster represents hemato- expression of CD73 before treatment. However, in the
gones (CD19+, CD34+, CD10+, CD45+ and CD20 ) of the healthy
above stability analysis, the number of cases and
donor in the left column. In the right column, the black cellular
cluster represents the mixed hematogones (CD19+, CD34+, detecting time points was limited, therefore more
CD10+, CD45+ and CD20 ) from both the healthy donor and the cases would be needed in the future.
MRD B-ALL patients. By using flow cytometry, false-positive In our study, B-ALL cases with BCR-ABL1 expressed
events of MRD were not detected based on CD73high, CD38low higher CD73 than other B-ALL without the fusion gene,
and CD58high. LAP, leukemia-associated phenotype.
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
healthy donors. As shown in Fig. 5, MRD events were and the sub-type of B-ALL, it is too early to draw any
not detected. 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
In this study, we analyzed the expression of CD73 in mature B cells was bimodal, for part of the mature B
B-ALL patients with or without MRD. Unlike other studies cells expressed a high level of CD73, while the rest

expressed a low level. For this reason we recommend in acute lymphoblastic leukemia: a French multicenter
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