Annals of Hematology

https://doi.org/10.1007/s00277-020-04296-9

ORIGINAL ARTICLE

Long-term outcomes of 172 children with severe aplastic anemia

treated with rabbit antithymocyte globulin and cyclosporine
Yang Lan 1 & Lixian Chang 1 & Meihui Yi 1 & Yuli Cai 1 & Jing Feng 1 & Yuanyuan Ren 1 & Chao Liu 1 & Xiaoyan Chen 1 &
Shuchun Wang 1 & Ye Guo 1 & Aoli Zhang 1 & Lipeng Liu 1 & Jingliao Zhang 1 & Xiaofan Zhu 1

Received: 27 May 2020 / Accepted: 3 October 2020

# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
This study retrospectively analyzed the clinical outcome of 172 children with newly diagnosed severe aplastic anemia (SAA) between
January 2008 and April 2018, who received rabbit antithymocyte globulin (ATG) and cyclosporine (CsA) as first-line treatment. The
median age at diagnosis was 5 years (range, 1–14). The overall response rates were 22.7%, 45.3%, and 61% at 40 days, 3 months, and
6 months, respectively, after rabbit ATG. In multivariate analysis, mild disease severity was the only predictor of favorable response at
6 months (P = 0.006). In the present study, median follow-up period was 63 months (range, 1–135). The 5-year overall survival (OS)
and failure-free survival (FFS) rates were 90.5% and 70.4%. Multivariate analysis showed that erythroid burst-forming units (BFU-E)
> 2/105 bone marrow mononuclear cell (BMMNC) (P = 0.037) and time interval before IST ≤ 30 days (P = 0.017) were independent
positive predictors for OS, meanwhile BFU-E > 2/105BMMNC (P = 0.029) was the only favorable prognostic factor for FFS.

Keywords Antithymocyte globulin . Child . Immunosuppressive therapy . Predictor . Severe aplastic anemia

Abbreviations aAA Acquired aplastic anemia

IST Immunosuppressive therapy HSCs Hematopoietic stem cells
ATG Antithymocyte globulin HSCT Hematopoietic stem cell transplantation
CsA Cyclosporine ALG Antilymphocyte globulin
SAA Severe aplastic anemia AA Aplastic anemia
HLA Human leukocyte antigen ANC Absolute neutrophil count
vSAA Very severe aplastic anemia PLT Platelet
OS Overall survival ARC Absolute reticulocyte count
FFS Failure-free survival CR Complete response
BFU-E Erythroid burst-forming units HGB Hemoglobin
BMMNC Bone marrow mononuclear cell NR Nonresponse
PB Peripheral blood PR Partial response
BM Bone marrow IMDM Iscove’s Modified Dulbecco’s Medium
FBS Fetal bovine serum
PNH Paroxysmal nocturnal hemoglobinuria
Electronic supplementary material The online version of this article CFU-GM Granulocyte-macrophage
(https://doi.org/10.1007/s00277-020-04296-9) contains supplementary
material, which is available to authorized users. colony-forming units
AML Acute myelogenous leukemia
* Xiaofan Zhu MDS Myelodysplastic syndrome
xfzhu@ihcams.ac.cn CFU-E Erythroid colony-forming units
NIH National Institutes of Health
1
Division of Pediatric Blood Diseases Center, State Key Laboratory of WBC White blood cell
Experimental Hematology, National Clinical Research Center for
ALC Absolute lymphocyte count
Blood Diseases, Institute of Hematology & Blood Diseases Hospital,
Chinese Academy of Medical Sciences & Peking Union Medical CFU-GEMM Granulocyte erythrocyte macrophage
College, 288 Nanjing Road, Heping District, Tianjin 300020, China megakaryocyte colony-forming units

Introduction
Severe aplastic anemia (SAA) is a rare but heterogeneous
disorder. It is characterized by peripheral blood (PB) pancy-
topenia and bone marrow (BM) hypoplasia [1]. The underly-
ing pathogenesis of acquired aplastic anemia (aAA) is be-
lieved to be immune-mediated damage of hematopoietic stem
cells (HSCs), with activated type 1 cytotoxic T cells implicat-
ed. For patients with SAA who lack a human leukocyte anti-
gen (HLA)-compatible sibling donor for hematopoietic stem
cell transplantation (HSCT), the mainstay of treatment is im-
munosuppressive therapy (IST) [2].
The IST regimen usually involves antithymocyte globulin
(ATG)/antilymphocyte globulin (ALG) and cyclosporine
(CsA). In many countries, horse ATG along with CsA has
been regarded as the standard IST for SAA patients, with a
60–75% hematologic response and 85% 5-year overall surviv-
al (OS) rate [3–5]. However, horse ATG is unavailable in
some countries including China. In China, both rabbit ATG
and porcine ALG are currently used as first-line treatment.
The outcome of 70 newly diagnosed SAA children treated
with porcine ALG and CsA at our institution was previously
reported [6]. Multiple studies indicated that the application of
horse ATG yields better outcomes in comparison with rabbit
ATG [7–9]. Hence, we retrospectively analyzed the clinical
features, hematologic response, and long-term outcome of pe-
diatric patients with newly diagnosed SAA who received rab-
bit ATG and CsA in our center last decade. We also explored
the predictive factors for response and survival.
Materials and methods
Study population
We conducted a single-center study at the Division of
Pediatric Blood Diseases Center, Institute of Hematology
and Blood Diseases Hospital (Tianjin, China). A total of 243
patients younger than 18 years with newly diagnosed SAA
were admitted from January 2008 to April 2018. Among
them, 70 patients were treated with porcine ALG and CsA,
and one did not have sufficient data. Therefore, 172 cases with
complete clinical information, receiving rabbit ATG plus CsA
as first-line treatment, and follow-up period over a minimum
of 6 months were included in the retrospective study. The
severity of aplastic anemia (AA) was classified by the pro-
posed criteria of Camitta et al. [10] and Bacigalupo et al.
[11]. SAA was defined as BM cellularity < 25% (or 25–50%
with < 30% residual hematopoietic cells) and at least two pe-
ripheral cytopenias: (1) absolute neutrophil count (ANC) <
0.5 × 109/L, (2) platelet (PLT) count < 20 × 109/L, (3) absolute
reticulocyte count (ARC) < 20 × 109/L. Very severe aplastic
anemia (vSAA) was considered if patients satisfied the criteria
Ann Hematol
for SAA, and ANC was < 0.2 × 109/L. Complete history and
physical examination, routine cytogenetic analysis, mitomy-
cin C testing, comet assay, and if possible, next-generation
sequencing methods were performed to exclude inherited
bone marrow failure syndrome. The patients were diagnosed
as SAA when they finished BM investigation and met the
criteria. So, the time point for diagnosis and assessment of
severity mainly based on BM investigation. None of the 172
patients had a HLA-matched sibling donor for HSCT at
diagnosis.
The study protocol conformed to the ethical guidelines of
the Declaration of Helsinki and was approved by the Ethics
Committee and Institutional Review Board of Chinese
Academy of Medical Sciences and Peking Union Medical
College. All legal guardians of pediatric patients provided
informed consent before participation.
Treatment protocol
IST consisted of rabbit ATG (Thymoglobuline®, Genzyme)
intravenously 2.5–3.5 mg/kg/day for 5 consecutive days and
oral CsA 5 mg/kg/day for at least 2 years. For the prevention
of serum sickness, intravenous methylprednisolone (1 mg/kg/
day) was administered on days 1–5 followed by oral prednis-
olone (1 mg/kg/day) for 2 weeks, and then tapered until with-
drawal on day 28. The dosage of CsA was adjusted to achieve
a trough whole-blood level at 100–200 ng/mL. Blood counts
and serum biochemistry were supervised accordingly.
Response criteria and follow-up
Hematologic responses were assessed at 40 days, 3 months,
and 6 months after the initiation of IST. Complete response
(CR) was defined as ANC ≥ 1.5 × 109/L, PLT ≥ 100 × 109/L,
and hemoglobin (HGB) normal for age and gender; nonre-
sponse (NR) was defined as persistence of severe disease;
and partial response (PR) was defined as transfusion indepen-
dent and not meeting the criteria for SAA [12, 13]. Relapse
was considered if the peripheral blood counts of patients de-
clined to a level requiring blood products transfusion, further
intensive immunotherapy, or HSCT [14, 15]. OS referred to
the time from initiation of IST until death from any cause or
last follow-up. Failure-free survival (FFS) was defined as the
time from IST until the date of treatment failure, relapse, clon-
al evolution, or death, whichever came first. For non-
responders who did not receive HSCT and finally died or
maintained alive in NR, 6 months were considered as FFS.
Culture assay of hematopoietic progenitor cells
BM samples were treated with ammonium chloride solution
and separated using Ficoll-Paque® PLUS gradient centrifuga-
tion. Then, we utilized CD34 + cell sorting method to enrich
Ann Hematol

precursor cells from BM samples. The prepared cells were Table 1 Baseline characteristics of 172 pediatric patients with SAA
diluted 1:1 with Iscove’s Modified Dulbecco’s Medium Characteristics Value
(IMDM) and 2% fetal bovine serum (FBS). Next, cell samples
were added to the MethoCult® medium H4434, shaken, and Median age at diagnosis, years (range) 5 (1–14)
inoculated into a petri dish. These cells were cultured for Gender, male (%) 74 (43%)
14 days in a 37 °C and 5% CO2 incubator. Finally, an inverted Median duration from diagnosis to IST, days (range) 31 (3–187)
microscope and a grid counting dish were used to count and PNH clone detected
judge the colony type. Minor, n (%) 58 (33.7%)
Major (> 10%), n (%) 0
Statistical analysis Disease severity
SAA, n (%) 82 (47.7%)
The statistical analysis was performed using the SPSS 18.0 vSAA, n (%) 90 (52.3%)
(SPSS, Chicago, IL, USA) and GraphPad Prism 7.04 software Peripheral blood count
(GraphPad, La Jolla, CA, USA). The chi-square test was used Median ANC, × 109/L (range) 0.18 (0–1.03)
for categorical variables. Multivariate logistic regression mod- Median HGB, g/L (range) 64 (32–117)
el was applied to evaluate which factors could predict re- Median PLT, × 109/L (range) 8 (0–41)
sponse. The OS and FFS rates were analyzed utilizing the Median ARC, × 109/L (range) 13.1 (1.5–62.5)
Kaplan-Meier method. Log-rank test was used for the com- Median MCV, fl (range) 83 (67.5–111.1)
parison between subgroups. Cox proportional hazard models Bone marrow morphology
were applied to assess risk factors for survival. P value < 0.05 Median marrow granulocyte, % (range) 8 (0–68)
was considered statistically significant. Median marrow erythrocyte, % (range) 8 (0–64)
Median marrow lymphocyte, % (range) 77 (19–99.5)
Hematopoietic progenitor cells culture of BM
Results Median CFU-E, /105BMMNC (range) 10 (0–88)
Median BFU-E, /105BMMNC (range) 2 (0–25)
Patient characteristics Median CFU-GM, /105BMMNC (range) 2 (0–31)

A total of 172 pediatric patients (74 males, 98 females) were
included in the study. The median age at diagnosis was 5 years
(range, 1–14). Ninety (52.3%) patients were diagnosed as
vSAA. The median interval from diagnosis to initiation of
IST was 31 days (range, 3–187). Flow cytometry of PB for
paroxysmal nocturnal hemoglobinuria (PNH) antigens
(CD55, CD59) was performed for all patients at diagnosis
and a minor PNH clone (less than 10%) was found in 58
(33.7%) patients. The demographic and clinical features at
baseline are shown in Table 1.
Hematologic response and response predictors
Table 2 shows the hematologic response to IST at different
time points. Two (1.2%) children died within 3 months after
treatment. Both of them were vSAA patients and died of seri-
ous infection. At 40 days, 3 months, 6 months, and 12 months
after IST, the overall response rates of all patients were 22.7%,
45.3%, 61%, and 67.4%, respectively. The SAA group had a
significantly better response rate than the vSAA group at
40 days (31.7 versus 14.4%, P = 0.007) and 6 months (72
versus 51.1%, P = 0.005). At 6 months post-IST, a total of
67 patients showed no response. Owing to financial and per-
sonal reasons, only 26 non-responders underwent HSCT, and
the remaining non-responders received high-dose CsA and
supportive care which mainly in the form of blood transfusion
SAA, severe aplastic anemia; IST, immunosuppressive therapy; PNH,
paroxysmal nocturnal hemoglobinuria; vSAA, very severe aplastic ane-
mia; ANC, absolute neutrophil count; HGB, hemoglobin; PLT, platelet;
ARC, absolute reticulocyte count; MCV, mean corpuscular volume; BM,
bone marrow; CFU-E, erythroid colony-forming units; BMMNC, bone
marrow mononuclear cell; BFU-E, erythroid burst-forming units; CFU-
GM, granulocyte-macrophage colony-forming units
and antibiotics. Additional 11 patients responded within 6–
12 months after IST. At the time point of last follow-up, 4
new patients achieved delayed response, and 11 patients were
maintained alive in NR. Therefore, the overall response rate
was 69.8% (120 responders) during the observation time.
To identify predictive factors for response to IST at
6 months, we compared the baseline variables between re-
sponders and non-responders. Univariate analysis showed that
disease severity, baseline ARC, marrow granulocyte %, and
marrow lymphocyte % were correlated with the response rate
(Table 3). We further performed multivariate logistic regres-
sion analysis which included the following baseline variables:
gender, disease severity, ARC, marrow granulocyte %, mar-
row lymphocyte %, erythroid burst-forming units (BFU-E),
and granulocyte-macrophage colony-forming units (CFU-
GM). We confirmed that mild disease severity (P = 0.006)
was a significant predictor of favorable response at 6 months
(Supplementary Table S1).
 Ann Hematol

Table 2 Hematologic response to

IST at different time points Total (n = 172) SAA (n = 82) vSAA (n = 90) P value

Response at 40 days after IST

CR 1 (0.6%) 0 (0%) 1 (1.1%)
PR 38 (22.1%) 26 (31.7%) 12 (13.3%)
NR 133 (77.3%) 56 (68.3%) 77 (85.6%)
ORR (CR/PR) 39 (22.7%) 26 (31.7%) 13 (14.4%) 0.007*
Response at 3 months after IST
CR 13 (7.5%) 8 (9.7%) 5 (5.6%)
PR 65 (37.8%) 35 (42.7%) 30 (33.3%)
NR 94 (54.7%) 39 (47.6%) 55 (61.1%)
ORR (CR/PR) 78 (45.3%) 43 (52.4%) 35 (38.9%) 0.075
Response at 6 months after IST
CR 47 (27.3%) 29 (35.4%) 18 (20%)
PR 58 (33.7%) 30 (36.6%) 28 (31.1%)
NR 67 (39%) 23 (28%) 44 (48.9%)
ORR (CR/PR) 105 (61%) 59 (72%) 46 (51.1%) 0.005*
Response at 12 months after IST
CR 57 (33.1%) 38 (46.3%) 19 (21.1%)
PR 59 (34.3%) 23 (28.1%) 36 (40%)
NR 56 (32.6%) 21 (25.6%) 35 (38.9%)
ORR (CR/PR) 116 (67.4%) 61 (74.4%) 55 (61.1%) 0.063

IST, immunosuppressive therapy; SAA, severe aplastic anemia; vSAA, very severe aplastic anemia; CR, complete
response; PR, partial response; NR, nonresponse; ORR, overall response rate
*
Represents P value < 0.05

Long-term outcome and survival analysis
For all patients, the follow-up endpoint was July 2019. The me-
dian follow-up was 63 months (range, 1–135). A total of 16
(9.3%) patients died, including 2 (1.2%) early deaths and 14
(8.1%) late deaths. Among the 14 late deaths, 6 died of serious
bacterial and fungal infection, 6 died of hemorrhage, 1 died of
secondary acute myelogenous leukemia (AML), and 1 died of
pulmonary fibrosis after HSCT. The median time of late deaths
was 10 months (range, 5–110). Relapse was observed in 7 of the
120 responders at a median of 49 months (range, 40–98) follow-
ing IST, and the cumulative incidence of relapse was 5.8%. Five
of the relapsed children were treated with increasing doses of
CsA and the other two received HSCT. At the time point of last
follow-up, 51 (42.5%) of the 120 patients with response contin-
ued to receive CsA treatment. Two (1.2%) patients who had
normal chromosomes before IST suffered from clonal evolution
at the follow-up endpoint. Between them, the one who
progressed to myelodysplastic syndrome (MDS) underwent
HSCT and was evaluated as CR at last follow-up while another
one who developed AML received chemotherapy and died dur-
ing the treatment. A total of 29 (16.9%) patients underwent
HSCT as salvage settings due to lack of response to IST (n =
26), relapse (n = 2), or clonal evolution (n = 1). Except one pa-
tient died of pulmonary fibrosis at 1 year after HSCT, the remain-
ing 28 patients were all alive at the follow-up endpoint.
The 5-year overall survival (OS) rate was 90.5% and the 5-
year FFS rate was 70.4% for all patients (Fig. 1a, b). Prognostic
factors correlated with OS and FFS were evaluated in univariate
analysis (Supplementary Table S2). Log-rank test indicated that
BFU-E (P = 0.042) and time interval before IST (P = 0.022)
were associated with OS, while BFU-E (P = 0.025) and ery-
throid colony-forming units (CFU-E) (P = 0.045) showed influ-
ence on FFS. Patients with BFU-E > 2/105 bone marrow mono-
nuclear cell (BMMNC) or time interval before IST ≤ 30 days
had a significantly higher OS rate (Fig. 2a, b). Meanwhile, pa-
tients with BFU-E > 2/105 BMMNC or CFU-E > 10/105
BMMNC had a significantly higher FFS rate (Fig. 2c, d). Cox
proportional hazard models were also used to evaluate risk fac-
tors for OS and FFS. BFU-E > 2/105 BMMNC (P = 0.037) and
time interval before IST ≤ 30 days (P = 0.017) were independent
positive predictors for OS in multivariate analysis
(Supplementary Table S3). We also confirmed that BFU-E >
2/105 BMMNC (P = 0.029) was the only favorable prognostic
factor for FFS (Supplementary Table S4).
Discussion
IST with ATG/ALG and CsA is often regarded as the initial
therapy for SAA, since most patients lack an available HLA-
matched sibling donor or are not suitable for HSCT. In China,
Ann Hematol

Table 3 Univariate analysis for

response to IST at 6 months after Factors Responders (n = 105) Non-responders (n = 67) χ2 value P value
treatment
Gender 3.385 0.066
Male 51 23
Female 54 44
Age 1.727 0.189
> 5 years 45 22
≤ 5 years 60 45
Time interval before IST 0.024 0.876
> 30 days 53 33
≤ 30 days 52 34
PNH clone 0.038 0.845
Positive 36 22
Negative 69 45
Disease severity 7.836 0.005*
SAA 59 23
vSAA 46 44
Baseline HGB 0.007 0.932
> 60 g/L 62 40
≤ 60 g/L 43 27
Baseline PLT 2.376 0.123
> 10 × 109/L 35 15
≤ 10 × 109/L 70 52
Baseline ARC 4.058 0.044*
> 10 × 109/L 68 33
≤ 10 × 109/L 37 34
Baseline MCV, fl 1.018 0.313
> 80 fl 78 45
≤ 80 fl 27 22
Baseline marrow granulocyte % 5.733 0.017*
> 10% 54 22
≤ 10% 51 45
Baseline marrow erythrocyte % 3.114 0.078
> 10% 52 24
≤ 10% 53 43
Baseline marrow lymphocyte % 6.598 0.010*
> 80% 39 38
≤ 80% 66 29
Baseline CFU-E 1.828 0.176
> 10/105BMMNC 42 20
≤ 10/105BMMNC 63 47
Baseline BFU-E 3.680 0.055
> 2/105BMMNC 45 19
≤ 2/105BMMNC 60 48
Baseline CFU-GM 3.281 0.070
> 2/105BMMNC 54 25
≤ 2/105BMMNC 51 42

IST, immunosuppressive therapy; PNH, paroxysmal nocturnal hemoglobinuria; SAA, severe aplastic anemia;
vSAA, very severe aplastic anemia; HGB, hemoglobin; PLT, platelet; ARC, absolute reticulocyte count; MCV,
mean corpuscular volume; CFU-E, erythroid colony-forming units; BMMNC, bone marrow mononuclear cell;
BFU-E, erythroid burst-forming units; CFU-GM, granulocyte and macrophage colony-forming units
*
Represents P value < 0.05

rabbit ATG was the only ATG formulation available and was
approved in 2003 as a first-line agent to treat SAA. A previous
study showed an obvious difference in 6-year OS rate among
patients of different age receiving ATG and CsA: < 20 years (n =
31, 100%), 20–40 years (n = 51, 92%), 40–60 years (n = 51,
71%), and > 60 years (n = 59, 56%) [16]. This finding indicates
that the survival rates of children and adults who receive the same
treatment differ significantly. So, the results of IST for adult and
pediatric patients with SAA should be analyzed separately.
Our study retrospectively analyzed a large cohort of 172
SAA children treated with rabbit ATG and CsA at a single
center. In our work, the overall response rates at 3 months and
6 months after IST were 45.3% and 61%, respectively. It was
comparable with the study of Jeong et al. [9] and Chen et al.
[17] in which rabbit ATG along with CsA was used as first-
line therapy for childhood SAA. However, a recent study by
North American Pediatric Aplastic Anemia Consortium
showed a higher response rate of 71.2% in subjects treated
 Ann Hematol

(a)
with horse ATG and CsA [18]. Considering baseline clinical
and laboratory data may put a salient impact on the response to
IST, we further performed univariate and multivariate analy-
ses on the baseline factors between responders and non-re-
sponders. The pre-treatment variables involved in our study
were comprehensive, which not only covered basic informa-
tion, blood counts, and PNH clone detection, but also included
the findings of BM aspiration such as BM morphology and
hematopoietic progenitor cells culture.
Several studies showed a favorable outcome in patients (b)

with AA who were PNH clone positive [19–21]. In the work

of Tutelman et al. [19], 73% of pediatric AA patients had a
PNH clone positivity and a high proportion (94%) of them
achieved either CR or PR to IST. Meanwhile, the patients
who were PNH negative had a lower response rate of 33%.
In our pediatric cohort, 58 (33.7%) patients had a PNH clone
positivity at diagnosis. However, the PNH clone detected at

(c)
(a)

(d)

(b)

Fig. 1 a Overall survival (OS) of 172 children with severe aplastic ane-
mia (SAA) who received rabbit antithymocyte globulin (ATG) and cy-
closporine (CsA). Five-year OS rate was 90.5% for all patients. b Failure-
free survival (FFS) of 172 children with severe aplastic anemia (SAA)
who received rabbit antithymocyte globulin (ATG) and cyclosporine
(CsA). Five-year FFS rate was 70.4% for all patients
Fig. 2 a Overall survival (OS) of 172 children with severe aplastic anemia
(SAA) who received rabbit antithymocyte globulin (ATG) and cyclospor-
ine (CsA) categorized by erythroid burst-forming units (BFU-E). Five-year
OS rate was significantly higher in patients with BFU-E > 2/105 bone
marrow mononuclear cell (BMMNC), 96.6 versus 87.0% (P = 0.042). b
Overall survival (OS) of 172 children with severe aplastic anemia (SAA)
who received rabbit antithymocyte globulin (ATG) and cyclosporine
(CsA) categorized by time interval between diagnosis and IST. Five-year
OS rate was significantly higher in patients with time interval ≤ 30 days,
96.5 versus 83.8% (P = 0.022). c Failure-free survival (FFS) of 172 chil-
dren with severe aplastic anemia (SAA) who received rabbit antithymocyte
globulin (ATG) and cyclosporine (CsA) categorized by erythroid burst-
forming units (BFU-E). Five-year FFS rate was significantly higher in
patients with BFU-E > 2/105 bone marrow mononuclear cell (BMMNC),
83.2 versus 63.8% (P = 0.025). d Failure-free survival (FFS) of 172 chil-
dren with severe aplastic anemia (SAA) who received rabbit antithymocyte
globulin (ATG) and cyclosporine (CsA) categorized by erythroid colony-
forming units (CFU-E). Five-year FFS rate was significantly higher in
patients with CFU-E > 10/10 5 bone marrow mononuclear cell
(BMMNC), 78.8 versus 65.6% (P = 0.045)
Ann Hematol
baseline was not correlated with an increased response rate at
6 months following IST. This outcome was consistent with
the work of Yoshida et al. [22] and the National Institutes of
Health (NIH) group [23] which demonstrated the absence of
prognostic value with PNH clone.
A previous multicenter study in Japan reported that gender,
duration from diagnosis to treatment, and white blood cell
(WBC) count were confirmed as predictive factors for hema-
tologic response [24]. Another Asian study also indicated that
male gender and shorter time interval before IST were inde-
pendent predictors of better response in childhood SAA [9]. In
our study, male group displayed better response than female
group, although the difference was not of statistical signifi-
cance (P = 0.066). Besides, the response rates were not signif-
icantly different grouped by time interval before IST.
In univariate analysis, we found that disease severity, base-
line ARC, marrow granulocyte %, and marrow lymphocyte %
were significantly associated with the response rate.
Moreover, we identified that disease severity was the only
predictive factor of response in multivariate analysis. As for
pediatric patients with SAA, baseline ARC and marrow gran-
ulocyte % can reflect the status of hematopoiesis. Thus, a
higher pre-treatment ARC and marrow granulocyte % may
show better residual hematopoiesis function and the ability
to support blood cell production following IST. The NIH
group [25] showed that baseline ARC was the predominant
factor for predicting the response at 6 months. When it comes
to the correlation between response and disease severity, con-
troversial results have been reported. A prospective multicen-
ter trial in Europe reported favorable response rates in pediat-
ric patients with vSAA compared with SAA [26], but several
studies indicated the opposite results [11, 27–29]. Our data
were consistent with those published studies which demon-
strated that SAA patients had a better response than their
vSAA counterparts. In our work, the overall response rates
of SAA children and vSAA children were 72% and 51.1%
at 6 months post-IST, respectively.
The long-term survival of responders is mainly influenced
by disease relapse and clonal evolution to MDS or AML.
Besides, the occurrence of relapse and clonal evolution are
associated with CsA tapering strategy [30] and residual hema-
topoietic cell biology [31]. In our work, the median follow-up
period was 63 months, and the actuarial risk of relapse was
only 5.8%, which was much lower than previous studies [32,
33]. The implementation of slow CsA tapering schedule and
continuous CsA treatment in nearly half of the responders at
last follow-up may account for the low relapse rate. The pres-
ent study reported that 2 (1.2%) patients experienced clonal
evolution, and the incidence of evolution was similar to rabbit
ATG cohort in the study of Jeong et al. [9].
In our pediatric cohort treated with rabbit ATG and CsA,
the 5-year OS and FFS rates were 90.5% and 70.4%, which
were obviously higher than previous report [32]. And the 5-
year OS rate was comparable with horse ATG cohort in the
study of North American Pediatric Aplastic Anemia
Consortium [18]. Regarding the association between prognos-
tic factors and survival, we further performed univariate and
multivariate analyses. Higher absolute lymphocyte count
(ALC) and ARC were previously reported to have a signifi-
cant connection to a higher survival rate [25] while in another
study, only higher ARC was of prognostic value [27]. A mul-
ticenter study in Europe and Asia reported that patients age,
disease severity, and time interval between diagnosis and
treatment were predictors of survival [29]. In our study, uni-
variate analysis showed that BFU-E and time interval before
IST were correlated with OS, while BFU-E and CFU-E
showed influence on FFS. Our multivariate analysis showed
that BFU-E > 2/105 BMMNC and time interval before IST ≤
30 days were independent positive predictors for OS, mean-
while BFU-E > 2/105 BMMNC was the only favorable prog-
nostic factor for FFS. Hematopoietic progenitor cells are ex-
istent in the blood, as well as in the BM. Changes in the
numbers of hematopoietic progenitor cells reveal alteration
of pluripotent stem cells. In AA, the most consistent finding
is the very low numbers of hematopoietic progenitor cells
including CFU-GM, BFU-E, CFU-E, and granulocyte eryth-
rocyte macrophage megakaryocyte colony-forming units
(CFU-GEMM) [34]. In the present study, the median numbers
of CFU-E, BFU-E, and CFU-GM were much decreased in the
BM of children with SAA. Besides, BFU-E > 2/105 BMMNC
was verified as a good predictor for both OS and FFS. The
study of Rosenfeld et al. [35] had described in adults levels of
residual hematopoiesis as a predictor of response to immuno-
suppressive therapy, which was in line with our work. The 5-
year OS rates of children with intervals more than 30 and of
30 days or less were 83.8% and 96.5%, respectively. Yoshida
et al. [24] reported that AA children with long-standing dis-
ease may experience irreversible destruction to hematopoietic
progenitor cells or stromal components as time passed by.
Therefore, we suggested the administration of IST as soon
as possible in all pediatric patients with SAA who lack a
matched sibling donor.
Although our study is limited by its retrospective design
such as potential confounding factors and data availability, it
supplies a contemporary analysis of the diagnostic assessment
and treatment outcome for childhood SAA. Considering that
horse ATG is unavailable in China, we cannot directly com-
pare the efficacy of rabbit ATG and horse ATG.
With the application of rabbit ATG, the result of our study
is comparable with those reported results using horse ATG as
first-line treatment.
In conclusion, our study has demonstrated that rabbit ATG
along with CsA is an effective first-line therapy for pediatric
patients with SAA, with a hematologic response rate of 61%
at 6 months following IST, a 5-year OS rate of 90.5%, and a 5-
year FFS rate of 72.1%. Mild disease severity was the only

predictor of favorable response to IST. Independent prognos-
tic factors for OS were BFU-E and time interval before IST,
while the only predictor for FFS was BFU-E.
Acknowledgments The authors would like to sincerely thank the patients
that participated in the follow-up and the support of AiYou Foundation.
Authors’ contributions Yang Lan designed the study, collected and ana-
lyzed the data, and wrote the article; Lixian Chang designed the study,
collected the data, and reviewed the article; Meihui Yi, Yuli Cai, and Jing
Feng collected and analyzed the data; Chao Liu, Xiaoyan Chen, Aoli
Zhang, and Lipeng Liu analyzed the data; Yuanyuan Ren, Shuchun
Wang, Ye Guo, and Jingliao Zhang collected the data; Xiaofan Zhu
designed the study and reviewed the article. All authors read and ap-
proved the final manuscript.
Funding This study was funded by the National Key Research and
Development Program of China (grant No.2016YFC0901503).
Data availability The datasets generated during and/or analyzed during
the current study are available from the corresponding author on reason-
able request.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethics approval This study was performed in line with the principles of
the Declaration of Helsinki. Approval was obtained from the Ethics
Committee and Institutional Review Board of Chinese Academy of
Medical Sciences and Peking Union Medical College.
Consent to participate Informed consent was obtained from legal
guardians.
Consent for publication Patients signed informed consent regarding
publishing their data.
Code availability Not applicable.
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