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Low-dose PTCy plus low-dose ATG as GVHD prophylaxis after
UD-PBSCT for hematologic malignancies: a prospective,
multicenter, randomized controlled trial
© The Author(s) 2023
Blood Cancer Journal (2023)13:10 ; https://doi.org/
10.1038/s41408-022-00771-w
Dear Editor,
For unrelated donor hematopoietic stem cell transplantation
(UD-HSCT), graft-versus-host disease (GVHD) and non-relapse
mortality (NRM) remain the main factors in success [1]. It is
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undeniable that anti-thymocyte globulin (ATG) is the standard of
care of GVHD prophylaxis for patients undergoing UD-HSCT, due
to preventing acute GVHD (aGVHD) and chronic GVHD (cGVHD)
effectively [2]. However, the use of ATG is frequently accompanied
by high rates of GVHD and infections, especially cytomegalovirus
(CMV) and Epstein-Barr virus (EBV) infection [3], affecting the long-
term survival of the patients. Post-transplant cyclophosphamide
(PTCy) has been a milestone in haploidentical HSCT (haplo-HSCT)
by mitigating bidirectional alloreactivity and promoting graft
tolerance to mitigate the risk of GVHD [4] and become prevalent
in UD-HSCT [5, 6]. It is worth mentioning that the superiority of
PTCy has been predominantly reflected in bone marrow (BM) as
the source of stem cells and peripheral blood stem cell (PBSC)
graft displayed high risk of GVHD compared to BM graft [7].
Consequently, the joint regimen of PTCy and ATG at different
dose was progressively implemented in haplo-HSCT, and
extended to UD-HSCT to improved long-term outcomes[8, 9].
The combination of low-dose ATG (5 mg/kg) and low-dose PTCy
(one dose, 50 mg/kg) as GVHD prophylaxis reduced the risk of
GVHD in haplo-HSCT and matched unrelated donor peripheral
blood stem cell transplantion (MUD-PBSCT) [8, 10]. In addition, the
joint use of low-dose PTCy (14.5 mg/kg on days 3 and 4) plus
standard-dose ATG as well as low-dose ATG (4.5 mg/kg) plus
standard-dose PTCy (50 mg/kg on days 3 and 4) demonstrated
significant improvements in the rates of GVHD and NRM in haplo-
HSCT and UD-HSCT [9, 11].
Although those joint strategies are effective as GVHD prophy-
laxis, there is paucity of comparative data between low-dose
PTCy-ATG versus quadruplet ATG based regimen in UD-PBSCT. We
initiated a prospective, multicenter, randomized controlled clinical
trial to assess the efficacy of the novel regimen for GVHD
prophylaxis based on low-dose rabbit ATG (6 mg/kg, Sanofi-
Aventis) and low-dose PTCy (20 mg/kg on days 3 and 4),
cyclosporin A (CsA) and mycophenolate mofetil (MMF) initiating
on day +5 in UD-PBSCT (ChiCTR2200056979). The patients of
control cohort were performed with quadruplet ATG based
regimen, ATG 2.0 mg/kg/day on days −5 to −1 and short-course
methotrexate (MTX) 10 mg/m2 on day +1 followed by 7 mg/m2 on
days +3, +6, and +11, with CsA and MMF on day −1.
Received: 8 November 2022 Revised: 7 December 2022 Accepted: 8 December 2022
www.nature.
From January 2019 to March 2022, a total of 162 patients with
hematological malignancies at ages 14–62- years from 3 institu-
tions were eligible in the study and a total of 160 patients were
enrolled. The patients were randomly one-to-one assigned to the
low-dose PTCy-ATG and the quadruplet ATG cohorts (Supplemen-
tary Fig. 1). All patients were eligible for adequate baseline
laboratory values. This study had ethical approval from ethical
committees of each center and in accordance with the Declaration
of Helsinki. All patients provided informed consent prior HSCT. The
conditioning regimens for all patients were based on fludarabine
30 mg/m2/day on days −6 to −2, busulfan at cumulative dose of
51.2 mg/kg divided into 16 doses on days −5 to −2, cytarabine
(Ara-C) 2.0 g/m2/day on days −6 to −2. The patients with donor
specific antibody-positive (with a median fluorescent intensity ≥
10,000) were administered 2.5–3 Gy of total body irradiation on
day −7. Rituximab at dose of 100 mg on day +5 was given to
patients in the low-dose PTCy-ATG cohort as EBV prophylaxis.
There was no difference in the baseline characteristics between
both groups except that the patients receiving a graft that was
allele mismatches in the low-dose PTCy-ATG cohort were more
than those in the quadruplet ATG cohort (48.8% vs. 30.0%,
P = 0.047) (Supplementary Table 1). The median follow-up time
was 457 (44-1376) days in the low-dose PTCy-ATG cohort as
compared to 424 (8-1394) days in the quadruplet ATG cohort
(P = 0.325).
Primary graft failure was documented in two patients in each
cohort. One recipient in each of the two groups had mixed
chimerisms at day +30 (Table 1). Two patients underwent second
salvage allo-HSCT in the low-dose PTCy-ATG cohort. No significant
difference in mononuclear cells and CD34+ cells was observed in
both cohorts. The median times to neutrophil and platelet
recovery in cohort low-dose PTCy-ATG were 2 days shorter
compared to those in cohort quadruplet ATG (12 days vs. 14 days;
P = 0.000; and 12 days vs. 14 days; P = 0.000, respectively)
(Table 1), which inconsistent with that the reconstitution
advantage of PBSC might be negated [12]. Accelerated the
hematopoietic reconstitution of addition to low-dose ATG in the
low-dose PTCy-ATG cohort and delayed engraftment due to use of
MTX in the quadruplet ATG cohort probably explained the finding.
On competing risk analysis, the 100-day cumulative incidences
(CIs) of grade II–IV and III–IV aGVHD in the low-dose PTCy-ATG
cohort were significantly lower as compared with those in the
quadruplet ATG cohort (23.8% vs. 40.0%, P = 0.026; 6.3% vs.
17.5%, P = 0.029, respectively) (Table 1) (Fig. 1A; Fig. 1B). The
decrease was also noted in 2-year CI of cGVHD compared to
quadruplet ATG cohort (14.7% vs. 26.7%, P = 0.047) (Fig. 1C). We
found no difference in moderate to severe cGVHD at 2 years
between the study cohorts (Fig. 1D). Furthermore, an exploratory
post-hoc subgroup analysis of grade III–IV aGVHD is depicted in
 Correspondence
2
Table 1. Outcomes and complications of two cohorts.
Variables PTCy-ATG group(N = 80) Quadruplet ATG group(N = 80) P values
Time to ANC recovery (Median,days) 12(10–15) 14(9–31) <0.001
Time to platelets recovery (Median,days) 12(9–22) 14(9–66) <0.001
Chimerism at day +30(n, %)
Full donor chimerism 79(98.8) 79(98.8) 1.000
Cumulative incidence GVHD % (95% CI)
Grade II–IV aGVHD at day +100 23.8(15.1–33.6) 40.0(39.2–50.6) 0.026
Grade III–IV aGVHD at day +100 6.3(2.3–13.0) 17.5(10.1–26.6) 0.029
cGVHD at 2 years 14.7(7.7–23.7) 26.7(17.4–36.8) 0.047
Moderate/Severe cGVHD at 2 years 4.2(1.1–10.7) 11.5(5.6–19.8) 0.073
Cumulative incidence % (95%CI)
Non-relapse mortality at 2 years 19.5(11.0–29.8) 28.7(18.2–40.1) 0.162
Relapse at 2 years 5.5(1.8–12.6) 3.8(1.0–9.7) 0.668
Disease-free survival at 2 years 75.0(69.3–80.3) 67.4(61.5–73.3) 0.228
Overall survival at 2 years 79.4(74.3–84.5) 68.4(61.6–73.2) 0.055
GVHD and relapse-free survival at 2 years 66.1(60.4–71.8) 49.3(42.9–55.7) 0.032
Cumulative incidence % (95%CI)
CMV reactivation at day 180 53.6(44.9–66.8) 40.0(29.2–50.6) 0.053
CMV disease at day 180 5.0(1.6–11.4) 2.5(0.5–7.9) 0.407
EBV reactivation at day 180 11.3(5.5–19.3) 68.8(57.2–77.8) <0.001
Other complications (n, %)
PTLD 1(1.3) 0(0.0) 0.316
Pulmonia 32(40.0) 39(48.8) 0.265
Bacteremia 5(6.3) 9(11.3) 0.263
Hemorrhagic cystitis 34(42.5) 47(58.8) 0.040
TMA 5(6.3) 2(2.5) 0.246
PRES 4(5.0) 3(3.8) 0.699
VOD 2(2.5) 2(2.5) 1.000
ANC absolute neutrophil count, aGVHD acute graft-versus-host disease, cGVHD chronic graft-versus-host disease, CI cumulative incidence, CMV
cytomegalovirus, EBV Epstein-Barr virus, PTLD posttransplantation lymphoproliferative disorders, TMA thrombotic microangiopathy, PRES posterior reversible
encephalopathy syndrome, VOD veno-occlusive disease of the liver.

Supplementary Fig. 2, which was not powered to show an effect
between two groups. However, it was a trend that HLA
mismatched patients preferred to the novel regimen compared
to HLA-matched patients in III–IV grade aGVHD end point. HLA-
matched degree was the foremost element related to GVHD
presence. Previous research showed that the CIs of aGVHD and
cGVHD in patients receiving UD-HSCT were comparable with
standard PTCy-based regimen or low-dose PTCy-ATG regimen for
GVHD prophylaxis [10, 13]. Though those results suggested HLA-
matched intensity might not affect the incidences of GVHD under
the platforms of PTCy or PTCy-ATG, the results should be
interpreted with caution due to limited sample size. In this study,
the DQ mismatch in HLA 9/10 mismatched unrelated donor PBSCT
was no difference between two cohorts (Supplementary Table 1).
Due to limited number of patients, we need to enlarge the sample
size to further analyze the effect of different mismatch HLA loci on
the GVHD.
Worth mentioning is that the CI of CMV reactivation at day 180
had a trend to increase for patients with low-dose PTCy-ATG, even if
there was no significant difference (53.6% vs. 40.0%, P = 0.053)
(Table 1). A recent Center for International Blood and Marrow
Transplant Research study demonstrated that PTCy doubled the risk
of CMV infection for CMV seropositive recipients. PTCy could give
priority to sparing and recover suppressive regulatory T-cells by
selectively impairing allo-reactive T-cells [14], which may affecting
cellular immunity against CMV. EBV reactivation is another common
complication of infection. Our results showed that the risk of EBV
reactivation was significantly reduced in the low-dose PTCy-ATG
cohort (11.3% vs. 68.8%, P < 0.001), which may be due to the
administration of prophylactic rituximab, and it was a small flaw due
to mismatch on rituximab between the two groups. However, the
dose and timing of rituximab as given is mostly empirical. What is
more, it is controversial whether rituximab administration early has
an impact on the occurrence of GVHD [15]. In other complications,
no significant difference was observed in the incidence of pulmonia,
bacteremia, transplant-associated thrombotic microangiopathy
(TMA), posterior reversible encephalopathy syndrome and veno-
occlusive disease of the liver except that the significant lower was
noted in the incidence of hemorrhagic cystitis with BK virus compare
to quadruplet ATG cohort (42.5% vs. 58.8%, P = 0.040) within the
duration of follow-up (Table 1). It was worth mentioning that the rate
of TMA was 6.3%. Whether it related to the administration of PTCy
deserves further study.
In terms to the outcomes, the 2-yaer CIs of NRM and CIs of
relapse (CIR) between two cohorts were comparable (P = 0.162;
P = 0.668, respectively)(Supplementary Fig. 3A, B). Interesting that
the CIR did not associated with the decrease in GVHD and NRM.
Then, we observed a significantly better GVHD-free/relapse-free
survival (GRFS) in the low-dose PTCy-ATG cohort (66.1%)
compared with the quadruplet ATG cohort (49.3%) (P = 0.032)
(Fig. 1E), which indicating the combination of low-dose PTCy and
low-dose ATG had the potential to assist patients to gain an

Blood Cancer Journal (2023)13:10

 Correspondence
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Fig. 1 Cumulative incidences (CIs) of graft-versus-host-disease (GVHD) and clinical outcomes between low-dose post-transplant
cyclophosphamide (PTCy) combined with low-dose anti-thymocyte globulin (ATG) and quadruplet ATG cohorts. A The CI of grade II–IV
acute GVHD (aGVHD); B The CI of grade III–IV aGVHD; C The 2-yaer CI of chronic GVHD (cGVHD); D The 2-yaer CI of moderate to severe cGVHD;
E The 2-yaer CI of GVHD-free, relapse-free survival (GRFS); F The 2-yaer probability of overall survival (OS).

improved quality of life. The 2-year probability of disease-free
survival was comparable (P = 0.228) (Supplementary Fig. 3C).
Finally, low-dose PTCy-ATG had a trend to improve OS for patients
after UD-PBSCT (79.4% vs. 68.4%; P = 0.055) (Fig. 1F), albeit no
statistical difference. The causes of death is depicted in
Supplementary Table 2.
In conclusion, this is a pilot study to establish a low-dose PTCy
and low-dose ATG regimen to effectively prevent GVHD in UD-
HSCT. Further studies with high methodological quality, such as
further investigations of the optimal dose and schedule of the
combination of ATG and PTCy are warranted in the future to verify
the feasibility of the strategy.
1 1 1 1
Yingling Zu , Ruirui Gui , Zhen Li , Juan Wang , Yanli Zhang ,
Fengkuan Yu1, Huifang Zhao1, Xinrong Zhan2, Zhongliang Wang2,
✉ equivalent to those of contemporaneous HLA-matched related and unrelated
Pengtao Xing , Xianjing Wang3, Huili Wang3, Yongping Song 4
2
1✉
and Jian Zhou
1
Department of Hematology, Affiliated Cancer Hospital of
Zhengzhou University and Henan Cancer Hospital, Zhengzhou
450000 Henan, China. 2Department of Hematology, Central Hospital
of Xinxiang, Xinxiang 453000 Henan, China. 3Department of
Hematology, The Third People’s Hospital of Zhengzhou, Zhengzhou
450000 Henan, China. 4Department of Hematology, the First
Affiliated Hospital of Zhengzhou University, Zhengzhou 450052
Henan, China. ✉email: songyongping001@126.com;
zhoujiandoctor@163.com
DATA AVAILABILITY
The datasets used and/or analyzed during the current study are available from the
corresponding author on reasonable request.
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ACKNOWLEDGEMENTS
We are grateful to all of the patients and the co-investigators for their cooperation in
this study.
AUTHOR CONTRIBUTIONS
YLZ analyzed the data, wrote the paper and takes responsibility for the integrity of
the work as a whole. JZ and YPS designed the research. Other authors assessed
patients for eligibility, collected data, and critically revised the paper. All authors
contributed to the paper and approved the submitted version.
FUNDING
This work was partly supported by grants from the Henan Province Medical Science
and Technology Project (grant number SBGJ202103034).
COMPETING INTERESTS
The authors declare no competing interests.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The study protocol was approved by the Ethics Committee of Affiliated Cancer
Hospital of Zhengzhou University, Central Hospital of Xinxiang and the Third People’s
Hospital of Zhengzhou. The study is registered at http://www.chictr.org.cn/
ChiCTR2200056979. Informed consent was obtained from each patient.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41408-022-00771-w.
Correspondence and requests for materials should be addressed to Yongping Song
or Jian Zhou.
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© The Author(s) 2023
Blood Cancer Journal (2023)13:10