Impacts of post-transplantation cyclophosphamide treatment after allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia

Abstract

Post-transplant cyclophosphamide has become a promising medical option after allogeneic HSCT. In this study we aimed to evaluate the efficacy of cyclophosphamide and cyclosporine combination in acute and chronic graft-versus-host disease (GvHD) prophylaxis in acute myeloid leukemia (AML) cases scheduled for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Retrospective analysis of data from 40 cases who underwent allogeneic HSCT under GvHD prophylaxis with cyclophosphamide and cyclosporine combination between April 2016 and August 2017 was made. Cyclophosphamide was given at daily doses of 50 mg/kg on post-transplant 3rd and 4th days, and cyclosporine was applied at daily doses of 3 mg/kg/day starting from the 5th post-transplant day. Cyclosporine dose was tapered beginning from the 45th postoperative day and completely discontinued on the 90th post-transplant day. Mean age was 38.25 ± 15.25 years. Posttransplant median follow-up was six months (6–17 months). Post-transplant, the number of deaths and mortality rates related and unrelated to transplantation were 5 (12.5%), and 2 (5%), respectively. Acute GvHD was diagnosed in 7 cases (17.5%), and relapse was noted in 9 cases (22.5%). Myeloablative or reduced intensity conditioning was performed in 22 (55%) and 18 (45%) patients, respectively. The distribution of the donors was as follows: match-related (n = 26; 65%), match-unrelated (n = 9, 22.5%) and haploidentical donors (n = 5; 12.5%). There was no statistically significant correlation between the transplant-related and unrelated mortality and parameters under investigation.Cyclophosphamide use appears to be a highly effective and promising strategy for acute GvHD prophylaxis in non-haploidentical allogeneic HSCT cases. Identification of the impact of cyclophosphamide use on the development of chronic GvHD needs further investigation.

Full text

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Impacts of post-transplantation
cyclophosphamide treatment after
allogeneic hematopoietic stem cell
transplantation in acute myeloid
leukemia
Sinem Namdaroglu, Ali Hakan Kaya, Hikmettullah Batgi, Omur Kayikci, Mehmet Sinan Dal,
Dicle Iskender, Merih Kizil Cakar, Emre Tekgunduz & Fevzi Altuntas
Post-transplant cyclophosphamide has become a promising medical option after allogeneic HSCT. In
this study we aimed to evaluate the ecacy of cyclophosphamide and cyclosporine combination in
acute and chronic graft-versus-host disease (GvHD) prophylaxis in acute myeloid leukemia (AML) cases
scheduled for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Retrospective analysis of
data from 40 cases who underwent allogeneic HSCT under GvHD prophylaxis with cyclophosphamide
and cyclosporine combination between April 2016 and August 2017 was made. Cyclophosphamide was
given at daily doses of 50 mg/kg on post-transplant 3rd and 4th days, and cyclosporine was applied at
daily doses of 3 mg/kg/day starting from the 5th post-transplant day. Cyclosporine dose was tapered
beginning from the 45th postoperative day and completely discontinued on the 90th post-transplant day.
Mean age was 38.25 ± 15.25 years. Posttransplant median follow-up was six months (6–17 months).
Post-transplant, the number of deaths and mortality rates related and unrelated to transplantation
were 5 (12.5%), and 2 (5%), respectively. Acute GvHD was diagnosed in 7 cases (17.5%), and relapse
was noted in 9 cases (22.5%). Myeloablative or reduced intensity conditioning was performed in 22
(55%) and 18 (45%) patients, respectively. The distribution of the donors was as follows: match-related
(n = 26; 65%), match-unrelated (n = 9, 22.5%) and haploidentical donors (n = 5; 12.5%). There was
no statistically signicant correlation between the transplant-related and unrelated mortality and
parameters under investigation.Cyclophosphamide use appears to be a highly eective and promising
strategy for acute GvHD prophylaxis in non-haploidentical allogeneic HSCT cases. Identication of the
impact of cyclophosphamide use on the development of chronic GvHD needs further investigation.
Transplantation of hematopoietic stem cells from any source (bone marrow, peripheral blood, umbilical cord
blood) is a treatment not only for hematopoietic system diseases but also for metabolic and immunological disor-
ders. Patients with hematopoietic stem cell transplantation (HSCT) carry a high risk of transplant-related mortal-
ity and morbidity due to immunological mechanisms, toxicity due to drugs used in the preparation regimens, and
long hospitalization times1. In addition to early complications of HSCT, particularly allogeneic transplant patients
are exposed to long-term consequences that require lifelong follow-up and treatment2.
Transplantation-related mortality has gradually decreased in recent years with the development of supportive
therapies, preventive treatments, and early diagnosis facilities. However, HSCT, in addition to its therapeutic
properties, faces us with its many complications. HSCT can be classied according to the source of the progenitor
cell used. Although both of these sources have advantages and disadvantages, both infectious and non-infectious
complications are more likely to occur in allogeneic transplantations1,2. In a cohort study involving 1479 patients
with at least two years survival aer allogeneic HSCT, relapse of the primary disease was the most frequent
Department of Hematology, Stem Cell Transplantation Unit, University of Health Sciences,Ankara Oncology Training
and Research Hospital, Ankara, Turkey. Correspondence and requests for materials should be addressed to S.N.
(email: drsinemnamdaroglu@gmail.com)
Received: 5 October 2018
Accepted: 20 December 2018
Published: xx xx xxxx
OPEN
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cause of mortality (29%), while the most common causes of non-relapse mortality were gra-versus-host disease
(GvHD) (22%), infections (11%), secondary malignancies (7%), pulmonary complications (5%), cardiac toxicity
(2%) and other treatment-related events (8%)3.
In a similar retrospective analysis performed in autologous stem cell transplantation treated diuse large β
cell patients, causes of mortality apart from relapse of the disease were found to be respiratory failure (31%),
infections (13%), cardiac toxicity (15%) and secondary malignancies (15%)4. Allogeneic HSCT is a treatment
option with the potential to cure many malignant and non-malignant hematological disorders. As results are
improved with preventive and supportive therapies, indications are also developing. Alternative stem cell sources
have increased the likelihood of nding donors, and even haploidentical transplantation have yielded acceptable,
successful outcomes.
Post-transplant cyclophosphamide has become a promising medical option aer allogeneic HSCT. is
method has gained popularity due to its safety prole and ecacy for reduction of GvHD5. HLA-haploidentical
HSCT using post-transplant high-dose cyclophosphamide is becoming a more popular alternative strategy for
allogeneic HSCT6.
e aim of the present study was to investigate the eect of posttransplant cyclophosphamide use on mortality,
relapse and acute or chronic GvHD formation in acute myeloid leukemia (AML) patients with allogeneic HSCT.
Materials and Methods
Study design. In this retrospective study, data were extracted from the les of 40 AML patients treated with
allogeneic HSCT in a tertiary center who were also receiving immunosuppressive therapy with cyclophosphamide
and cyclosporine during the post-transplant period. is study was conducted by the Declaration of Helsinki and
was approved by Ankara Oncology Training and Research hospital ethics committee. Written informed consent
was obtained from all patients.
In our center, medical records of allogeneic HSCT patients under prophylaxis for GvHD with cyclophospha-
mide during the post-transplant period were retrospectively evaluated between April 2016 and August 2017. A
total of 40 patients (14 female, 26 male) had a mean age of 38.25 ± 15.25 years in our series. In all cases, cyclo-
phosphamide at daily doses of 50 mg/kg was given on 3rd and 4th days aer transplantation, and cyclosporine
at daily doses of 3 mg/kg/day starting from the 5th postoperative day was administered. Cyclosporine dose was
tapered beginning from the 45th postoperative day, and completely discontinued on the 90th postoperative day.
Acute GvHD detected on the cases was staged according to standard criteria.
Patients were divided into two groups according to pre-transplant risk class, chemotherapy regimen applied
(myeloablative or reduced intensity monitoring), date of transplantation, GvHD, the presence of a genetic anom-
aly, transplant-related and unrelated 100-day mortality rates and relapses were recorded.
Cytogenetic risk classication was performed according to standard criteria7. Conditioning intensity was
dened as myeloablative or reduced intensity using the consensus criteria8. e day of platelet recovery was
termed as the rst day on which the platelet count reached or exceeded 20 × 109/L without transfusion. e
day of neutrophil recovery was dened as the rst of 3 consecutive days on which the absolute neutrophil count
was more than 0.5 × 109/L. e International Bone Marrow Transplant Registry criteria were used for acute
GvHD staging and grading, and the National Institute of Health criteria were used to determine chronic GvHD
severity9–11.
Initial neutrophil engraftment was defined as the first of 3 consecutive days with an ANC of more than
0.5 × 109/L. Platelet engrament was dened as the rst of 3 consecutive days with a PLT count of more than
20 × 109/L without transfusion support. GVHD was graded by the consensus criteria12–14.
e inclusion criteria for this study consisted of: (1) using G-CSF mobilized peripheral blood as gra source,
(2) using a matched related/unrelated or haploidentical donor and (3) using PT-Cy (50 mg/kg on days +3 and
+4), a calcineurin inhibitor and mycophenolate mofetil as GvHD prophylaxis. Filgrastim 5 μg/kg daily was
administered starting day +5 until neutrophil recovery. e lack of complete remission on the last bone marrow
biopsy performed within 4 weeks before conditioning initiation was accepted as active disease.
Donor engrament was determined by a PCR-based assay for STRs from bone marrow samples or peripheral
blood mononuclear cells9, and dened as donor chimerism >95%, and gra failure as <5% not due to relapse.
Gra failure, relapse or death (all without GvHD) were considered competing risks for GvHD; relapse was
considered a competing risk for nonrelapse mortality; death without relapse was considered competing risk for
relapse; and death without count recovery was considered a competing risk for count recovery.
e allogeneic stem cell transplantation was performed with a conditioning regimen including Busulfan s3.2 mg/
kg/day for three days (BU3), Fludarabine 40 mg/m²/day for four days (FLU4), rabbit Anti-thymocyte globulin
2.5 mg/kg/day for three days.(ATG). Patients were hydrated starting fron 24 hours prior to the stem cell infusion.
Premedication consistent of chlorfenoxamine hydrochloride (10 mg) in 100 ml of isotonic saline and dexamethasone
(8 mg) in 100 ml isotonic saline was administered within 30 minutes approximately 1 hour before stem cell infusion.
e infusion volume and the number of stem cells (CD34+ or nucleated cells) were noted. Irradiation or the uses of
infusion pump or leukocyte lter were omitted. e rate of stem cell infusion was 2 ml/min. In case of the absence
of any reaction in 30 minutes, infusion was carried on at a rate of 150–200 ml/hour. e infusion is completed max-
imally within 4 hours. In case of major incompatibility, stem cell transfusion was initiated at a rate of 20 ml/hour in
the rst 30 minutes and at a rate of 40 ml/hour in the second 30 minutes. Aer the rst hour, infusion was carried on
at a rate of 150–200 ml/hour. For major incompatibility, the rates of infusion were 60 ml/hour and 90 ml/hour for the
rst and second 30 minutes, respectively. Aer the rst hour, the rate was kept at 150–200 ml/hour.
Statistical analysis. SPSS 21 program (Chicago, USA) was used for data analysis. As a descriptive analysis,
the mean ± standard deviation was given when assumptions were met for numerical variables, and when not met
the median and the smallest-largest values were given. For categorical variables, numerical and percentage values
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were given. e t-test was used in independent groups when assumptions were met in intergroup comparisons
of numerical measurements, when not met the Mann-Whitney U test was used. For the comparisons between
the categorical variables, the chi-square test was used. A value of p < 0.05 was considered statistically signicant.
Results
Posttransplant median follow-up was 6 (range, 1 to 17) months. Most frequently detected genetic
anomalies encountered in this series combined WT1 (n = 9, 22.5%), FLT3 ITD and WT1 positivity (n = 4; 10%).
Myeloablative or reduced intensity conditioning was performed in 22 (55%) and 18 (45%) patients, respectively.
e distribution of the donors was as follows: (MRD) (n = 26; 65%), match-unrelated (n = 9, 22.5%) and haploi-
dentical (n = 5; 12.5%).
As for the number of posttransplant deaths, the mortality rates related, and unrelated to bone marrow trans-
plantation were determined as 5 (12.5%) and 2 cases (5%), respectively.
Acute GvHD has been diagnosed in 7 cases (17.5%) as skin, liver, and gastrointestinal tract involvement can
occur. Chronic GvHD was not observed in any case. Relapse was seen in 9 cases (22.5%).
Correlation analysis between parameters studied (aGvHD, relapse, gender, genetic anomaly, pre-transplant
protocol, type of donor) and mortality status related or unrelated to transplantation on the 100th postoperative
day revealed no statistically signicant dierence (Tables1 and 2).
Correlation analysis between parameters studied (relapse, gender, genetic anomaly, pre-transplant protocol,
type of donor) and aGvHD revealed no statistically signicant dierence (Table3).
Correlation analysis between parameters studied (gender, genetic anomaly, pre-transplant protocol, type of
donor) and relapse revealed no statistically signicant dierence (Table4).
ere was no statistically signicant relationship between age-related variables and the transplant-related
mortality (p = 0.905), the presence of aGvHD (p = 0.304) and relapse (p = 0.483).
Discussion
Allogeneic HSCT is a complex procedure which is used to treat many hematological and immunological entities.
In spite of its therapeutic potential, toxicities linked with its application usually restrict the utility of allogeneic
HSCT against high-risk diseases or if other less toxic therapies have failed. ere are mainly two complications
related to the procedure: conditioning regimen toxicity and immunological complications15. e development
of immunological co-morbidities aer allogeneic HSCT is related to the diversity in human leukocyte antigen
(HLA) status between the donor and the recipient. Eorts have been spent to improve HLA donor typing and use
of immunosuppressive agents following allogeneic HSCT.
ere is an increasing interest in the use of agents such as cyclophosphamide and cyclosporine for GvHD
prophylaxis, both in the haploidentical and HLA-identical settings. is method oers advantages such as the
improved eect on GvHD control without any complex procedures like gra manipulation5,6. e safety prole of
this method is satisfactory compared to previous protocols, and this is evident not solely from the very low rate of
gra failure but also in an acceptable incidence of infective complications. ere are still many questions awaiting
to be replied related to the ways for improvement of these settings. Even if the use of immunosuppressive agents
is feasible, the relatively high rate of aGvHD and vagueness on the incidence of cGvHD constitute important
challenges. erefore, keeping aGvHD rate lower with dierent immunosuppressive protocols is important. In
this aim, the use of cyclosporine together with cyclophosphamide have yielded promising results16. Consequently,
an acceptable toxicity prole and a fast immune reconstitution must be targeted to establish an immunological
platform capable for reduction of relapse aer transplantation.
Var ia bl e
Mortality status on the 100th
postoperative day related to
transplantation
p-valueSurvive (n, %) Dead (n, %)
aGvHD Present 28 (71.8) 4 (10.2) 1.000
Absent 6 (15.4) 1 (2.6)
Relapse Present 26 (66.7) 5 (12.8) 0.570
Absent 9 (23.1) 0
Gender Female 12 (30.8) 2 (5.1) 1.000
Male 23 (59) 3 (7.7)
Genetic anomaly Present 4 (10.2) 01.000
Absent 30 (76.9) 5 (12.8)
Pre-transplant protocol Myeloablative 18 (46.2) 4 (10.2) 0.355
RIC 17 (43.6) 1 (2.6)
Type of donor
MRD 24 (61.5) 2 (5.1)
0.220Haploidentical 3 (7.7) 2 (5.1)
MUD 8 (20.5) 1 (2.6)
Table 1. Correlation between parameters studied and mortality status related to transplantation on the 100th
postoperative day. (Abbreviations: aGvHD: acute gra versus host disease; RIC: reduced intensity conditioning;
MRD: match related donor; MUD: match-unrelated donor).
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Ruggeri et al. evaluated the eect of stem cell source in haploidentical transplantation with posttransplan-
tion cyclophosphamide in 451 patients transplanted for AML and revealed that the use of peripheral blood
stem cells increases the risk of acute GVHD, whereas survival outcomes are comparable17. Experience using
post-transplant cyclophosphamide as GVHD prophylaxis in allogeneic HSCT from matched sibling or unrelated
donors is limited and with controversial results. Ruggeri et al. analyzed 423 patients with acute leukemia who
Var ia bl e
Mortality status on the 100th
postoperative day unrelated
to transplantation
p-valueSurvive (n, %) Dead (n, %)
aGvHD Present 28 (71.8) 4 (10.2) 1.000
Absent 6 (15.4) 1 (2.6)
Relapse Present 26 (66.7) 5 (12.8) 0.404
Absent 9 (23.1) 0
Gender Female 12 (30.8) 2 (5.1) 0.533
Male 23 (59) 3 (7.7)
Genetic anomaly Present 4 (10.2) 01.000
Absent 30 (76.9) 5 (12.8)
Pre-transplant protocol Myeloablative 18 (46.2) 4 (10.2) 0.196
RIC 17 (43.6) 1 (2.6)
Type of donor
MRD 24 (61.5) 2 (5.1)
0.301Haploidentical 3 (7.7) 2 (5.1)
MUD 8 (20.5) 1 (2.6)
Table 2. Correlation between parameters studied and mortality status unrelated to transplantation on the 100th
postoperative day. (Abbreviations: aGvHD: acute gra versus host disease; RIC: reduced intensity conditioning;
MRD: match related donor; MUD: match-unrelated donor).
Var ia bl e
aGvHD
p-value+ −
Relapse Present 24 (61.5) 6 (15.4) 1.000
Absent 8 (20.5) 1 (2.6)
Gender Female 10 (25.6) 4 (10.2) 0.225
Male 22 (56.4) 3 (7.7)
Genetic anomaly Present 3 (7.7) 1 (2.6) 1.000
Absent 28 (71.8) 6 (15.4)
Pre-transplant protocol Myeloablative 20 (51.3) 2 (5.1) 0.205
RIC 12 (30.8) 5 (12.8)
Type of donor
MRD 21 (55.3) 4 (10.2)
0.424Haploidentical 3 (7.7) 2 (5.1)
MUD 8 (20.5) 1 (2.6)
Table 3. e correlation between acute gra-versus-host disease (aGvHD), and the parameters analyzed.
(Abbreviations: aGvHD: acute gra versus host disease; RIC: reduced intensity conditioning; MRD: match
related donor; MUD: match unrelated donor).
Var ia bl e
Relapse
p-value+ −
Gender Present 12 (30.8) 2 (5.1) 0.453
Absent 19 (48.7) 7 (17.9)
Genetic anomaly Female 3 (7.7) 1 (2.6) 1.000
Male 28 (71.8) 7 (17.9)
Pretransplant protocol Myeloablative 18 (46.2) 4 (10.2) 0.705
RIC 13 (33.3) 5 (12.8)
Type of donor
MRD 19 (48.7) 7 (17.9)
0.243Haploidentical 5 (12.8) 0
MUD 7 (17.9) 2 (5.1)
Table 4. Correlation between relapse and the parameters analyzed. (Abbreviations: RIC: reduced intensity
conditioning; MRD: match related donor; MUD: match unrelated donor).
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received cyclophosphamide alone or in combination with other immunosuppressive drugs as GVHD prophylaxis
and stated that the addition of other immunosuppressive drugs enhances its eect and reduces the risk of severe
cGVHD, reducing mortality and improving survival18.
Gra-versus-host disease is a leading cause of non-relapse mortality aer allogeneic HSCT19. More recently,
high dose administration of cyclophosphamide aer transplantation has been introduced as an eective method
of prophylaxis for GvHD20. is protocol is based on the logic that alloreactive donor T lymphocytes can be elimi-
nated, which are induced to proliferate soon aer transplant, which induces tolerance. Trials on non-myeloablative
conditioning haploidentical HSCT with cyclophosphamide aer transplantation displayed obvious tolerability
with low rates of GvHD, infection, and mortality unrelated to relapse16. e relapse of malignancy is still the
predominant cause of treatment failure. To reduce the risk of relapse in patients with high-risk hematologic malig-
nancies, the feasibility of cyclophosphamide aer transplantation administration utilizing reduced intensity or
myeloablative conditioning regimens can be used. Our results indicated that none of the parameters under inves-
tigation were signicantly linked with mortality, relapse, and GvHD. Owing to the success of cyclophosphamide
aer transplantation tolerance in the setting of haplo-HCT, this regime has been extended to the patients who
underwent allogeneic HSCT from HLA-matched donors. Acute GvHD is a challenge that must be managed, and
its incidence can be reduced using the utility of one or more immunosuppressive agents21. In this perspective, we
recommend the use of cyclophosphamide and cyclosporine as prophylactic agents for GvHD. Currently, sucient
evidence for its ecacy and safety aer transplantation has accumulated under both reduced intensity and myelo-
ablative settings. e timing of transplantation is supposed to be critical for a successful outcome for patients with
hematological malignancies. Cyclophosphamide aer transplantation allows expansion of the donor pool, making
marrow transplantation applicable for patients with stem cell donor. is method is associated with a low risk of
complications, even with haploidentical related donors. Factors such as the experience of the center, facilities and
the availability of clinical trials must be remembered to set the suitable treatment algorithm. Allogeneic HSCT
with cyclophosphamide aer transplantation, as an immunological platform, provides the chance to safely explore
innovative approaches to diminish the risk of relapse following transplant5,6.
An ideal immunosuppressive regimen needs to be eective for patients with standard or high-risk myeloid
malignancies, who are going to receive HSCT. It is expected to have reduced extramedullary toxicity, low rate of
relapse, acute and chronic GvHD rate, and improved survival.
Stem cell transplantation is an important treatment modality and cure for malignant or non-malignant bone
marrow diseases. However, considering the causes of mortality other than the relapse of the primary disease,
long-term multidisciplinary and close follow-up of the patients is needed, because of the early or long-term
comorbidities and complications that may arise. In a recent study, Chiusolo et al. suggested that post-transplant
cyclophosphamide regimen provided protection against GVHD, low toxicity, and encouraging low relapse inci-
dence in AML patients who received unmanipulated haploidentical marrow transplants along with a myeloabla-
tive regimen21. Bacigalupo et al. implied that a myeloablative conditioning regimen followed by unmanipulated
haploidentical bone marrow transplantation with post-transplant cyclophosphamide was associated with a low
risk of acute and chronic GVHD and encouraging rates of mortality and overall survival22. Another recent publi-
cation demonstrated that an MA conditioning regimen followed by haploidentical bone marrow transplantation
with BMT with post-transplant cyclophosphamide resulted in a low risk of acute and chronic GVHD and more
promising rates of transplant-related mortality and disease-free survival23. Klein et al. reported excellent rates of
engrament, GVHD, and TRM in pediatric/young adult patients treated with post-transplant cyclophosphamide
regimen. ey suggested that this approach was a widely available, safe, and feasible option for pediatric and
young adult patients with high-risk hematologic malignancies, including those with a prior history of myeloab-
lative bone marrow transplantation and/or those with comorbidities or organ dysfunction24. us, the results of
relevant contemporary publications are mainly consistent with our ndings.
We could not determine any signicant relationship between relapse, gender, type of donor, genetic anomaly,
pre-transplant protocol and mortality, relapse and GvHD. is may be associated with limitations of our study
such as retrospective design, single-centered construct, relatively low number of cases and short-term follow-up.
Conduction of multi-centered, controlled, prospective relevant studies with larger series and longer duration of
follow-up is needed.
In the last decade, the most important development in AML has been the demonstration of a relationship of some
cytogenetic anomalies with prognosis. e cytogenetic properties of the patients are the most important factors for
determining the time of transplantation for most leukemia specialists. Follow-up aer consolidation is recommended
for patients with a good cytogenetic prognostic prole, and those who developed remission aer induction therapy.
In patients having favorable prognostic criteria, patients with PR or relapses, following induction and consolidation
of chemotherapies in patients with moderate or poor prognosis allogeneic HSCT should be performed. If appropriate
HLA -matched unrelated donors are available, HSCT should be performed using these donors.
To conclude, cyclophosphamide use appears to be a highly eective and promising strategy for acute GvHD
prophylaxis in allogeneic HSCT cases. Due to the short duration of median follow-up, it is too early to make a
healthy interpretation concerning the impact of this approach on the development of chronic GvHD, and factors
related to mortality and relapse.
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Author Contributions
Sinem Namdaroglu wrote the main manuscript text. Ali Hakan Kaya reviewed literature. Hikmettullah
Batgi,Omur Kayikci, Dicle Iskender prepared Tables 1–4. Mehmet Sinan Dal, Merih Kizil Cakar reviewed the
article before submission not only for spelling and grammar but also for its intellectual content. Emre Tekgunduz
planned methodology to reach the conclusion. Fevzi Altuntas constructed an idea for manuscript.
Additional Information
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