Reduced-toxicity conditioning regimen with low dose post-transplantation cyclophosphamide and low-dose anti-thymocyte globulin as graft-versus-host disease prophylaxis for haploidentical stem cell transplantation in older patients

Reduced-toxicity conditioning (RIC) regimens are used for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in older patients. However, successful outcomes are still hindered by graft-versus-host disease (GVHD), treatment-related mortality (TRM) and relapse, especially in the setting of haploidentical donor (HID) hematopoietic cell transplantation (HCT). The aim of this study was to evaluate the effectiveness of a RIC conditioning regimen that included a combination of lower doses of post-transplantation cyclophosphamide (PTCy) (40 mg/kg on day +3) and anti-thymocyte globulin (ATG) (7.5 mg/kg) as GVHD prophylaxis, followed by haplo stem cell transplantation (SCT) in older patients. Method: We retrospectively analyzed 55 patients ≥ 55 years of age with hematologic malignancies treated with �udarabine, Cytarabine, busulfan, and low dose cyclophosphamide as the conditioning regimen between January 1, 2019 and November 30, 2023. Results: Neutrophil engraftment was successful in all the patients within 28 days, with 54 (98.2%) patients achieving complete donor chimerism. The cumulative incidence of non-relapse mortality was 0 at 30 days, 7.5% at 100 days and 19% at 1-year. The cumulative incidence of grade II-IV aGVHD was 25% (95%CI,15%−38%), while grade III-IV aGVHD was 9.1% (95%CI,3.3%−19%). The cumulative incidence of extensive cGVHD at 1 year was 3.6% (95%CI,0.66%-11%). The cumulative incidence of relapse (CIR), overall survival (OS) and GVHD-free/relapse-free survival (GRFS) at 1-year were 9.0%, 71.6%, and 67.1%, respectively. Conclusions: These results suggested that a RIC conditioning regimen including a combination of lower PTCy/ATG as GVHD prophylaxis, followed by haplo-SCTmight be a promising option for appropriately selected older patients.


INTRODUCTION
The incidence of hematologic malignancies is expected to rise in elderly people due to aging of the world population [1,2].Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has the potential to cure or improve outcomes of many of these diseases [1,3].However, historically the use of allo-HSCT has been hampered in older adults due to the lack of an HLA-matched donor, age-associated comorbidities, and lower performance status in this age group of patients, in addition to a more aggressive nature of the disease [3].An increasing number of studies have demonstrated that a reduced intensity conditioning (RIC) regimen combined with a haploidentical donor (HID) is a feasible option when a matched sibling donor or unrelated donor is unavailable for older adults [4][5][6].
It is well established that the risk of graft-versus-host disease (GVHD) increases with age and the use of a peripheral blood stem cell (PBSC) graft [7][8][9][10][11].In the last decade, several reports have described that the outcome of older patients undergoing haplo-SCT has improved due to effective prophylaxis strategies for acute or chronic GVHD.A regimen of post-transplantation cyclophosphamide (PTCy) for GVHD prophylaxis has recently become a feasible option in patients older than 55 years or even 76 years receiving a haplo-SCT [12][13][14][15].Nevertheless, the cumulative incidence (CI) of II-IV acute GVHD (aGVHD) has remained at the high levels of 37-40% [16-18], while chronic GVHD (cGVHD) has a CI of 26-42% [15,18].It is worth noting that a high risks of aGVHD and cGVHD are associated with the source of PBSC graft rather than bone marrow (BM) graft in haplo-SCT with PTCy used for GVHD prophylaxis [19].
Compared with anti-thymocyte globulin (ATG)-based regimens for GVHD prophylaxis, PTCy has been shown to be associated with lower incidences of viral and fungal infections [20].Luznik et al. [21] retrospectively analyzed the effects of different doses of PTCy (two doses of PTCy on day + 3, +4 vs. one dose of PTCy on day + 3) on the results of transplantation in haplo-SCT with PTCy.The data indicated that one dose of PTCy had the same effect on GVHD prophylaxis as two doses of PTCy, although there was a trend towards a higher risk of cGVHD, especially serious cGVHD.
In recent years, a total ATG dose of 10 mg/kg has been used for prevention of GVHD, followed by haplo-SCT and has been shown to be bene cial for reducing acute and chronic GVHD in older patients.
However, the use of ATG increases the chances of cytomegalovirus (CMV) infections due to a delay in immune reconstitution, with the 100-day CIs of CMV and EBV viremia being 68% and 50%, respectively [5].Over the last decade, the e cacy and safety of modi ed combinations of PTCy and ATG have reduced the risk of GVHD in haplo-SCT without in uencing other outcomes [20,[22][23][24][25].Our previous study reported that the use of a combination of PTCy (50 mg/kg on day + 3) with ATG (7.5 mg/kg) as GVHD prophylaxis for HID in high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) had a better 2-year GRFS rate, lower 180-day incidence of II-IV acute GVHD (aGVHD), lower 1-year incidence of moderate to severe cGVHD and comparable CIR rates than that observed in the standard prophylaxis regimen group [26].Although the combination of PTCy and ATG has dramatically improved the outcomes of HID in younger patients [27,28], evidence regarding its e cacy with HID is scarce in older patients.To decrease the risk of aGVHD or cGVHD and lower CMV or EBV reactivation, the current study retrospectively investigated the effect of a novel RIC regimen that included a combination of lower doses of PTCy and ATG on transplant outcomes in patients aged > 55 years who underwent a haplo-HCT.The study showed encouraging results for this novel regimen in older patients undergoing haplo-SCT.

Study design and data collection
The study was a retrospective design based on the transplantation database of the A liated People's Hospital of Ningbo University.The inclusion criteria were patients aged ≥ 55 years diagnosed with hematologic malignancies who underwent a haplo-SCT and received an RIC regimen for GVHD prophylaxis of low dose PTCy and low-dose ATG.The study was approved by the institutional review board and was carried out in accordance with the principles of the Declaration of Helsinki.The variables collected from the medical records of the patients included clinical features, pre-transplant-related parameters, transplant-related parameters, post-transplant outcomes, and adverse events.Written informed consent for all aspects of the study was obtained when a patient was admitted to our center.

Evaluation points
The study focused mainly on transplant-related mortality (TRM), engraftment, GVHD, infection, relapse, overall survival (OS), and GVHD-free/relapse-free survival (GRFS).TRM was de ned as any cause of death other than relapse.Time to neutrophil engraftment and platelet engraftment were de ned as the rst three consecutive days when the absolute neutrophil count was ≥ 0.5 × 10 9 /L or the platelet count was ≥ 20 ×10 9 /L without transfusion requirements, respectively.Hematopoietic chimerism was de ned as the presence of 95% donor hematopoietic cells evaluated by Short Tandem Repeat analysis.AGVHD and cGVHD were graded based on previously described criteria [29].Disease relapse (if complete remission had been achieved before allo-HSCT) was de ned by reappearance of signs of disease, con rmed by either cytology or histology.GRFS was calculated from the allograft date to the last date of follow-up or development of either III-IV acute GVHD, extensive chronic GVHD, relapse, or death [30].

Statistical analysis
Continuous variables were expressed as the median and categorical variables as percentages.The chisquare test was used to analyze categorical variables, while the quantitative data were analyzed using the rank sum test.Transplant outcomes were measured in terms of TRM, engraftment, GVHD, CIR, OS and GRFS.The curves for TRM, GVHD, and CIR were plotted using estimates of CI by the competing risk model.We considered death without engraftment as the competing event for engraftment, death without GVHD as the competing event for GVHD, relapse without TRM as the competing event for TRM, and TRM without relapse as the competing event for CIR.The survival curves for OS and GRFS were plotted using the Kaplan-Meier method.
As this retrospective analysis was designed as an exploratory investigation, no statistical sample size (i.e., power) calculation was conducted.The endpoint of the last follow-up for all the surviving patients was November 30, 2023.SPSS25.0 (IBM, Chicago, IL, USA) was used for the statistical analyses and R version 4.3.0(http://www.r-project.org)software was used for the competing risks analysis.A P value < 0.05 was considered statistically signi cant.

Patient and donor characteristics
From January 1, 2019 to November 30, 2023, a total of 55 consecutive patients 55 years or older were included in the study, with a median follow-up period of 11.7 (1.0−57.2) months.The median age of the 55 patients was 59 (55−71) years old, with 8 (14.5%) having an HCT−CI ≥3.The most common diagnoses were AML and MDS.The details of the pre-transplant patients, disease, transplant characteristics, and donor characteristics are shown in Table 1.

Engraftment and chimerism
All 55 patients achieved hematopoietic reconstitution within 28 days.Neutrophil reconstruction occurred at a median of 12 (9−23) days.One patient did not achieve full donor chimerism and was rescued with a donor lymphocyte infusion on day 46 and thereafter achieved complete donor chimerism.Fifty-three patients (96.4%) achieved platelet engraftment with a median time of 12 (8−106) days after HSCT.The CI of platelet reconstruction at 100 days was 94.5%.Two patients did not achieve platelet engraftment due to a TRM.A secondary graft failure (SGF) without evidence of relapse was observed in one case on day 31.

Discussion
This study, for the rst time, evaluated the feasibility and effectiveness of haplo-SCT with PTCy/ATG retrospectively in the 55 older patients with hematological malignancies (AL 72.7%).The patients received a novel RIC regimen of udarabine, Cytarabine, busulfan, and low dose of cyclophosphamide.We showed that patients up to the age of 71 years can achieve an acceptable TRM, GVHD, relapse rate, GRFS, and survival using this novel approach.
GVHD historically has been considered the most common and potentially life-threatening transplantrelated complication.Several studies have con rmed that the risk of developing GVHD is higher in older patients [7,8,31,32] and to address this problem, various strategies for GVHD prophylaxis have been developed.ATG has been used widely to reduce GVHD in an HLA-mismatched allo-HSCT in elderly patients [5,[33][34][35].Sun et al. [5] reported the results for ATG (10 mg/kg) combined with CsA as GVHD prophylaxis in 50 older patients with hematological diseases, with 80% receiving a combination of bone marrow and peripheral blood as grafts.The results showed a lower CI for grades II − IV aGVHD and cGVHD.However, there are reports that using PBSC might lead to higher rates of GVHD compared to the use of bone marrow stem cells [23,36,37].This led us to speculate that the use of bone marrow in 80% of patients in Sun et al.'s study may have contributed to the low incidence of GVHD.Most studies have reported that ATG-based regimens for preventing GVHD in HID transplantation in older patients were associated with relatively high risks of aGVHD, with the incidence of grades II-IV ranging between 29.5-45.6%[33][34][35], Taken together, these results highlight the importance of optimizing ATG-based regimens in elderly patients receiving GVHD prophylaxis using haplo-SCT, especially in HID-PBSCT.Many studies have investigated the use of PTCy-based regimens including HID for allo-HSCT to decrease the risk of GVHD in older patients.However, the results of these studies are unsatisfactory with an incidence of II − IV aGVHD and cGVHD of 29 − 35% and 11 − 33%, respectively [38][39][40].Another retrospective report of a variety of donors who underwent an allo-HSCT using ATG (4.5mg/kg) combined with PTCy (50mg/kg, on days + 3, +4 ) for GVHD prophylaxis, showed that the CIs for grade II − IV aGVHD at day 100 was 11 (5 − 21)%, and for 2 − year cGVHD was 26 (16 − 38)% [41].While this encouraging result appears to offer a promising method for GVHD prophylaxis only 12 (19%) cases of haplo-SCT were included in the report.To date, no studies have investigated older patients who underwent a haplo-SCT and received lower doses of PTCy combined with ATG for GVHD prophylaxis.Despite the fact that up to 81.8% of the patients in our study received PBSC as a graft, we showed that the CIs for grades II − IV aGVHD and grades III − IV aGVHD at 100 days post-transplantation were 25.0% (95%CI,15%−38%) and 9.1% (95%CI,3.3%−19%),respectively using haplo-SCT and low-dose PTCy and ATG regimen for GVHD prophylaxis.We also showed a lower incidence of moderate-severe cGVHD at 1 year in older individuals who received the PTCy-ATG combination.Moreover, the incidence of CMV reactivation and EBV reactivation was acceptable.These results suggested that low-dose PTCy-ATG effectively and safely prevents aGVHD and cGVHD in elderly patients undergoing a haplo-SCT.
Traditionally, haplo-SCT in older individuals has a higher TRM because of the patient's accumulation of co-morbidities, poor performance status, and acute or chronic GVHD.In a previous study Raiola et al. [39] investigated 634 patients who underwent unmanipulated haploidentical bone marrow transplantation and showed a correlation between age and TRM, with those older than 60 years having a 4.5-fold increased risk of TRM compared to that of younger patients.Three retrospective studies in older patients who received RIC or nonmyeloablative HLA-haploidentical transplantation also demonstrated that the incidence of non-relapse mortality was 6 − 21% at 100 days and 12 − 30% at 1-year [4,5,19,31,42].The results of the current study also showed relatively low CIs for TRM in patients of 7.5% at 100 days, and 19.0% at 1-year.In contrast, a report from the Fred Hutchinson Cancer Research Center [43] showed the rate of TRM appeared to increase with higher HCT-CI scores.The reason our study had relatively lower rates of TRM might be because the patients had relatively lower HCT-CI scores, with only 18.4% of patients having a score ≥ 3. Most reports of allo-HSCT in older patients have considered udarabinebased conditioning regimens historically have lower transplant-related mortality compared to that of busulfan plus cyclophosphamide [5,44].We also used a novel RIC regimen consisting of low doses of Cy (1,200 mg/m 2 /day, on day − 3) and udarabine, and this may be another reason for the lower TRM we observed.Furthermore, GVHD represents the main source of NRM in transplant patients.For example, only one patient in our study died from GVHD, whereas seven (43.8%) died from infection.Of note, nearly half of the HSCT recipients in the group died of COVID-19 pneumonia in the early post-transplant phase due to the absence of a pathogen-speci c adaptive immune response.These data imply that immune recovery was slower in our study, with the results showing there is room for further improvement in our novel conditioning regimen, such as reducing the total dosage of ATG.
To our knowledge, a relapse of malignancy is the predominant cause of treatment failure and death in patients receiving the RIC regimen.At our center, we used udarabine -intensi ed RIC regimens and a combination of ATG and PTCy for GVHD prophylaxis.Interestingly, despite one-third of patients in this series not being in complete remission at the time of transplant, we observed a low and encouraging 1year CIR after transplantation of only 9.0% that compared favorably with the incidence reported in previous studies [4,5,34,45].A reasonable interpretation of this relatively lower CIR is that the combination of udarabine with high dosage Ara-C in the preconditioning regimen for AML and MDS may have enhanced the intensity of the pretreatment regimen and reduced later recurrence.This possibility agrees with the ndings of Cao et al [26].Several studies have also shown that HID with PBSC as the stem cell source rather than bone marrow grafts has stronger graft-versus-leukemia (GVL) and results in lower relapse rates [19,[46][47][48].The association between the level of GVHD prevention and the risk of relapse has been debated previously.A report on 133 older patients undergoing allo-HSCT showed that the risk of relapse was like that observed between the combination of PTCy/ATG for GVHD prophylaxis and other regimens [41].These data suggest that potent suppression of GVHD can be accomplished with dual in vivo TCD without a corresponding increase in the risk of relapse.The patient cohort in our study differed from other studies in that it was limited to patients older than 55 years and included more recently treated patients in whom our novel conditioning regimen became more focused on PBSC as the graft type.However, the heterogeneity and relatively small number of patients, the short follow-up time, and possible selection bias when choosing candidates could not be excluded as potential confounding factors in the study and may have contributed to the low CIR we observed.
The current study had some limitations as it was carried out at a single center on a small number of patients with heterogeneous characteristics, making it di cult to perform a multivariable analysis.In addition, post-transplant immune reconstitution data was not monitored routinely in the 55 patients and therefore we could not assess whether administration of PTCy/ATG as the GVHD prophylaxis regimen affected immune reestablishment.
In conclusion, the ndings of this study suggest that a low dose PTCy/ATG regimen following a RIC regimen is an effective therapeutic strategy for appropriately selected patients aged 55 years and older with hematologic malignancies.