Splenic irradiation combined with plasmapheresis and rituximab: a new option reducing donor-specific antibody in haploidentical hematopoietic stem cell transplantation

TO THE EDITOR: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative treatment for various malignant and nonmalignant hematopoietic diseases [1]. However, human leukocyte antigen (HLA) donor-specific antibodies (DSAs) found in patients before and/or after present a major problem in the producing positive patient outcomes following haplo-identical hematopoietic stem cell transplantation (haplo-HSCT) [2]. Previous studies have shown that pre-existing DSAs in patients before transplantation were associated with graft failure, delayed hematopoietic recovery, and poor graft function [3]. When patients are found to have DSAs, they usually receive desensitization treatment to reduce DSAs, commonly consisting of plasmapheresis, immunoglobulin therapy, and monoclonal antibodies [4]. Sometimes patients do not respond well to desensitization treatment and DSAs persist, especially in patients with high mean fluorescence intensity (MFI) levels of DSAs before treatment [3]. There are some studies suggesting that the spleen is a major source of DSAs during antibody-mediated rejection (AMR) in solid organ transplantation (SOT) [5]. Splenectomy before transplantation may help eliminate the would-be contribution of newly generated DSAs originating from splenic lymphoid tissue, thus improving transplantation outcomes [6]. Since splenectomy has a long recovery time, it is not always a feasible treatment option in patients in need of haplo-HSCT. Thus, splenic irradiation may be an effective alternative treatment which has already been reported in the treatment of kidney transplantation patients with DSAs [7]. To our knowledge, it is currently unknown as to whether splenic irradiation can eliminate pre-existing DSAs before haplo-HSCT. We have conducted a prospective, single-arm study (chictr.org.cn: ChiCTR2100051456) where we combined low-dose splenic irradiation with plasmapheresis and rituximab as a new treatment protocol to reduce DSAs before haplo-HSCT. Our aim is to explore the safety and efficacy of the outlined treatment protocol including splenic irradiation. This study was designed as shown in Supplementary Fig. 1. In this study, 265 consecutive patients who had received haplo-HSCT at the First Affiliated Hospital of Soochow University from December 2019 to June 2021 were enrolled. Finally, 38 patients who were detected to have a high MFI (>= 8000) of DSAs were recruited, among which 19 patients signed their informed consents to join this single arm clinical trial and were defined as the splenic irradiation group. They received the combined treatment protocol containing low-dose splenic irradiation with plasmapheresis and rituximab to reduce DSAs before haplo-HSCT, and the other 19 patients were defined as the control group who received the same protocol, but without splenic irradiation. In the splenic irradiation protocol, the total mass of the spleen’s volume was defined as the irradiation target. The total dose was 200 cGy, which was divided into four fractions. The patients were irradiated 50 cGy per day. Medical linear accelerator was used for radiotherapy and the photon line energy was >= 6MV. We always verified the location parameters used to direct radiation output before the first irradiation treatment to ensure accuracy. After DSA desensitizing treatment, modified busulfan and cyclophosphamide (BU/CY) was used as the transplant conditioning regimen for patients undergoing HSCT [8]. Anti-HLA antibodies were screened at three time points: the time at which DSAs were detected before desensitization treatment (T0), 7 days after desensitization treatment (T1), and 14 days after haplo-HSCT (T2). De novo DSAs were defined as DSAs detected after haplo-HSCT that were not present before. Characteristics of the patients are shown in Supplementary Table 1. The diversity of specific anti-HLA antibodies (SAbs) and the MFI for the DSAs did not differ from each other of both groups at enrollment. Most patients in both groups responded to antibody desensitization treatment, however the significant decrease in the MFI of anti-HLA-II DSAs at T1 was only observed in the splenic irradiation group DSA reduction in the splenic irradiation group was more significant than the control group, especially for anti-HLA-II DSAs (Fig. 1, Supplementary Table 2). Four patients (21.1%) from the control group were found to have increased diversity of SAbs targeting HLA-I and 1 (5.3%) targeting HLA-II after transplantation, compared with none in splenic irradiation group. Besides, a total of five patients (26.3%) in the control group had de novo DSAs detected after transplantation, compared with none in the splenic irradiation group (Supplementary Table 3, P < 0.05). No extra toxicities were observed in the splenic irradiation group during the transplantation (Supplementary Fig. 2). There were 3 (15.8%) patients that experienced primary graft failure (PGF) or secondary graft failure (SGF) in the control group, all of which were detected de novo DSAs, while no patient in the splenic irradiation group experienced primary or secondary graft failure. During the median follow up of 418 days (range 64–884), the estimated overall survival (OS) and progress-free survival (PFS) of the splenic irradiation group was observed to be superior to the control group (74.1% vs. 43.1%, P= 0.007; 73.5% vs. 48.8%, P= 0.048, respectively). No significant difference in the cumulative incidence of relapse was observed, while a higher transplantrelated mortality (TRM) rate seemed to be found in the control group (Supplementary Fig. 3, Supplementary Table 4).


TO THE EDITOR:
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative treatment for various malignant and nonmalignant hematopoietic diseases [1]. However, human leukocyte antigen (HLA) donor-specific antibodies (DSAs) found in patients before and/or after present a major problem in the producing positive patient outcomes following haplo-identical hematopoietic stem cell transplantation (haplo-HSCT) [2]. Previous studies have shown that pre-existing DSAs in patients before transplantation were associated with graft failure, delayed hematopoietic recovery, and poor graft function [3]. When patients are found to have DSAs, they usually receive desensitization treatment to reduce DSAs, commonly consisting of plasmapheresis, immunoglobulin therapy, and monoclonal antibodies [4]. Sometimes patients do not respond well to desensitization treatment and DSAs persist, especially in patients with high mean fluorescence intensity (MFI) levels of DSAs before treatment [3]. There are some studies suggesting that the spleen is a major source of DSAs during antibody-mediated rejection (AMR) in solid organ transplantation (SOT) [5]. Splenectomy before transplantation may help eliminate the would-be contribution of newly generated DSAs originating from splenic lymphoid tissue, thus improving transplantation outcomes [6]. Since splenectomy has a long recovery time, it is not always a feasible treatment option in patients in need of haplo-HSCT. Thus, splenic irradiation may be an effective alternative treatment which has already been reported in the treatment of kidney transplantation patients with DSAs [7].
To our knowledge, it is currently unknown as to whether splenic irradiation can eliminate pre-existing DSAs before haplo-HSCT. We have conducted a prospective, single-arm study (chictr.org.cn: ChiCTR2100051456) where we combined low-dose splenic irradiation with plasmapheresis and rituximab as a new treatment protocol to reduce DSAs before haplo-HSCT. Our aim is to explore the safety and efficacy of the outlined treatment protocol including splenic irradiation.
This study was designed as shown in Supplementary Fig. 1. In this study, 265 consecutive patients who had received haplo-HSCT at the First Affiliated Hospital of Soochow University from December 2019 to June 2021 were enrolled. Finally, 38 patients who were detected to have a high MFI (>= 8000) of DSAs were recruited, among which 19 patients signed their informed consents to join this single arm clinical trial and were defined as the splenic irradiation group. They received the combined treatment protocol containing low-dose splenic irradiation with plasmapheresis and rituximab to reduce DSAs before haplo-HSCT, and the other 19 patients were defined as the control group who received the same protocol, but without splenic irradiation.
In the splenic irradiation protocol, the total mass of the spleen's volume was defined as the irradiation target. The total dose was 200 cGy, which was divided into four fractions. The patients were irradiated 50 cGy per day. Medical linear accelerator was used for radiotherapy and the photon line energy was >= 6MV. We always verified the location parameters used to direct radiation output before the first irradiation treatment to ensure accuracy. After DSA desensitizing treatment, modified busulfan and cyclophosphamide (BU/CY) was used as the transplant conditioning regimen for patients undergoing HSCT [8]. Anti-HLA antibodies were screened at three time points: the time at which DSAs were detected before desensitization treatment (T0), 7 days after desensitization treatment (T1), and 14 days after haplo-HSCT (T2). De novo DSAs were defined as DSAs detected after haplo-HSCT that were not present before.
Characteristics of the patients are shown in Supplementary  Table 1. The diversity of specific anti-HLA antibodies (SAbs) and the MFI for the DSAs did not differ from each other of both groups at enrollment. Most patients in both groups responded to antibody desensitization treatment, however the significant decrease in the MFI of anti-HLA-II DSAs at T1 was only observed in the splenic irradiation group DSA reduction in the splenic irradiation group was more significant than the control group, especially for anti-HLA-II DSAs (Fig. 1, Supplementary Table 2). Four patients (21.1%) from the control group were found to have increased diversity of SAbs targeting HLA-I and 1 (5.3%) targeting HLA-II after transplantation, compared with none in splenic irradiation group. Besides, a total of five patients (26.3%) in the control group had de novo DSAs detected after transplantation, compared with none in the splenic irradiation group (Supplementary Table 3, P < 0.05). No extra toxicities were observed in the splenic irradiation group during the transplantation (Supplementary Fig. 2).
There were 3 (15.8%) patients that experienced primary graft failure (PGF) or secondary graft failure (SGF) in the control group, all of which were detected de novo DSAs, while no patient in the splenic irradiation group experienced primary or secondary graft failure. During the median follow up of 418 days (range 64-884), the estimated overall survival (OS) and progress-free survival (PFS) of the splenic irradiation group was observed to be superior to the control group (74.1% vs. 43.1%, P = 0.007; 73.5% vs. 48.8%, P = 0.048, respectively). No significant difference in the cumulative incidence of relapse was observed, while a higher transplantrelated mortality (TRM) rate seemed to be found in the control group ( Supplementary Fig. 3, Supplementary Table 4).
In recent years, splenic irradiation has become an exciting tool in the treatment of antibody-mediated rejection (AMR) associated with renal transplantation [7,9]. Our study in this small cohort shows a promising result. Although a decrease in the diversity of anti-HLA SAbs was detected in both groups, splenic irradiation significantly degraded the MFI of DSAs compared with routine methods alone. A greater decrease in the MFI of anti-HLA-II DSAs has been observed at T1, an even earlier time point, which was not seen in the control group (Fig. 1). These findings indicate that splenic irradiation might be more effective in the degradation of anti-HLA-II DSAs than routine desensitization protocols. Although routine desensitization therapy for DSA treatment may play a role in eliminating pre-existing DSA to a certain extent, there is still a lack of research on preventing the development of de novo DSAs. The development of de novo DSAs is often significantly associated with poor graft function [10]. In our report, we found no de novo DSAs in the splenic irradiation group. These results suggest that splenic irradiation might play a role in preventing de novo DSAs.
Besides, patients in splenic irradiation group are more likely to have good results than patients in the control group, however the precise mechanism driving the therapeutic effects observed following splenic irradiation is currently unclear. A previous study has indicated that B cell elimination after stem cell transplantation is associated with low cGVHD incidence [11]. In a report on kidney transplant patients by Lan Zhu et al., although DSA levels in patients eventually rebounded to some extent after splenic irradiation, the serum levels of creatinine remained stable for a long time [9]. As is reported before, low-dose splenic irradiation may kill B cells in the spleen [12]. Thus, the better prognosis observed in the study group may be due to depletion of B cells following spleen irradiation.
Our research demonstrated that adding low dose splenic irradiation to a routine anti-HLA antibody-eliminating protocol seems effective in reducing graft failure and improving prognosis after transplantation. Due to the small size of our cohort, clearly more studies are required to define the role of low-dose splenic irradiation for patients planning to undergo haplo-HSCT.  T0 T1 T2 T0 T1 T2   T0 T1 T2 T0 T1 T2  T0 T1 T2 T0 T1 T2   T0 T1 T2 T0 T1 T2   0   25000   20000   15000 10000 5000 Fig. 1 Change of DSAs at each time point. The MFI of DSAs for each patient and mean MFI was compared between two groups for anti-HLA-I (a) and HLA-II (b) DSAs. Reduction of the MFI was compared between the two groups of anti-HLA-I (c) and HLA-II (d) DSAs at each time point. *Indicates that the difference was statistically significant.