Early withdrawal immunosuppression improved mixed chimerism in hematopoietic stem cell transplantation for pediatric aplastic anemia

doi:10.21203/rs.3.rs-4670642/v1

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Early withdrawal immunosuppression improved mixed chimerism in hematopoietic stem cell transplantation for pediatric aplastic anemia

https://doi.org/10.21203/rs.3.rs-4670642/v1

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Mixed chimerism (MC) occurs frequently with the risk of graft reject (GR) for aplastic anemia (AA) patients undergoing matched sibling donor (MSD) hematopoietic stem cell transplantation (HSCT). So far, no one knows how to adjust immunosuppression (IS) during MC. We retrospectively analyzed 87 consecutive pediatric patients. Early withdrawal (EW) IS and donor lymphocyte infusion (DLI) were attempted to reverse MC. The rate of MC was 26% (n = 23). Low dose cyclophosphamide (CY) (120–150 mg/kg) is an independent risk factor for MC (P = 0.0002) and increase dosage of Fludarabine (FLU) cannot eliminated it. Patients receiving 200 mg/kg CY had the lowest MC rate (8%) and best 3-year graft-versus-host disease (GVHD)/failure free survival (GFFS; 95%). Chimerism in T cells is more sensitive than that in whole blood (P = 0.001). In 17 patients with early-onset MC (ratio of DLI: 83% versus 82%), EW IS strategy is helpful to improving complete chimerism (CC) (63 vs. 295 days, P = 0.008). Our study shows that FLU is necessary to intensify CY + ATG conditioning to maintain the engraftment and 200 mg/kg CY + 150 mg/m2 FLU is recommended as a basic conditioning regimen. EW IS strategy should be considered as an important option to improve donor chimerism in early-onset MC.

Biological sciences/Immunology/Adaptive immunity

Biological sciences/Immunology/Bone marrow transplantation

pediatric

aplastic anemia

matched sibling donor

hematopoietic cell transplant

mixed chimerism

Aplastic anemia (AA) is a rare heterogeneous disorder characterized by pancytopenia and bone marrow failure resulting from immune-mediated destruction of hematopoietic stem cells ^1–3 and allogeneic hematopoietic stem cell transplantation (HSCT) remains an important treatment in children ^4–6.

The combination of cyclophosphamide (CY) and anti-thymocyte globulin (ATG) recommended by severe aplastic anemia working party (SAAWP) of the European society for blood and marrow transplantation (EBMT) has been considered the standard conditioning regimen for human leukocyte antigen (HLA)-matched sibling donors (MSDs) and presented encouraging outcomes ^7–9. Fludarabine (FLU) was added to the regimen to enhance the immunosuppressive action of CY + ATG and offer comparable survival ¹⁰. However, mixed chimerism (MC) associated with transfusion-dependent cytopenia or secondary graft rejection (GR) has also been observed ^11,12. Donor lymphocyte infusion (DLI) and adjustments to the immunosuppression (IS) strategy are limited choices to avoid graft loss ^11,13. Guidelines published in 2009 suggested not reducing or withdrawing IS to use against later-onset MC ¹, but the optional IS strategy is still unclear, especially for early-onset MC.

Cohort and Ethics

This cohort consisted of patients under 18 years old who received MSD HSCT for AA between July 2002 and December 2022 at Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine. Data on 87 consecutive children were collected retrospectively. All patients received FLU to intensify the CY + ATG conditioning. Busulfan (BU) or total body irradiation (TBI) was used in patients with an absolute neutrophil count (ANC) > 1.0×10⁹/L, disease duration > 3 years, or received > 20 transfusion units. DLI and reduced IS were managed for early-onset MC patients according to the valley and time of the MC. The need for informed consent was waived by the institutional review board of Shanghai Children’s Medical Center (SCMCIRB-K2023187-1). All methods were performed in accordance with the Declaration of Helsinki

Chimerism monitor, EW IS, and DLI intervention for mixed chimerism

Whole blood total donor chimerism was mainly monitored by fluorescent in situ hybridization with sex chromosome probes in patients with opposite sex donors, otherwise by short tandem repeat (STR) analysis. STR analysis was also performed in samples selected by flow cytometry to check the chimerism status in T, B, and NK cell lineages. Whole blood or lymphoid donor chimerism between 5% and 95% occurring before 100 days after HSCT was defined as early-onset MC. EW IS was defined as reducing IS at least 90% within 100 days when patients had early-onset MC. Dose and timing of DLIs were prescribed by the attending for patients with MC and without obvious graft-versus-host disease (GVHD). Donor lymphocytes were either frozen aliquots at the time of the original hematopoietic stem cell harvest or collected peripherally before DLI.

Definitions, Statistics and outcomes

Neutrophil engraftment was defined as the first of 3 consecutive days with an ANC ≥ 0.5×10⁹/L. Platelet engraftment was defined as the first of 3 consecutive days with a platelet count ≥ 20×10⁹/L without platelet transfusion within the previous 7 days. Poor graft function (PGF) was defined as multi-lineage cytopenia (ANC < 0.5×10⁹/L, platelet count < 20×10⁹/L and/or hemoglobin < 70 g/L) with complete donor chimerism in the absence of other explanations, such as drugs, infection, GVHD, transplant-associated thrombotic microangiopathy (TA-TMA) or immune cytopenia ¹⁴. Acute GVHD (aGVHD) and chronic GVHD (cGVHD) were defined according to standard criteria ^15,16.

Overall survival (OS) was calculated from the time of transplantation until death from any cause. Failure-free survival (FFS) was calculated from the time of transplantation until GR, PGF, or death. GVDH/failure-free survival (GFFS) was calculated from the time of transplantation until GR, PGF, aGVHD of grade III-IV, severe cGVHD or death. The probability of survival was estimated by log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test. The data cut-off for this analysis was 31 December 2022.

Patient characteristics

Eighty-seven children with AA underwent MSD HSCT (n = 53 [60%] males) and 85 received stem cells from GCSF-mobilized peripheral blood (PBSC) and 2 received bone marrow. Patient characteristics are summarized in Table 1. The median patient age was 9 years (range 1.5–16 years). Among the patients, 44 (51%) had severe aplastic anemia (SAA), 30 (34%) had very severe aplastic anemia (VSAA), and another 13 (15%) had transfusion-dependent non-severe aplastic anemia (TD-NSAA). The median age of donors was 10 years (range 1–38 years). Thirty-four (39%) patients received a graft from a same-sex donor. The median time from diagnosis to HSCT was 2 months (range 1–96 months) and 19 (22%) patients experienced up to a 3-year interval from diagnosis to HSCT. The median number of packed red blood cell (PRBC) and platelet transfusions before HSCT was 4 (range 0–45) and 8 (range 0–50), respectively. Twenty-two (26%) patients were transfused with up to 20 units before HSCT.

Conditioning regimen and transplantation procedure

All 87 patients received FLU + CY + ATG (FCA) ± BU/TBI intensified conditioning regimen, with CY 120–150 mg/kg + FLU 150 mg/m² for 4 patients, CY 120–150 mg/kg + FLU 180–200 mg/m² for 25 patients, CY 200 mg/kg + FLU 150 mg/m² for 17 patients, and CY 200 mg/kg + FLU 180–200 mg/m² for 21 patients. Fourteen patients received additional BU (3.2–4.8 mg/kg/day) for 2 days. Six heavy transfusion patients received additional 3GY TBI. Until December 2010, ATG-F (Anti-human T Lymphocyte Rabbit Immunoglobulin, Fresenius S, Germany) was administered at 15–40 mg/kg in 10 earlier patients. After that, ATG (Rabbit Anti-human Thymocyte Immunoglobulin, thymoglobulin, USA) became available in China and was administered at 7.5 or 10 mg/kg in 8 and 69 patients, respectively.

A median dose of 3.9×10⁶ cells/kg (range 1.3–10.5 ×10⁶ cells/kg) of CD34 + cells and 14.1×10⁸ cells/kg (range 3.9–36.2 ×10⁸ cells/kg) of total nucleated cells (TNCs) were given. Cyclosporine (CsA) and short-term methotrexate (MTX; 15 mg/m² on day 1, 10 mg/m² on days 3 and 6) were used for GVHD prophylaxis with the target blood trough level of CsA of 150–200 ng/ml.

The cumulative incidence of MC in the CY120-150 + FLU150, CY120-150 + FLU180-200, CY200 + FLU150, and CY200 + FLU180-200 cohorts was 25%, 56%, 0%, and 14%. The P value of the Mantel-Cox test in these four cohorts was 0.0002, and the P value between the CY120 + FLU180-200 and CY200 + FLU180-200 cohorts was 0.004 (Fig. 1a). A low dose of CY was an independent risk factor for MC (P value = 0.0002, Fig. 1b).

BU was intensified for 14 patients, and 5 of them (36%) still developed MC. TBI was added for 6 patients, all with complete chimerism (CC).

GVHD and viral reaction

Eight patients (9%) developed aGVHD, 6 grade II and 2 grade III. There was no significant difference between the cumulative incidence of aGVHD in the MC cohort and CC cohort (P = 0.89; Table 1, Fig. 2e). Thirteen (15%) patients developed cGVHD, 6 with mild cGVHD and 7 cGVHD. The cumulative incidence in the MC and CC cohorts is shown in Fig. 2f (P = 0.33). Both aGVHD and cGVHD resolved completely with IS.

The total incidence of hemorrhagic cystitis was 9% (N = 8). The cytomegalovirus (CMV) reaction incidence was 49% (N = 43), and the incidence of Epstein-Barr virus (EBV) reaction was 56% (N = 43, data unavailable in 10 patients). We found no significant difference between the MC and CC cohorts. Three patients were pathologically diagnosed with EBV-related post-transplant lymphoproliferative disorder (PTLD), and all of them received anti-CD20 monoclonal antibody. Finally, two patients recovered, and one died of central nervous system PTLD.

Engraftment, chimerism, intervention, and outcome

Among these 87 MSD HSCT patients, 86 (99%) patients achieved neutrophil engraftment with a median time of 13 days (range 6–33 days) and 82 (92%) patients achieved platelet engraftment with a median time of 13 days (range 0–49 days). As shown in Table 2, 23 (26%) patients presented with MC in a median 56 days (range 13–736 days). Seventeen patients were in early-onset post-HSCT before 100 days, 6 patients had later onset after 100 days. The median chimerism rate the first time in whole blood was 90.4% (range 47.5–94.9%), and the median valley was 86.9% (range 47.53–94.9%). Chimerism was also identified in T cells in 11 patients 53 times. The median rate and valley were 83.6% (range 32.8–93.8%) and 81.1% (range 30.4–92.0%), respectively. All of the rates in T cells were lower than in whole blood at the same time point during MC (P = 0.001, Fig. 1d, Supplement Figure). Nineteen patients (83%) received DLI 1 to 4 times with a median maximum dose of 10×10⁶/kg (range 2.7–90 ×10⁶/kg, Supplement Table).

For the 17 early-onset MC patients, 6 received an early withdrawal IS strategy (EW cohort) and 11 did not (non-EW cohort). In the EW cohort, after reduced IS at a median day + 42 (range day + 18 to + 74), 5 patients stopped IS at a median day + 66 (range day + 39 to + 80). For case 8, after reducing CsA 90%, the full dose IS had to be re-started because of aGVHD, which gradually stopped oo day + 365. Another 5 (82%) patients had a median maximum dose of 9.3 ×10⁶/kg (range 4–90 ×10⁶/kg) DLI. Finally, with the exception of GR in case 2, 5 of 6 (83%) cases recovered to CC within a median of 63 days (range 36–91 days). After reducing IS at a median 45 days (range 33–86 days), 4 patients reused CsA (3 for GVHD and 1 for GR) and another 2 patients completely stopped IS at day 62 and day 70, even with DLI. Detailed information on withdrawal IS, DLI, and the MC outcome of these 6 EW IS patients is provided in Fig. 3. In the non-EW cohort, 82% (9/11) of patients received DLI at a median maximum dose of 14 ×10⁶/kg (range 3.3–63 ×10⁶/kg). Finally, 7 (64%) patients had CC with a median duration of MC of 295 days (range 40–612 days), 1 (9%) patient experienced GR, and 3 (27%) patients exhibited stable mixed chimerism (SMC). The median time for MC recovery to CC in the EW and non-EW groups was 63 days (range 36–91 days) and 295 days (range 40–612 days), respectively. Thus, EW IS will benefit from the quick return to CC (P = 0.008; Fig. 1c).

For the 23 MC patients, 69% (N = 16) returned to CC, 22% (N = 5) exhibited SMC, and 9% (N = 2) suffered GR on day + 118 and day + 120. Among patients who experienced GR, one finally recovered autologously and another was rescued by second HSCT.

After a median follow-up of 35.3 months (range 2.3-146.8 months), 2 patients died (1 from fungal pneumonia and 1 from CNS PTLD), and 4 patients developed PGF at 43 days, 117 days, 158 days, and 2 years after HSCT. Two patients suffered from secondary GR. The entire cohort obtained 3-year 98% OS, 91% FFS, and 89% GFFS. After active intervention, there was no difference in the OS, FFS, and GFFS between the MC and CC cohorts (P = 0.29, 0.12, and 0.08, respectively; Table 1, Fig. 2a-c). Patients who received a conditioning regimen with CY120-150 had significantly decreased 3-year GFFS compared with CY200 (83% versus 95% P = 0.03) (Fig. 2d), also maybe because of the MC and intervention.

MSD HSCT has been the standard first-line treatment for children with SAA/VSAA ^10,17, with nearly 90% OS ^18,19. In our retrospective study of 87 pediatric AA patients undergoing MSD peripheral blood stem cell transplantation, we noted 98% OS. Two patients experienced GR, 4 PGF, and 2 died of infection, resulting in an FFS of 91%, which is comparable to previous Japanese reports ^20,21. Even with PBSC, the incidences of aGVHD and cGVHD in this cohort were only 9% and 15%, respectively, and all were controlled by IS, reaching a GFFS of 89%. This result is in line with previous reports of ex vivo T cell-depleted PBSC or bone marrow MSD HSCT ^22,23. Such a high survival rate can be attributed to an intensified conditioning regimen and timely MC intervention. To the best of our knowledge, this is the first study to explore the EW IS strategy for early-onset MC in AA HSCT.

In 2010, EBMT SAAWP recommended the combination of CY and ATG as a standard conditioning regimen for MSD HSCT ^8,24. In the early clinical practice of our center and other studies, CY + ATG was found to be insufficient to obtain engraftment. FLU is considered one of the choices to supplement CY + ATG to improve stable engraftment ^10,25. To decrease GR, FLU 150 mg/m² was routinely used to intensify the CY + ATG conditioning regimen for MSD HSCT at our center. Due to concerns about the cardiotoxicity ²⁶, for patients with heavy transfusion or potential cardiac insufficiency before HSCT, we tried to increase the dose of FLU to 180–200 mg/m² and tapered CY to 120–150 mg/kg. With this intensified conditioning regimen, the rate of MC in this study still reached 26%. We reported an FCA + TBI/BU based conditioning regimen for alternated donor HSCT for AA in 2021 ³. Until now, according to the severity of bone marrow failure, the number of transfusion, and degree of HLA matching, the risk-adapted conditioning regimen has been performed in 580 pediatric AA HSCT patients with a complete engraftment rate of 98% (data not shown). Thus, FLU is necessary to intensify CY + ATG and it is important to maintain sufficient engraftment for pediatric acute onset SAA with MSD HSCT.

This retrospective study shows that low-dose CY (CY 120–150 mg/kg) is an independent risk factor for MC (P = 0.0002), whereas increase dosage of FLU did not decrease the MC. Although some studies have shown that CY 150 mg/kg has higher mortality than CY 50 mg/kg or 100 mg/kg combined with FLU + ATG + TBI in old patients ^27,28. CY 200 mg/kg is still the main option for pediatric AA ¹⁰. In this group, CY 200 mg/kg was used in 38 (57%) patients, with slightly lower than 8% MC and no patient developed cardiac dysfunction. Two days of BU was added to further intensify FCA conditioning for 14 patients, though half of the TD-NSAA patients still developed MC. Even with DLI, three patients exhibited SMC finally (cases 10, 14, and 20). For case 10, who had a 4-year history of TD-NSAA and heavily transfused before HSCT, more intensified conditioning with CY 200 mg/kg + FLU 200 mg/m² + BU (2 days) was used, but she still had early-onset MC. Even with active DLI, the valley chimerism fluctuated approximately 69% with SMC to the present (post-HSCT 5 years). Although future studies with larger cohorts are needed, we recommend 200 mg/kg CY + 150 mg/m² FLU as a standard conditioning regimen for pediatric SAA patients undergoing MSD HSCT.

MC is linked to GR and treatment failure in AA ^29–31. Compared to late-onset MC, early-onset MC has a greater risk of GR ³². For patients with early-onset MC, reducing IS after non-myeloablative HSCT is a feasible and safe strategy for prophylaxis relapse ^33,34. However, EBMT guidelines suggested not reducing or withdrawing IS to protect against later-onset MC ¹. This suggestion was based on little clinical data ¹¹. Coexistence of donor and recipient hematopoietic cells in the recipient’s body imply the existence of bidirectional immune tolerance. Until now, how to manage IS for early-onset MC has been unclear ³⁵. In this study, we tried EW IS for 6 out of 17 early-onset MC patients. Except for one case of GR, five patients returned to CC in a median 63 days (range 36–91 days) and 2 completely stopped IS within 70 days post-HSCT (cases 6 and 7). Although most patients underwent DLI, the ratio of DLI was consistent in two cohorts. Thus, the EW IS strategy is helpful for quickly improving the CC transition, which will benefit stable engraftment (P = 0.008). Case 8 achieved CC by EW IS alone and more clearly reflects that early reduction of IS will benefit the recovery to CC. By contrast, Zhang reported that increased immunosuppressant intensity is only effective in 1 of 4 (25%) MC patients³². And our results also showed that timely monitoring and early reduction of IS is key to saving early MC. T cell chimerism is more sensitive than that of whole blood (P = 0.001), giving us more precise data to monitor the donor chimerism and detect MC early. In active EW IS and DLI, 88% of patients recovered to CC (12/17), and only 12% (2/17) experienced GR, which is significantly better than the 30% GR reported by Zhang ^36,37. Active intervention and T cell chimerism monitor for early-onset MC achieved an OS of 100%.

Although EW IS and DLI may increase the risk of aGVHD ^36,37, these iatrogenic GVHD cases are relatively easy to control. In this EW IS cohort, 50% (3/6) experience aGVHD, two cases related to EW IS and DLI, and one related to EW IS alone. Finally, all GVHD cases resolved, and only case 16 experienced grade III aGVHD and moderate cGVHD. The overall responsiveness to GVHD treatment did not seem to be affected in the patients who developed aGVHD after EW IS ^33,34. This may be because the donor's immunity does not prevail in the MC state.

This study has several limitations. This retrospective study was based on real-world treatment data for pediatric AA patients. Thus, the design of the conditioning regimen lacked prospective considerations. The dose of ATG may affect engraftment. In addition, DLI also affects the MC outcome. To find the best treatment for patients, future prospective studies with larger cohorts are needed.

This study shows FLU is necessary to enhance the immunosuppression for SAA patients undergoing MSD HSCT with CY + ATG conditioning. Low dose CY is considered an independent risk factor for MC and it cannot be eliminated by increasing FLU dose. We recommend 200 mg/kg CY and 150 mg/m² FLU as a basic conditioning regimen for pediatric AA patients undergoing MSD HSCT. The EW IS strategy should be considered as an important option for improving MC in early-onset MC.

Funding: This study was supported by the Shanghai Hospital Development Center (SHDC12014118) and National Natural Science Foundation of China (81970114, 8160010893).

Conflict of interest: No financial or non-financial benefits have been received or will be received from any party related directly or indirectly to the subject of this article.

Ethical appproval: The need for informed consent was waived by the institutional review board of Shanghai Children’s Medical Center (SCMCIRB-K2023187-1).

Permission to reproduce material from other sources: Not applicable.

Declaration of generative AI: No Use.

Clinical trial registration: URL: https://www.chictr.org.cn; ChiCTR-1900023509. (Retrospective registration in 2019/5/31)

Contributors' statement

XW and XQ: research design, analysis and interpretation of data, screening potentially eligible studies, edrafting the article or revising it critically for important intellectual content; Chengjuan L, Changying L, JW, XH, QM and YL: acquisition of data, recruiting the patients and collecting specimens, revising the article critically for important intellectual content; JC: taking the responsibility for the integrity of the entire work as guarantor of the paper. All authors gave final approval of the version to be published.

Corresponding author:

Jing Chen, M.D., Ph.D.

1678 Dong Fang Road

Shanghai, CN 200127

Phone No: +86-21-3862-6161

Fax No: +86-21-3862-6161

Email Address: [email protected]

Acknowledgements

The authors would like to thank all of the clinicians, nurses, and families who made this study possible by providing samples.

Data availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

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Table 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

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07 Jul, 2024
First submitted to journal
01 Jul, 2024

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Early withdrawal immunosuppression improved mixed chimerism in hematopoietic stem cell transplantation for pediatric aplastic anemia

Status:

Version 1

Abstract

Figures

Introduction

Methodology

Cohort and Ethics

Chimerism monitor, EW IS, and DLI intervention for mixed chimerism

Definitions, Statistics and outcomes

Results

Patient characteristics

Conditioning regimen and transplantation procedure

GVHD and viral reaction

Engraftment, chimerism, intervention, and outcome

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1