T‐cell replete haploidentical stem cell transplantation with low dose anti‐thymocyte globulin for relapsed/refractory Ewing sarcoma family tumors

Abstract Background Despite intensive multimodal therapies, the prognosis of relapsed/ refractory Ewing sarcoma family tumors (RR‐ESFTs) is dismal. Some case reports using allogeneic stem cell transplantation (allo SCT) for RR‐ESFTs have been reported, however, the efficacy of allo SCT is yet to be established. Aim The purpose of this study was to evaluate the response and toxicity of T‐cell replete haploidentical SCT (TCR‐haplo‐SCT) in RR‐ESFTs. Methods and results In this study, we retrospectively analyzed six patients with RR‐ESFTs who received TCR‐haplo‐SCT. Four patients had relapsed and two patients had refractory Ewing sarcoma. Before the TCR‐haplo‐SCT, all patients received a reduced intensity‐conditioning regimen containing fludarabine, melphalan, and low‐dose rabbit anti‐thymocyte globulin (2.5 mg/kg), as well as graft‐versus‐host disease (GVHD) prophylaxis, which consisted of tacrolimus, methotrexate, and prednisolone. Primary neutrophil engraftment was achieved in all the patients. Four patients developed acute GVHD (aGVHD) (grade I, 1; grade II, 1; grade III, 2), and two patients developed chronic GVHD (cGVHD). Among the four that developed aGVHD, three survived for 14, 116, and 129 months without relapse, while one died due to a transplant‐related complication. In contrast, the two patients who did not develop aGVHD experienced relapse early after TCR‐haplo‐SCT. Conclusions In this study, three of the six patients with RR‐ESFTs survived for more than one year without relapse, and the treatment toxicity was considered acceptable even for patients who underwent high‐intensity pretreatment. TCR‐haplo‐SCT could be a potential therapeutic option for patients with RR‐ESFTs.


| INTRODUCTION
The 5 year survival rate of patients with relapsed/refractory Ewing sarcoma family tumors (RR-ESFTs) is approximately 10%-20% despite intensive multimodal therapies. [1][2][3][4][5] The age at relapse, time to relapse, pattern of relapse, and response to second-line chemotherapy are considered as prognostic factors for survival. The reported 5 year overall survival rate for patients with relapse within 2 years of diagnosis was poor at 5%, while the 5 year overall survival rate for patients with relapse after 2 years was relatively good at 34.9%. 1 Achieving second complete remission (CR) after relapse has been reported to be an important prognostic factor for survival. 6 The reported probability of post-relapse progression-free survival at 2 years is around3% for patients who did not achieve a second CR. 6 Allogeneic stem cell transplantation (allo SCT) has been presented as a potential curative approach based on the graft-versus-tumor (GVT) effect for intractable hematological malignancies. Even though there are case reports exploring and reporting the use of allo SCT in RR-ESFTs, 7-9 the efficacy of allo SCT for RR-ESFTs is yet to be established. However, these reports suggested that allo SCT could improve the survival rates of RR-ESFTs through cellular immunotherapy. Haploidentical SCT, which is associated with a stronger allogeneic immune response than conventional SCT, is rarely performed for solid tumors because of the high risk of transplantation-related mortality (TRM) from graft-versushost disease (GVHD) and the opportunistic infections. 10 The curative role of haploidentical SCT and its impact on survival remains unclear. 11 However, the authors confirmed that allo SCT was not associated with increased death from complications. In the current article, we report the outcomes of T-cell replete haploidentical SCT (TCR-haplo-SCT) after reduced intensity conditioning for pediatric patients with RR-ESFTs.

| METHODS
Six patients with RR-ESFTs who received TCR-haplo-SCT between 2010 and 2020 at Fukushima Medical University Hospital were retrospectively analyzed. Among the six patients, five were diagnosed with Ewing sarcoma, and one with primitive neuroectodermal tumor (PNET) ( Table 1). The institutional review board approved the protocol, and written informed consent was obtained from the patients or their guardians, as well as their donor family members.
Human leukocyte antigen (HLA) genotyping was conducted using PCR-Luminex (Luminex Corporation, Austin, Texas), based on reverse sequence-specific oligonucleotide (PCR-rSSO) technology (Genosearch HLA, Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) in Fukushima Medical University hospital. Peripheral blood stem cells (PBSCs) were collected from related donors using standard mobilization protocols. Granulocyte colony-stimulation factor (G-CSF) (400 μg/m 2 /day; Filgrastim, Kyowa Hakko Kirin Pharma Inc., Japan) was administered to the donors for five consecutive days to mobilize stem cells into the peripheral blood. PBSC harvesting was initiated on days 4 and 5 after G-CSF administration. PBSC collection was performed using COBE Spectra or Spectra Optia (Terumo BCT, Tokyo, Japan).
The conditioning regimen for all patients consisted of fludarabine (30 mg/m 2 /day, days À9 to À5), melphalan (70 mg/m 2 /day, days À4 to À3), and low-dose rabbit anti-thymocyte globulin (thymoglobulin 1.25 mg/kg/day, days À2 to À1) ( Table 2). GVHD prophylaxis was given with a combination of tacrolimus, short-term methotrexate, and prednisolone. 12 Methotrexate was administered intravenously, 10 mg/m 2 on day +1, and 7 mg/m 2 on days +3 and + 6 after transplantation. Prednisolone was started on day +0 at an initial dose of 1 mg/kg/day. If the patient showed no signs of acute GVHD (aGVHD), the initial dose was tapered every week from day +29 and was discontinued 2 months after transplantation. aGVHD and chronic GVHD (cGVHD) were graded using standard criteria. 13,14 Transplantation-related toxicities were evaluated using the Common Terminology Criteria for Adverse Events (CTCAE version 4.0) outlined by the National Cancer Institute. 15 3 | RESULTS

| Patient characteristics
Patient characteristics are shown in Table 1. The median age was 15 years (range, 6-23 years). The initial diagnoses included localized Ewing sarcoma (n = 4), metastatic Ewing sarcoma (n = 1), and PNET (n = 1). In the initial treatment, radiation therapy was administered in all cases, and all but one case (Patient 3) with primary pelvic tumor underwent surgical resection and chemotherapy. Disseminated relapse occurred during the initial treatment in one case (Patient 3).
Two patients (Patients 2 and 6) did not achieve CR at the end of the initial treatment and were considered to have a poor prognosis; therefore, we decided to perform TCR-haplo-SCT. The other three patients achieved CR at the end of initial treatment, however, all of them relapsed with multiple lesions, and four relapsed patients (Patients 1, 3, 4, and 5) were treated with chemotherapy. Four patients (Patients 3, 4, 5, and 6) received high-dose chemotherapy with busulfan and melphalan as consolidation therapy before TCR-haplo-SCT.

| TCR-haplo-SCT related information
Detailed information regarding TCR-haplo-SCT is provided in Table 2.
The disease status at the time of TCR-haplo-SCT was non-CR in all patients. One patient had a large residual tumor mass in the lung and mediastinum that were unresponsive to chemotherapy. One patient had a microscopic residual tumor with positive post-surgical margins.
The remaining four patients responded to second-line chemotherapy and experienced tumor shrinkage; however, metastatic tumors remained.
PBSCs were used as the stem cell source in all patients and were collected from family donors (four from mothers, one from a father, and one from an uncle). The patients received a median of 5.3 Â 10 6 CD34 positive hematopoietic cells/kg (range, 3.6 to 12.4 Â 10 6 cells/kg).
We used unmanipulated PBSCs to avoid attenuation of the GVT effect.

| HLA disparities and GVHD
The HLA disparities to graft versus host directions were 2/8 in one

| Complications within 100 days after the second TCR-haplo-SCT
Five patients had infectious complications within 100 days after the TCRhaplo-SCT, including cytomegalovirus antigenemia, Epstein-Barr virus reactivation, human herpes virus 6 encephalitis, and hemolytic cystitis due to BK virus. All were successfully treated with antiviral agents or by tapering their immunosuppression. Patient 1 developed pancreatitis (grade 2), and Patient 6 had a generalized convulsion (grade 2) of unknown origin.

| GVT effect
Results of imaging studies before and after the TCR-haplo-SCT in three patients with confirmed GVT effects are presented in Figure 1.
The GVT effect was confirmed in iliac bone metastasis in Patient 3, lung metastasis in Patient 5, and tibia metastasis in Patient 6. Patient 2 was difficult to evaluate through imaging because of a residual microscopic lesion. Patient 4 died due to a transplantation-related complication; therefore, we were unable to fully evaluate the case.
3.6 | Transplantation-related mortality, relapse, and outcome One patient (Patient 4) had a severe respiratory syncytial virus infection after TCR-haplo-SCT and died on day 134 due to pneumonia and T A B L E 1   In fact, there was no significant difference in the frequency of aGVHD between HLA-mismatch and HLA-matched groups in this study. They also found that disease status prior to hematopoietic allo SCT was the strongest prognostic factor with a significantly lower hazard ratio (0.4) for patients who achieved CR at transplantation. In our study, no GVT effect was observed in Patient 1, who had a large tumor burden, suggesting that pre-transplant disease status might be the greatest effect on transplantation outcome.
Therefore, for successful TCR-haplo-SCTs, it is essential to first reduce the tumor volume through multidisciplinary treatments after relapse. While our method did not exhibit a therapeutic effect on large residual tumors, it may be possible to increase the anti-tumor effects by combining it with drugs that inhibit the immune escape mechanism of the tumor, such as immune checkpoint inhibitors.
Although the sample size of this study was small, our data suggests that TCR-haplo-SCTs for RR-ESFTs are feasible and demonstrated three evaluable patients with GVT effect. We believe that TCR-haplo-SCT could be a potential therapeutic option for patients with RR-ESFTs. Further clinical studies are required to determine the efficacy of this novel approach.

ACKNOWLEDGMENTS
We would like to thank Dr. Tadashi  International Hospital for providing patient data.

CONFLICT OF INTERESTS
The authors declare that they have no conflicts of interest related to this study.