Successful Allogeneic Hematopoietic Cell Engraftment after a Minimal Conditioning Regimen in Children with Relapsed or Refractory Solid Tumors

Biol Blood Marrow Transplant 19 (2013) 291e297

Successful Allogeneic Hematopoietic Cell Engraftment after a Minimal Conditioning Regimen in Children with Relapsed or Refractory Solid Tumors

ASBMT

American Society for Blood and Marrow Transplantation

David R. Shook 1, 2, *, Brandon M. Triplett 1, 2, Ashok Srinivasan 1, 2, Christine Hartford 1, Mari H. Dallas 1, 2, Asha Pillai 1, 2, Joseph Laver 1, 2, Wing Leung 1, 2 1 2

Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, Tennessee Department of Pediatrics, University of Tennessee Health Science Center, College of Medicine, Memphis, Tennessee

Article history: Received 3 July 2012 Accepted 3 October 2012 Key Words: Pediatric soild tumor Allogeneic stem cell transplantation Graft-versus-tumor Reduced-intensity transplantation

a b s t r a c t Children with relapsed or refractory solid tumors face dismal prognoses, and novel therapies are desperately needed. Allogeneic hematopoietic cell transplantation (HCT) offers potential for cell-based therapy, but the toxicity of myeloablation limits this approach in heavily pretreated patients. We sought to determine the feasibility of HCT in a cohort of 24 children with incurable solid tumors using human leukocyte antigene matched sibling or unrelated donors and a minimal conditioning regimen. Before stem cell infusion, all patients received 3 daily doses of 30 mg/m2 fludarabine followed by 2 Gy of total body irradiation. Hematopoietic cell recovery was rapid and reliable. Median time to neutrophil engraftment was 13.5 days for sibling donors and 12 days for unrelated donors. Donor lymphocyte infusions were used safely in 4 patients, all of whom had either improved chimerism or apparent tumor response. Graft-versus-host disease was comparable across donor sources and did not affect survival. Relapse remains a substantial barrier, although objective graft-versus-tumor effect was observed in several patients. Four patients with detectable disease before HCT achieved a complete response for at least 30 days after HCT, and two remain long-term survivors. Three patients were in complete response before HCT and remained in remission for 3, 6, and 74 months after HCT. Early disease response was associated with improved survival. Allogeneic HCT using this conditioning regimen offers a potential platform for novel immunotherapies. Ó 2013 American Society for Blood and Marrow Transplantation.

INTRODUCTION Although substantial progress has been made in the treatment of pediatric malignancies, the prognosis for children with solid tumors has lagged behind that of acute leukemia [1]. Those with relapsed or refractory disease have exceptionally poor outcomes. Recurrent Ewing sarcoma and rhabdomyosarcoma are fatal in more than 80% of cases [2,3]. Children with metastatic or persistently unresectable hepatoblastoma have similarly dismal prognoses [4]. Relapsed neuroblastoma is generally considered to be beyond salvage [5]. Even malignancies such as lymphoma and Wilms tumor, which may have favorable prognoses at first relapse, have markedly decreased survival with chemorefractory disease [6,7]. Autologous stem cell transplantation (ASCT) offers an option for some patients, although success is limited [8-10]. Along with the risk of transplantation-related mortality [11], disease recurrence primarily limits the effectiveness of this approach [12]. Tumor contamination of autologous stem cell grafts is frequent, and purging does not improve survival [13]. Children who fail autologous transplantation have few options and face dismal prognoses. The effectiveness of cell-mediated cytotoxicity against leukemia has long been recognized [14,15], perhaps best demonstrated by reduced-intensity hematopoietic cell transplantation (HCT) conditioning regimens that exploit

Financial disclosure: See Acknowledgments on page 296. * Correspondence and reprint requests: David R. Shook, MD, 262 Danny Thomas Place, Mail Stop #1130, Memphis, TN 38105-3678. E-mail address: [email protected] (D.R. Shook).

a graft-versus-leukemia effect in lieu of myeloablative chemotherapy [16]. There is also evidence for potential graft-versus-tumor (GVT) effects in a number of pediatric solid tumors [17-21]. Children with relapsed or refractory solid tumors represent a heavily pretreated cohort facing very high-risk malignancies. Reduced-intensity conditioning (RIC) transplantation may offer the antitumor benefit of allografting without myeloablation, the excessive treatmentrelated toxicity of which can potentially abrogate the benefit afforded by GVT. Allogeneic HCT does not carry the risk of graft contamination with tumor cells and offers a substantial platform for additional cell therapy, such as donor lymphocyte infusions (DLI) and immunotherapy for purposes of tumor control [22]. Although allogeneic HCT is increasing among adult solid tumor patients [23], HCT for children with solid tumors has not been extensively described, especially using an RIC approach. Early allogeneic HCT efforts with myeloablative conditioning offered no advantage compared with autologous grafts, largely because of complications such as graft-versus-host disease (GVHD) and other regimen-related toxicities [24]. However, transplantationrelated mortality has been reduced significantly in modern pediatric HCT, even with unrelated or haploidentical donors [25]. Further, children not eligible for myeloablative HCT can be successfully transplanted using RIC [26]. We evaluated the feasibility of treating children with relapsed or refractory solid tumors with allogeneic HCT using a minimal conditioning regimen and a matched sibling donor (MSD) or a matched unrelated donor (MUD).

1083-8791/$ e see front matter Ó 2013 American Society for Blood and Marrow Transplantation. http://dx.doi.org/10.1016/j.bbmt.2012.10.001

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MATERIALS AND METHODS Patients Between March 2001 and July 2008, 24 patients were accrued to the St. Jude Children’s Research Hospital (St. Jude) STSIB protocol in Memphis, Tennessee. The protocol recruited patients to two strata: 12 patients transplanted using an MSD and 12 transplanted with an MUD. The protocol was approved by the St. Jude Institutional Review Board, and written informed consent was obtained from all patients and their families. The study was completed on September 30, 2010, and both survivors had at least 1 year of follow-up, including over 5 years since HCT. Two patients treated on this protocol were reported previously [27,28]. Patients 21 years old or younger with a histologically confirmed solid malignancy including lymphoma, for which standard curative therapy had failed or was unavailable, were eligible for the study. Children with primary brain tumors were not eligible. Detectable disease at the time of transplantation was not required. All patients required either an available human leukocyte antigen (HLA) identical sibling or a 6/6 HLA-matched unrelated donor. Both bone marrow and peripheral blood apheresis were acceptable as hematopoietic stem cell sources. Exclusion criteria included an estimated life expectancy of less than 8 weeks and autologous transplantation in the preceding 3 months. Prior radiation therapy was permissible, but those patients whose prior irradiation resulted in the inability to tolerate the prescribed radiation dose for this protocol were not enrolled. Severe infection at the time of enrollment was also not allowed. Organ-specific exclusion criteria included glomerular filtration rate <40 mL/min/1.73 m2, direct bilirubin >5 mg/dL, or serum glutamic pyruvic transaminase >500 U/L. Female patients were also excluded for pregnancy or lactation.

HLA Typing, Donor Selection, and Chimerism Donorerecipient pairs were selected based on HLA typing performed at St. Jude. All participants reported in this study were evaluated for HLA matching using family studies. HLA was typed using high-resolution at HLAA, -B, -C, -DRB1, and -DQB1 specificities. Peripheral whole blood chimerisms, performed using variable nucleotide tandem repeat polymerase chain reaction, were determined at approximately weekly intervals beginning at day þ14 until day þ100 and then approximately monthly for all patients who remained on study.

Treatment Protocol The same conditioning regimen was used for all patients, regardless of donor type. On days 4 to 2, patients received fludarabine at an intravenous dose of 30 mg/m2 once daily. On day 0, patients were treated with a single fraction of 2 Gy total body irradiation, followed by donor hematopoietic stem cell infusion. Granulocyte colony-stimulating factoremobilized peripheral blood stem cells, collected via apheresis, were the preferred stem cell source and were collected from sibling donors who were 16 years of age, as per institutional guidelines. Sibling donors <16 years old underwent bone marrow harvest. Nine of 12 patients in the MSD group received bone marrow as a stem cell source. The remaining 3 patients received a peripheral blood stem cell apheresis product. Unrelated donor requests were done in accordance with the guidelines of the National Marrow Donor Program. In the MUD group, 4 of 12 patients received a marrow product, and 8 received a peripheral blood stem cell apheresis product. GVHD prophylaxis consisted of mycophenolate mofetil and cyclosporine A. Mycophenolate mofetil was started on day 0 within 6 hours after the stem cell infusion at a dose of 15 mg/kg/dose twice daily. Mycophenolate mofetil was continued until approximately day þ27. Cyclosporine A was started at a dose of 2 mg/kg/dose intravenously every 12 hours on days 1. Beginning on day þ1, dosing was changed to 1 mg/kg/dose intravenously every 12 hours or 4 mg/kg/ dose by mouth every 12 hours. Doses were then adjusted to maintain therapeutic levels based on regular blood monitoring. For those patients with 90% to 100% donor chimerism at approximately day þ35, cyclosporine A was continued at a therapeutic level until approximately day þ60. Cyclosporine A was then tapered by 25% every 5 days until discontinued. For patients with <90% donor chimerism around day þ35, cyclosporine A was tapered more rapidly and discontinued by approximately day þ50. Patients with donor chimerism persistently <90% after withdrawal of immune suppression for 14 days or patients with stable or progressive disease were eligible for DLI if they were free of clinical GVHD. All patients received supportive care according to standard institutional policies and procedures, including regular outpatient follow-up with physical examination and laboratory testing. In addition to appropriate viral, fungal, and Pneumocystis prophylaxis, patients were monitored weekly by polymerase chain reaction assay for cytomegalovirus, Epstein-Barr virus, and adenovirus as well as by galactomannan assay for Aspergillus. Granulocyte colony-stimulating factor was not routinely given or required by protocol, but it was allowed intermittently at a dose of 5 mg/kg/day to maintain an absolute neutrophil count of at least 500/mm3.

The primary study endpoint was the induction of stable mixed or fulldonor hematopoietic chimerism using a minimal-intensity conditioning regimen in a heavily pretreated cohort of pediatric solid tumor patients. Acute and chronic GVHD was graded using the Seattle criteria [29,30], and toxicities were determined using the National Cancer Institute Common Toxicity Criteria, version 2.0, both institutional standards at the time of data collection. Tumor response evaluations were performed at approximately day þ30 and day þ60 for all surviving patients. Those patients with no detectable disease by any modality were designated as complete response (CR). Other definitions of tumor responses were responsive disease, defined as >50% regression of all tumor masses; progressive disease (PD), defined as an increase of 25% in the size of any tumor mass; and stable disease (SD), designated to those patients who failed to qualify for either responsive disease or PD. Study Design and Statistical Analysis The sample size of 12 patients for each stratum was chosen for preliminary evaluation of successful induction of mixed or full-donor chimerism. Stopping rules were in place for more than 2 failures in each stratum, giving confidence intervals of (0.52, 0.98), (0.62, 1.0), and (0.74, 1.0) for 10, 11, or 12 successes, respectively. The MSD and MUD groups were analyzed separately, and differences between donor groups were compared by Fisher’s exact test. Kaplan-Meier estimates were used to evaluate overall and event-free survival. The log-rank (Mantel-Cox) test was used to compare survival function of different groups.

RESULTS Patient Information Characteristics of the 24 patients enrolled are detailed in Table 1. Patient diagnoses included Ewing sarcoma family of tumors (n ¼ 3), Hodgkin lymphoma (n ¼ 3), non-Hodgkin lymphoma (n ¼ 1), hepatoblastoma (n ¼ 2), melanoma (n ¼ 1), neuroblastoma (n ¼ 7), rhabdoid renal tumor (n ¼ 1), rhabdomyosarcoma (n ¼ 4), and Wilms tumor (n ¼ 2). All patients were considered incurable with available therapy and were heavily pretreated. Sixteen patients had received prior myeloablative therapy; 6 of 12 patients who received an MSD transplantation and 8 of 12 patients who received an MUD transplantation had one previous ASCT. One additional patient in each group had two prior ASCTs. Only three patients had no detectable disease at the time of enrollment. All others were either in relapse after prior remission or refractory to all previous therapy. The median age of the MSD and MUD recipients was 14 and 8 years old, respectively. Twenty of 24 patients were white, including all 12 patients in the MUD group. Patients 1, 2, and 8 were African American, and patient 10 was of mixed ethnicity. All patients received fludarabine as an outpatient and were hospitalized on day 0 for total body irradiation and stem cell infusion. Most patients (n ¼ 20) were discharged the following day. Three patients were hospitalized 4 days, and 1 was an inpatient for 16 days. Median and mean lengths of initial hospitalization were 2 and 2.8 days, respectively.

Engraftment, Chimerism, and Additional Cell Product Infusion A summary of neutrophil and platelet recovery, separated by donor type, is shown in Figure 1. The graphs depict the percentage of patients achieving neutrophil and platelet recovery, as indicated by an absolute neutrophil count 500/ mm3 and platelet count 50,000/mm3. The median time to neutrophil recovery was 13.5 days (0e20) for those with sibling donors and 12 days (0e16) for unrelated donors. The median time to recovery to a platelet count of 20,000/mm3 was 0 days for both groups. Thus, platelet recovery was additionally evaluated using a threshold of 50,000/mm3. Median time to this level was 7.5 days (0e33) for those children with sibling donors and 16 days (0e36) for 11 of 12

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Table 1 Patient and Transplantation Characteristics with Disease Staging Disease

Disease Status

Prior ASCT

HSC Source

Day þ30 Staging

Day þ60 Staging

Days to PD

Patient Outcome

Matched sibling donors 1 12.6 2 13.4 3 7.7 4 17.6 5 14.6 6 17.3 7 17.9 8 5.8 9 13.0 10 16.2 11 12.3 12 17.7

NB NB WT RMS Melanoma Rhabdoid HL NB ESFT RMS ESFT HL

REF REF REL1 REL2 REF REL1 REL3 REL1 REL1 REF REF CR4

No Yes No Yes No No Yes Yes Yes Yes* No Yes

Marrow Marrow Marrow Blood Blood Marrow Marrow Marrow Marrow Blood Marrow Marrow

SD PD PD PD SD CR SD SD SD SD CR CR

PD SD PD SD SD PD CR PD SD PD CR CR

48 28 27 28 143 75 N/A 62 107 71 N/A 115

DPD DPD DPD DPD DPD DPD Alive DPD DPD DPD Alive DPD

Matched unrelated donors 13 20.7 14 10.2 15 6.3 16 21.2 17 8.6 18 20.4 19 8.1 20 14.1 21 3.2 22 7.7 23 7.5 24 4.1

NHL HBL NB HL NB RMS NB ESFT RMS NB WT HBL

REL2 REL3 REF CR5 REL1 REL2 CR2 REL2 REL1 REL1 REL1 REF

Yes Yes Yes Yes* Yes No Yes Yes No Yes Yes No

Blood Blood Marrow Blood Blood Blood Marrow Blood Blood Marrow Marrow Blood

N/A SD SD CR SD CR CR CR SD SD PD PD

N/A PD PD CR SD CR CR CR SD SD N/A PD

15 58 65 N/A 180 N/A 187 175 195 276 22 14

DPD DPD DPD DNED DPD DNED DPD DPD DPD DPD DPD DPD

Patient

Age (y)

REF indicates primary refractory; REL, relapse with number; CR, complete response with number; HSC, hematopoietic stem cell; SD, stable disease; PD, progressive disease; DPD, died from progressive disease; DNED, died with no evidence of disease; ESFT, Ewing sarcoma family of tumors; HL, Hodgkin lymphoma; NHL, non-Hodgkin lymphoma; HBL, hepatoblastoma; NB, neuroblastoma; RMS, rhabdomyosarcoma; WT, Wilms tumor. Time to progression is given as days after transplantation. * Patient had 2 prior ASCT.

children with unrelated donors. One child in the MUD cohort died (patient 13) at day þ15 before platelet count reached 50,000/mm3. Figure 2 summarizes the percentage of patients achieving various levels of whole blood chimerism, according to their donor source, at informative time points. Patient 13, who rapidly died from progressive disease, was not evaluated for donor chimerism at any time point. All tested patients had detectable donor chimerism after 2 weeks. Twenty-one of 24 patients (89%) achieved a high level of chimerism (75%) by 1 month, and surviving patients generally maintained that

chimerism at subsequent time points. Only one patient (patient 2) who had marrow-infiltrating neuroblastoma declined below 75% donor chimerism after reaching that level. Twenty patients were followed beyond day þ100. Eighteen of 20 patients maintained 100% donor chimerism, including patient 7, who remains 100%. Patient 6 fell to 95% donor shortly before death due to progressive disease, whereas patient 2 continues to have a stably mixed chimerism of approximately 95% donor. Four patients transplanted with an MSD received DLI. Patient 2 received a single DLI of 2.18  106 CD3þ cells/kg

Figure 1. Hematopoietic cell recovery by donor. Graphs show percentage of patients achieving (A) absolute neutrophil count recovery to 500/mm3 and (B) platelet count recovery to 50,000/mm3.

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Figure 2. Percentage of whole blood donor chimerism. SIB indicates matched sibling donor; MUD, matched unrelated donors. Averaged time points are used in the x-axis (2 weeks [days 11e17, median 14], 1 month [days 26e34, median 31], 2 months [days 55e64, median 61.5], and 100 days [95e123, median 101]).

on day þ71. Patient 3 received two DLI. The first was dosed at 5  106 CD3þ cells/kg on day þ63, and the second was 10  106 CD3þ cells/kg on day þ110. Both patients had temporary improvements in their chimerism with DLI that later reverted with disease progression. Patient 12 received two DLIs, each dosed at 10  106 CD3þ cells/kg. The first DLI on day þ137 converted her chimerism from 98% to 100%, where it remained until her eventual relapse, including before and after her second DLI on day þ182. Patient 5 also received two DLI for disease progression with a stable 100% chimerism: one at 5  106 CD3þ cells/kg on day þ169 followed by another at 45.83  106 CD3þ cells/kg on day þ391. All DLI were tolerated without GVHD or other adverse events in all patients. Supplemental Table 1 contains additional details regarding cell content of initial stem cell product.

Tumor Response All surviving patients were evaluated for disease response at day þ30 (n ¼ 23) and again at day þ60 (n ¼ 22). All responses were graded as PD, SD, or CR. Table 1 includes staging results as well as time to progression. One patient with relapsed disease died before day þ30 evaluation, and another died between day þ30 and day þ60 evaluations. Among the children who progressed by day þ30, none was in CR at day þ60, and none was an eventual long-term survivor. For the 11 children with disease stabilization at the first evaluation, 5 continued to have tumor control at day þ60. Seven patients were in CR at day þ30. Six remained in CR at the next evaluation, whereas only 1 had progressive disease. All 3 patients in CR before transplantation remained without evidence of disease at both evaluations.

Figure 3. Overall and event-free survival. (A) Overall survival by donor group, expressed as days after transplantation. Median overall survival 314 days in the SIB (sibling) group and 280 days in the MUD (unrelated) group. Log-rank test P ¼ .4215. (B) Event-free survival by donor group, expressed as days after transplantation. Events include death from any cause and receipt of nonprotocol therapy for progressive disease. Median event-free survival 211 days in the SIB group and 254 days in the MUD group. Log rank test P ¼ .6669. (C) Event-free survival by pretransplantation disease status. Log-rank test for relapsed disease vs complete response (CR), P ¼ .2200 refractory disease vs CR, P ¼ .4089. (D) Event-free survival by tumor stage at day þ30. Log-rank test for stable disease vs CR, P ¼ .432; progressive disease vs CR, P ¼ .0198.

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Table 2 Summary of Patient Outcome and GVHD

Patient outcome Death from disease Death with NED* Alive disease-free Acute GVHD Grade 1 or 2 Grade 3 or 4 Chronic GVHD Limited Extensive

Matched Sibling Donor (%)

Matched Unrelated Donor (%)

Total (%)

P Value

10 (83) d 2 (17)

10 (83) 2 (17) d

20 (83) 2 (8) 2 (8)

1.0

3 (25) 2 (17)

4 (33) 6 (50)

7 (29) 8 (33)

1.0 0.19

1 (8) 3 (25)

3 (25) 3 (25)

4 (17) 7 (29)

0.59 1.0

* Both died from aspergillosis complicating GVHD with no evidence of disease (NED).

Figure 3 illustrates overall and event-free survival, separated by donor source as well as by disease status. Twentytwo patients (92%) in this heavily pretreated cohort ultimately died from their underlying malignancy. Nine patients came off study to pursue additional therapy, all of whom died from progressive disease. There was no statistically significant difference in survival among donor types. Two patients remain long-term survivors. Patient 7 had multiply relapsed Hodgkin lymphoma with biopsy-confirmed malignant pulmonary nodules at the time of HCT, only partially responsive to salvage chemotherapy. Patient 11 completed intended therapy for metastatic Ewing sarcoma family of tumors, yet was found to have a persistent positron emission tomographyeavid mass adjacent to her previous primary tumor. Both patients are well with no evidence of disease. Of the patients that received DLI, patients 2 and 5 had disease progression despite DLI. Patient 3 had progression after her first DLI but had a mixed response after her second DLI. Patient 12 had multiply relapsed Hodgkin lymphoma that was in fourth CR at the time of HCT. Approximately 3 months after transplantation, she was found to have biopsyconfirmed recurrence in a thoracic vertebra that was treated with focal radiation and her second DLI. For almost 2 years, she was disease-free, until an asymptomatic relapse was detected on routine disease follow-up as a hepatic nodule. She received salvage chemotherapy but ultimately died 768 days after transplantation. GVHD and Toxicity A summary of GVHD as well as survival by donor source is contained in Table 2. Acute GVHD occurred in 15 patients, with low-grade (I or II) disease occurring in 3 patients transplanted using sibling donors and 4 who were transplanted using unrelated donors. Six children in the MUD group developed high-grade (III or IV) acute GVHD compared with two children transplanted with sibling donors. There were no statistical differences in rates of GVHD by donor source. In addition, there was no difference in survival among those who developed GVHD, as compared with those who did not. Both long-term survivors are off all immunosuppressive medications and have no evidence of GVHD. Patient-specific GVHD information is provided in Supplemental Table 1. In this extensively treated population, some grade IV toxicities occurred. Of these, the most commonly recorded toxicities were asymptomatic laboratory abnormalities (ie, electrolytes, blood counts). Infectious complications, most commonly catheter-related infection, occurred in 6 patients.

295

Two patients had their malignancy controlled but died from infectious complications. Patient 16 had multiply relapsed Hodgkin lymphoma that initially had a relatively uncomplicated post-HCT course until an orthopedic procedure incited significant, progressive sclerodermatous chronic GVHD approximately 18 months posttransplantation requiring systemic corticosteroids and cyclosporine A. Approximately 2 months after starting systemic immunosuppression, he presented with pulmonary aspergillosis from which he died on day þ887. Patient 18 had relapsed rhabdomyosarcoma whose post-HCT course was complicated by significant acute skin, liver, and gut GVHD as well as pulmonary Aspergillus infection. Despite antifungal therapy, she died from complications of disseminated aspergillosis on day þ250.

DISCUSSION Increasingly intensive chemotherapy in modern treatment protocols often leaves children affected by solid tumors with significant organ dysfunction [31,32]. Those that relapse face further toxic therapy, including myeloablation with ASCT [33]. Yet despite dose intensification, cure rates remain unacceptably low. With the impact of immunotherapy on outcomes for high-risk neuroblastoma [34], there has been renewed interest in novel solid tumor therapeutic approaches that bypass drug resistance. Allogeneic HCT offers the benefit of sustained alloreactivity, but conventional HCT approaches can be limited by toxicity. HCT using RIC has been effectively used for adults who cannot tolerate myeloablative therapy, and this study showed that a heavily pretreated pediatric population could also tolerate HCT using this approach. The conditioning regimen was well tolerated, and hematopoietic cell engraftment proved to be rapid and reliable. Combined with the ability to deliver the majority of the therapy as an outpatient, this approach could considerably decrease resource utilization, especially when compared with myeloablative HCT. All children had neutrophil recovery within 3 weeks, and some were never neutropenic. Most patients never developed thrombocytopenia below standard transfusion thresholds. In addition, the average length of hospitalization was less than 3 days. This compares quite favorably with a current 31-day average admission for allogeneic transplantation at our institution. Sibling and unrelated donors were analyzed separately; no significant differences between these two groups were detected, either in terms of toxicity or response, extending the feasibility of this therapy to those patients without matched siblings. Several mechanisms by which tumor cells may escape endogenous immune surveillance have been proposed, including altered antigen presentation and production of immunosuppressive cytokines [35]. Immune escape provides an important rationale for allogeneic HCT, where donor-immune effector cells may recognize tumor cells that host cells have spared. Unlike leukemia, where graft-versusleukemia has been well demonstrated, similar GVT effects in pediatric solid tumors have been difficult to consistently establish, and few such studies have been reported. Few tumor-specific antigens exist, and the mechanisms behind GVT remain poorly understood. This is likely due to the complex immunologic milieu within the tumor microenvironment, but tumor-infiltrating lymphocytes may play an important role, both in tumor invasion and recruitment of cytotoxic lymphocytes [36].

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The children in this study, representative of a wide variety of advanced pediatric tumors, were extremely high risk, eligible only if no alternative curative therapy was available. With little to no antineoplastic effect afforded by the conditioning regimen, all observed tumor responses support an active GVT effect. Four patients with evidence of disease before HCT achieved CR, and the 3 patients in CR before HCT remained so for 3, 6, and 74 months, respectively, after HCT. Two long-term survivors continue to be followed, and they are well without disease. Unfortunately, the two patients who died from fatal opportunistic fungal infections also both had no evidence of disease. Further strengthening the impact of alloreactivity, 4 patients with matched sibling donors received DLI, all with apparent clinical benefit. The infusions were well tolerated, and each patient had either improved donor chimerism or tumor response after DLI. One patient (patient 12) developed a distant recurrence associated with mixed chimerism that was treated with only local irradiation and DLI, yet she re-entered CR and had systemic disease control with 100% chimerism for nearly 2 years. GVHD rates were acceptable, and neither acute nor chronic GVHD statistically affected survival. Patient numbers were small, making it difficult to determine the impact of disease burden at the time of HCT on response or outcomes. However, other investigators using RIC HCT for pediatric sarcomas have recently described a survival benefit in children with no detectable disease before HCT [37]. One important observation was the impact of early disease progression. Complete response at day þ30 imparted a statistically significant increase in duration of survival over those with progressive disease. Thus, this subset of patients, representing approximately one-third of the cohort, would have survived long enough to allow subsequent novel consolidative therapy, such as cellular or monoclonal antibody therapy [34]. Allogeneic HCT is advantageous over ASCT in this regard, as donors can be reapproached for additional cell products for disease consolidation or to treat relapse. These cell products would also be expected to have improved in vivo survival and proliferation after allogeneic HCT, as compared with adoptive cellular therapy alone. Killer immunoglobulin-like receptor (KIR) typing was not performed on patients in this study, but the interaction between natural killer cell KIR and target cell HLA has been shown to affect outcomes on allogeneic transplantation [38]. A similar prognostic benefit to KIR-HLA mismatch has also been described in the autologous setting [39,40] as well as with immunocytokine therapy [41]. Even with the potential advantage of breaking self-tolerance through chemotherapy, the full immunologic benefit of ASCT can only be realized in a subset of fortuitous patients with appropriate natural killer cell reactivity. Allogeneic HCT offers the opportunity to select a potentially more appropriate KIR-HLA mismatched donor. In summary, this phase I study was able to demonstrate the feasibility of allogeneic HCT in children with relapsed or refractory solid tumors using HLA-matched sibling or unrelated donors and a minimal conditioning regimen. Although some disease control was achieved, disease progression remained the main cause of mortality. Improved biologic understanding of the interactions between patient tumor cells and donor immune effector cells may aid future studies. Engraftment was excellent and offers the opportunity for other cellular or antibody therapies, immune modulating agents [42], or additional adoptive cellular therapy with natural killer cells [43] or genetically modified T cells [44].

ACKNOWLEDGMENTS The authors thank our clinical, laboratory, and research office colleagues for data collection and the many patients and parents who participated in our transplantation and cellular therapy research program. Financial disclosure: Supported in part by the National Institutes of Health Cancer Center Support (CORE) grant P30 CA021765, a Center of Excellence Grant from the State of Tennessee, the Assisi Foundation of Memphis, and the American Lebanese Syrian Associated Charities (ALSAC). The authors have no financial relationships or other conflicts of interest to disclose. SUPPLEMENTARY DATA Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.bbmt.2012.10.001. REFERENCES 1. Pui CH, Pei D, Pappo AS, et al. Treatment outcomes in black and white children with cancer: results from the SEER Database and St Jude Children’s Research Hospital, 1992 through 2007. J Clin Oncol. 2012;30: 2005-2012. 2. Rodriguez-Galindo C, Billups CA, Kun LE, et al. Survival after recurrence of Ewing tumors: the St Jude Children’s Research Hospital experience, 1979e1999. Cancer. 2002;94:561-569. 3. Pappo AS, Anderson JR, Crist WM, et al. Survival after relapse in children and adolescents with rhabdomyosarcoma: a report from the Intergroup Rhabdomyosarcoma Study Group. J Clin Oncol. 1999;17: 3487-3493. 4. Katzenstein HM, London WB, Douglass EC, et al. Treatment of unresectable and metastatic hepatoblastoma: a pediatric oncology group phase II study. J Clin Oncol. 2002;20:3438-3444. 5. Garaventa A, Parodi S, De Bernardi B, et al. Outcome of children with neuroblastoma after progression or relapse. A retrospective study of the Italian neuroblastoma registry. Eur J Cancer. 2009;45:2835-2842. 6. Attarbaschi A, Dworzak M, Steiner M, et al. Outcome of children with primary resistant or relapsed non-Hodgkin lymphoma and mature B-cell leukemia after intensive first-line treatment: a population-based analysis of the Austrian Cooperative Study Group. Pediatr Blood Cancer. 2005;44:70-76. 7. Dome JS, Liu T, Krasin M, et al. Improved survival for patients with recurrent Wilms tumor: the experience at St. Jude Children’s Research Hospital. J Pediatr Hematol Oncol. 2002;24:192-198. 8. Gardner SL, Carreras J, Boudreau C, et al. Myeloablative therapy with autologous stem cell rescue for patients with Ewing sarcoma. Bone Marrow Transplant. 2008;41:867-872. 9. Stiff PJ, Agovi MA, Antman KH, et al. High-dose chemotherapy with blood or bone marrow transplants for rhabdomyosarcoma. Biol Blood Marrow Transplant. 2010;16:525-532. 10. Kasper B, Lehnert T, Bernd L, et al. High-dose chemotherapy with autologous peripheral blood stem cell transplantation for bone and soft-tissue sarcomas. Bone Marrow Transplant. 2004;34:37-41. 11. Foncillas MA, Diaz MA, Sevilla J, et al. Engraftment syndrome emerges as the main cause of transplant-related mortality in pediatric patients receiving autologous peripheral blood progenitor cell transplantation. J Pediatr Hematol Oncol. 2004;26:492-496. 12. Barrett D, Fish JD, Grupp SA. Autologous and allogeneic cellular therapies for high-risk pediatric solid tumors. Pediatr Clin North Am. 2010; 57:47-66. 13. Leung W, Chen AR, Klann RC, et al. Frequent detection of tumor cells in hematopoietic grafts in neuroblastoma and Ewing’s sarcoma. Bone Marrow Transplant. 1998;22:971-979. 14. Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood. 1990;75:555-562. 15. Kolb HJ. Graft-versus-leukemia effects of transplantation and donor lymphocytes. Blood. 2008;112:4371-4383. 16. Satwani P, Harrison L, Morris E, et al. Reduced-intensity allogeneic stem cell transplantation in adults and children with malignant and nonmalignant diseases: end of the beginning and future challenges. Biol Blood Marrow Transplant. 2005;11:403-422. 17. Perez-Martinez A, Leung W, Munoz E, et al. KIR-HLA receptor-ligand mismatch associated with a graft-versus-tumor effect in haploidentical stem cell transplantation for pediatric metastatic solid tumors. Pediatr Blood Cancer. 2009;53:120-124. 18. Koscielniak E, Gross-Wieltsch U, Treuner J, et al. Graft-versus-Ewing sarcoma effect and long-term remission induced by haploidentical stem-cell transplantation in a patient with relapse of metastatic disease. J Clin Oncol. 2005;23:242-244.

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