1 Pediatr Blood Cancer 2014;61: Pilot Trial of Risk-Adapted Cyclophosphamide Intensity Based Conditioning and HLA Matched Sibling and Unrelated Cord Blood Stem Cell Transplantation in Newly Diagnosed Pediatric and Adolescent Recipients with Acquired Severe Aplastic Anemia Catherine McGuinn, MD, 1 Mark B. Geyer, MD, 2 Zhezhen Jin, PhD, 3 James H. Garvin, MD, PhD, 4 Prakash Satwani, MD, 4 M. Brigid Bradley, MD, 4 Monica Bhatia, MD, 4 Diane George, MD, 4 Deirdre Duffy, BS, 5 Erin Morris, RN, BSN, 5 Carmella van de Ven, MA, 5 Joseph Schwartz, MD, 6 Lee Ann Baxter-Lowe, PhD, 7 and Mitchell S. Cairo, MD 5,8,9,10,11 * Background. Cyclophosphamide-based conditioning regimens and allogeneic hematopoietic stem cell transplantation (AlloHSCT) from matched related donors (MRD) has resulted in the highest survival rates in children and adolescents with acquired severe aplastic anemia (SAA). Time to transplant has consistently been associated with decreased overall survival. Reduced toxicity conditioning and AlloHSCT has been used successfully in other pediatric non-malignant diseases. Procedure. We piloted a riskadapted AlloHSCT approach, using fludarabine and anti-thymocyte globulin based conditioning with high (200 mg/kg) and low (60 mg/ kg) dose cyclophosphamide as upfront treatment in newly diagnosed pediatric patients with acquired SAA incorporating alternative donor sources, including cord blood. Average risk for non-engraftment patients with <10 transfusions received low dose cyclophosphamide (60 mg/kg); High Risk, those with 10 transfusions received conditioning regimen with higher intensity cyclophosphamide (200 mg/kg). Results. Seventeen patients were enrolled and underwent AlloHSCT including 12 males and 5 females with mean age of 8 years (range 3 16), and median follow-up time of 39 months (range 1 135). Donor sources included MRD BM (6/6 [n ¼ 9], 5/6 [n ¼ 2]) and unrelated CB (5/6 [n ¼ 4], 4/6 [n ¼ 2]). Five year OS was 67.6% ( ). Three secondary graft failures (17.6%) occurred in the low dose cyclophosphamide arm. Conclusions. Upfront treatment with risk-adapted cyclophosphamide conditioning AlloSCT is well tolerated for the management of newly diagnosed pediatric and adolescent patients with acquired SAA. However, the increased risk of graft rejection in the lower dose arm warrants additional research regarding the optimal intensity of cyclophosphamide-based conditioning regimen to reduce toxicity without increasing graft failure. Pediatr Blood Cancer 2014;61: # 2014 Wiley Periodicals, Inc. Key words: acquired severe aplastic anemia; adolescent; matched donors; pediatric; risk-adapted conditioning; unrelated donors INTRODUCTION Acquired severe aplastic anemia (SAA) is a life-threatening pancytopenia with risk for serious infections and transfusion dependence. Curative upfront therapy in newly diagnosed children and adolescents with SAA is allogeneic hematopoietic stem cell transplantation (AlloHSCT) from a human leukocyte antigen (HLA) matched sibling donor (MSD) . Patients lacking a wellmatched donor are often treated initially with immunosuppressive therapy (IST), anti-thymocyte globulin (ATG), and cyclosporine (CsA), despite a limited response and high failure rate (40 60%) . IST has a cumulative increased risk of clonal transformation [2,3] and potential increased risk of graft rejection [4 6] and graft-versus-host disease (GVHD) [4,5] with increasing transfusion exposure if salvage AlloHSCT is needed. Improvements in donor selection through HLA high resolution typing, optimized conditioning regimens, and supportive care, have resulted in comparable overall survival (OS) using well-matched unrelated donors [7,8]. Given the negative impact of previous IST and risk of transfusional alloimmunization, there is an increasing interest in exploring utilizing AlloHSCT as upfront therapy with well-matched unrelated donors . The challenges to this approach include timely identification of alternative donors, minimizing the risk of graft rejection and balancing the toxicity of transplantrelated mortality (TRM) and late effects. Unfortunately, greater than 70% of patients with SAA will not have a matched related donor (MRD), with decreasing likelihood in ethnically diverse populations. In these patients unrelated cord blood (UCB) presents the unique advantages of rapid availability, enhanced ability to cross donor-recipient mismatch and lower rates of GVHD [10 14]. UCB has been used successfully as a donor C 2014 Wiley Periodicals, Inc. DOI /pbc Published online 12 February 2014 in Wiley Online Library (wileyonlinelibrary.com). Additional Supporting Information may be found in the online version of this article. 1 Department of Pediatrics, Weill Cornell Medical College, New York, New York; 2 Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; 3 Department of Biostatistics, Columbia University, New York, New York; 4 Department of Pediatrics, Columbia University, New York, New York; 5 Department of Pediatrics, New York Medical College, Valhalla, New York; 6 Department of Pathology and Cell Biology, Columbia University, New York, New York; 7 Department of Surgery, University of California San Francisco, San Francisco, California; 8 Department of Medicine, New York Medical College, Valhalla, New York; 9 Department of Pathology, New York Medical College, Valhalla, New York; 10 Department of Microbiology and Immunology, New York Medical College, Valhalla, New York; 11 Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York Grant sponsor: Pediatric Cancer Research Foundation (PCRF); Grant sponsor: Brittany Barron Fund; Grant sponsor: Marisa Fund; Grant sponsor: Paul Luisi Foundation Conflict of interest: Nothing to declare. Correspondence to: Mitchell S. Cairo, Chief, Pediatric Hematology, Oncology and Stem Cell Transplantation, Director, Children and Adolescent Cancer and Blood Diseases Center, Medical and Scientific Director Cellular and Tissue Engineering Laboratory, Associate Chairman, Department of Pediatrics, Professor of Pediatrics, Medicine, Pathology, Microbiology & Immunology and Cell Biology & Anatomy, Maria Fareri Children s Hospital, Westchester Medical Center New York Medical College, Valhalla, NY Received 6 September 2013; Accepted 16 January 2014
2 1290 McGuinn et al. source in patients with refractory SAA [14 16] with the hurdles of non-engraftment and rejection [17,18] improved by optimizing cell dose  and conditioning regimens [14,15]. Risk-adapted conditioning is an alternative strategy in nonmalignant diseases to minimize the long- and short-term morbidity and TRM [19,20]. Conditioning regimens for SAA have focused on reducing radiation exposure, while maintaining engraftment through the judicious use of T-cell targeted immunosuppression . Based on these considerations, we piloted a risk-adapted cyclophosphamide-based conditioning intensity approach stratified according to prior transfusion history, as surrogate for risk of nonengraftment, in pediatric and adolescent patients with newly diagnosed acquired SAA. We compared cyclophosphamide standard dose 200 mg/kg with low dose 60 mg/kg, in combination with fludarabine and rabbit-atg in preparation for AlloHSCT. The objective of the study was to determine the feasibility, safety and efficacy of such a risk-adapted cyclophosphamide-based conditioning intensity approach, including timely identification of UCB units for alternative donor transplants. METHODS Seventeen patients underwent AlloHSCT after meeting diagnostic criteria for acquired SAA . The minimally transfused patients, defined as patients receiving 10 previous transfusions received low dose cyclophosphamide (60 mg/kg) (30 mg/kg, days 3to 2), fludarabine 180 mg/m 2 (30 mg/m 2, days 7to 2) and rabbit-atg (ratg) (Thymoglobulin) 8 mg/kg (2 mg/kg, days 4to 1) (n ¼ 7) as we have previously described . Patients with >10 transfusions, received higher dose cyclophosphamide (200 mg/kg) (50 mg/kg, days 5to 2), fludarabine 180 mg/m 2 (30 mg/m 2, days 7 to 2) and rabbit-atg (Thymoglobulin) 8 mg/kg (2 mg/kg, days 4 to 1) (n ¼ 10). Patients were eligible if they had a fully matched or one antigen-mismatched family donor (6/6 or 5/6) (MRD/mismatched related donor [MMRD]), or 6/6, 5/6 or 4/6 unrelated CB unit with a minimum cryopreserved cell dose of total nucleated cells (TNCs) per kg, washed and thawed [24 26]. All patients/parents signed informed consents and assents (when applicable). All protocols, informed consents and assents were approved by the Institutional Review Board in compliance with the Declaration of Helsinki. HLA-A and HLA-B typing was performed by intermediate resolution molecular testing, and HLA-DRB1 typing was determined by hybridization of PCR- amplified DNA with sequence specific oligonucleotide probes [27,28]. AGVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil (MMF) [29,30]. Tacrolimus was tapered starting on day þ180 if patient had Grade I agvhd. AGVHD and cgvhd were graded according to Seattle criteria . Isolation, transfusions, antibacterial, antifungal and antiviral prophylaxis and growth factor administration were performed according to institutional protocols as previously described [23,32 35]. Neutrophil recovery was defined as an ANC of >500/mm 3 for three consecutive days. Platelet recovery was defined as a platelet count of 20,000/mm 3 independent of transfusions for >7 days. Primary myeloid graft failure was defined as failure to achieve a donor-derived ANC 500/mm 3 by Day þ42 and/or 50% wholeblood donor chimerism by day þ60. Secondary graft failure was defined as achievement of neutrophil engraftment followed by 10 days of ANC 500/mm 3 and <50% whole-blood donor chimerism . Donor chimerism was measured on days þ30, 60, 100, 180 and 365 and yearly after transplant using a PCR-based method involving amplification of genes containing short tandem repeats . Opportunistic infections, viral and bacterial were defined as previously described [23,36,37]. Statistics Statistical analyses were performed in Prism 5.03 (GraphPad, La Jolla, CA). The cutoff date for analysis was December 5, Continuous variables were summarized as mean standard deviation (SD) and categorical variables as percentages. Continuous variables were compared between two groups using two-sample t-tests and categorical variables using chi-square tests. Probabilities of neutrophil and platelet engraftment, agvhd, cgvhd, post-transplantation lymphoproliferative disease (PTLD), day 100 TRM, and OS were estimated using the Kaplan Meier estimators, and comparisons between groups were carried out with log-rank tests. The Cox proportional hazards model was used to identify risk factors of agvhd and OS. A P-value less than 0.05 was considered significant. RESULTS Demographic data including age, prior treatments, time from diagnosis to transplant, donor source, cell source and dose, HLA concordance and period of transplantation are summarized in Tables I and II. Seven patients received 10 prior transfusions, classified as average risk for engraftment, received low dose cyclophosphamide (60 mg/kg) conditioning; 10 patients received >10 transfusion were classified as high risk and received a higher dose of cyclophosphamide (200 mg/kg) (Table I) with a median follow-up was 39 months (1 135). Recipients of higher dose compared with low dose cyclophosphamide did not differ significantly with respect to overall survival, time to neutrophil or platelet engraftment, whole-blood donor chimerism, probability of developing agvhd or cgvhd or incidence of bacterial and opportunistic infections (Fig. 1A C, Supplemental Figure 1A B, and Supplemental Tables I and II). Secondary graft failure occurred in three patients without a statistically significant difference based on donor type, cell source, prior IST, time to transplant, HLA match or conditioning regimen (Table III). However, all three secondary graft failures occurred in patients who received the low dose cyclophosphamide conditioning regimen with an HLA mismatched donor source. The OS in the study was 67.6% (CI 95 : ) (Fig. 2A and 2B). There was no statistically significant difference in OS according to donor type, cell source, conditioning intensity, sex or GVHD status (Table IV). There was a higher risk of overall mortality with a history of prior IST therapy (P ¼ ) and time >6 months from diagnosis to transplant (P ¼ 0.002) (Table IV). At the time of analysis, 12 patients remained alive with a median follow-up of 58 months for survivors. The causes of death in the remaining five patients were multi-organ failure (n ¼ 3), complications related to second AlloHSCT (n ¼ 1), and thrombotic microangiopathy (n ¼ 1). Three patients in our cohort developed PTLD following AlloHSCT. Two of three patients had an autoimmune hepatitis etiology of SAA. However, all three patients who developed PTLD occurred in the high risk group which received the higher dose cyclophosphamide conditioning regimen.
3 Risk-Adapted Upfront AlloHSCT in SAA 1291 TABLE I. Characteristics of Patients and AlloHSCT DISCUSSION All patients N ¼ 17 (%) Low dose Cy High dose Cy N ¼ 7 (41.2%) N ¼ 10 (58.8%) Age Mean 9.2 years 9.5 years 8.9 years Range 3 16 years 6 16 years 3 16 years Gender Male 12 (70.1%) 4 (57.1%) 8 (80%) Female 5 (29.4%) 3 (42.9%) 2 (20%) Prior treatment Immunosuppressive 2 (11.7%) 2 (20%) therapy (IST) No prior IST 15 (88.2%) 7 (100%) 8 (80%) Time from diagnosis to transplant Median 52 days 25 days 62.5 days Range 13 1,223 days days days <6 months 15 (83%) 7 (100%) 8 (80%) 6 months 2 (17%) 2 (20%) Donor source Related 11 (64.7%) 5 (71.4%) 6 (60%) Unrelated 6 (35.3%) 2 (28.6%) 4 (40%) Cell source Bone marrow 11 (64.7%) 5 (71.4%) 6 (60%) Unrelated cord blood 6 (35.3%) 2 (28.6%) 4 (40%) HLA match Related bone marrow Full Match (6/6) 9 (64.7%) 3 (42.8 %) 6 (60%) 1-antigen 2 (11.7%) 2 (28.5 %) mismatch (5/6) Unrelated cord blood 1-antigen 4 (23.5%) 2 (28.5%) 2 (20%) mismatch (5/6) 2-antigen 2 (11.7%) 2 (20%) mismatch (4/6) Date of transplant Before (47.1%) 4 (57.1%) 4 (40%) (52.9%) 3 (42.9 %) 6 (60%) AlloHSCT, allogeneic hematopoietic stem cell transplantation; Cy, cyclophosphamide; PBSC, peripheral blood stem cells; IST, immunosuppressive therapy; CsA, cyclosporine; ATG, anti-thymocyte globulin. Incorporating upfront use of alternative donor sources into the treatment of SAA, we extended the availability of potentially curative AlloHSCT in children and adolescents. The 5-year OS of 67.6% in this risk-adapted approach and AlloHSCT included patients with unrelated CB donor sources and HLA disparate grafts. The improvement in outcomes of alternative donor sources, reinforced by the increasing risk of poor outcomes with increasing time from diagnosis [8,38], pre-treatment with IST  and risk of clonal evolution to malignancy , support consideration for the role of AlloHSCT as first line therapy for all children diagnosed with acquired SAA. In our limited sample, a previous failed course of IST and prolonged disease duration prior to transplant (>6 months) was associated with a significant increase in mortality. In our series, 4/6 children with acquired SAA are alive following AlloHSCT from TABLE II. Outcomes of Transplantation HLA match Regimen agvhd cgvhd Graft failure Complications Disease status CD /kg TNC 10 8 /kg Donor source Related/ unrelated Time to transplant Prior IST Age/ sex Patient 1 4/M N 134 Unrelated Cord /6 Flu/Cy (200)/ATG N N Alive 2 12/F N 15 Related BM /6 Flu/Cy (60)/ATG N N Alive 3 8/F N 13 Related BM /6 Flu/Cy (60)/ATG N N Secondary Adenovirus/2nd AlloSCT Died 4 7/M Y 584 Related BM /6 Flu/Cy (200)/ATG Y N Multi-system organ failure Died 5 16/M Y 831 Unrelated Cord Flu/Cy (200)/ATG N N Multi-system organ failure Died 6 12/M N 45 Related BM /6 Flu/Cy (200)/ATG Y N Alive 7 11/M N 24 Unrelated Cord /6 Flu/Cy (60)/ATG Y N Secondary Autologous reconstitution Alive 8 6/M N 25 Related BM /6 Flu/Cy (60)/ATG Y N Alive 9 6/M N 52 Unrelated Cord /6 Flu/Cy (60)/ATG N N Alive 10 8/F N 43 Related BM /6 Flu/Cy (60)/ATG N N Secondary Pure RBC aplasia/2nd AlloSCT Alive 11 10/M N 53 Related BM /6 Flu/Cy (200)/ATG N N Thrombotic microangiopathy Died 12 15/F N 59 Related BM /6 Flu/Cy (200)/ATG N N Alive 13 16/F N 62 Related BM /6 Flu/Cy (60)/ATG N N Alive 14 8/M N 85 Unrelated Cord /6 Flu/Cy (200)/ATG N N Alive 15 3/M N 66 Unrelated Cord /6 Flu/Cy (200)/ATG Y Y PTLD/multi-system organ failure Died 16 5/F N 41 Related BM /6 Flu/Cy (200)/ATG N N PTLD Alive 17 9/M N 30 Related BM /6 Flu/Cy (200)/ATG N N PTLD Alive Age/sex indicates age in years; M, male; F, female; IST, immunosuppressive therapy; BM, bone marrow; Flu/Cy(60)/ATG, fludarabine (Flu) 180 mg/m 2 þ cyclophosphamide (Cy) 60mg/kg þ rabbit anti-thymocyte globulin (ratg) 8 mg/kg; Flu/Cy (200)/ATG, Cy 200 mg/kg þ Flu 180 mg/m 2 þ ratg 8 mg/kg.
4 1292 McGuinn et al. Fig. 1. A: Median time to neutrophil engraftment by Kaplan Meier method stratified by intensity of conditioning. B: Median time to platelet engraftment by Kaplan Meier method stratified by intensity of conditioning. C: Time to whole-blood donor chimerism stratified by intensity of conditioning compared using a students t-test. TABLE III. Predictors of Secondary Graft Failure Graft failure Engraftment Total P-value (2-tailed) N 3 18% 14 82% 17 Donor Related 2 18% 9 82% 11 Unrelated 1 17% 5 83% 6 Cell source Marrow 2 18% 9 82% 11 Cord blood 1 17% 5 83% 6 Conditioning intensity High dose cyclophosphamide 0 0% % 10 Low dose cyclophosphamide 3 43% 4 57% 7 Sex Male 1 8% 11 92% 12 Female 2 40% 3 60% 5 HLA match Fully matched 0 0% 9 100% 9 1-antigen mismatch 3 50% 3 50% 6 2-antigen mismatch (all 4/6 cords) 0 0% 2 100% 2 HLA match Fully matched 0 0% 9 100% 9 Not fully matched 3 38% 5 63% 9 Prior IST 1 No prior IST 3 20% 12 80% 15 Prior IST (CsA and/or ATG) 0 0% 2 100% 2 Time to transplant 1 <6 months from diagnosis 3 20% 12 80% 15 6 months from diagnosis 0 0% 2 100% 3 UCBT, umbilical cord blood transplantation; MRD, matched related donor; HLA, human leukocyte antigen; IST, immunosuppressive therapy; CsA, Cyclosporine; ATG, anti-thymocyte globulin.
5 Risk-Adapted Upfront AlloHSCT in SAA 1293 TABLE IV. Predictors of Overall Mortality HR 95% CI P-value Fig. 2. A: Probability of overall survival by Kaplan Meier method. B: Probability of overall survival by Kaplan Meier method stratified by intensity of conditioning. unrelated CB and there was no significant difference in OS based on donor stem cell source. Well-matched CB should be investigated as a potential donor source with acknowledgment of the importance of TNC dose and degree of HLA disparity in CB selection. In our series, the patients treated with two antigen-mismatched CB (4/6) had a trend towards decreased OS, consistent with reports highlighting the increased risk of TRM [39,40], GVHD  and infections  contributing to inferior outcomes with two antigenmismatched UCBT . Small prospective trials with UCBT in patients with acquired SAA have begun to show potentially encouraging results [15,20,41]. The largest series of UCBT for refractory SAA in 71 adults and pediatric patients from 23 EBMT centers showed a 51% engraftment rate and 38% 3-year OS, with a clear association of outcome with TNC dose . Moving from refractory disease to upfront treatment, a series of eighteen adult patients with newly diagnosed SAA treated with UCBT had an encouraging 2-year survival of 88.9% . This OS included an extremely high incidence of graft failure (16/18 patients) with the hypothesis that the immunosuppressive effect of the combination of T cell-directed conditioning regimen with autologous reconstitution mitigated the impact of graft failure on OS . We did not observe the same magnitude of effect in our pediatric population, but this represents an important concept in envisioning the potential roles for UCBT in management of SAA. OS was comparative between the low dose and higher dose cyclophosphamide groups with 6/7 and 6/10 patients surviving in each group. Importantly, there was a suggestion of increased rates of secondary graft failure with low dose cyclophosphamide conditioning, especially in the setting of mismatched donor sources. While these are small numbers, there is a suggestion that low doses Donor Related 1 Referent Unrelated Cell source Marrow 1 Referent Cord blood Conditioning intensity High dose 1 Referent cyclophosphamide Low dose cyclophosphamide Sex Male 1 Referent Female HLA match Fully matched 1 Referent 1-antigen mismatch antigen mismatch HLA match Fully matched 1 Referent Not fully matched GVHD status agvhd Grades 0 I 1 Referent agvhd Grades II IV Prior IST No prior IST 1 Referent Prior IST 2, (CsA and/or ATG) Time to transplant <6 months from diagnosis 6 months from diagnosis 1 Referent 2, MRD, matched related donor; UCBT, umbilical cord blood transplantation; GVHD, graft-versus-host disease; agvhd, acute graft-versushost disease; cgvhd, chronic graft-versus-host disease; IST, immunosuppressive therapy; CsA, cyclosporine; ATG, anti-thymocyte globulin. of cyclophosphamide may not be adequate for sustained engraftment. The optimal conditioning dose of cyclophosphamide for acquired SAA remains unclear. Preliminary analysis from BMT CTN 0301 (NCT ) identified that omitting cyclophosphamide potentially was associated with increased graft failures, but toxicity at escalating doses met safety stopping criteria; reinforcing the need for additional information to determine the optimal conditioning regimen in SAA AlloHSCT . Limitations of our study include small sample size, heterogeneous donor sources and surrogate, rather than biologic basis, for non-engraftment risk. Patient risk stratification was assigned solely on the number of preceding transfusions. Unfortunately, we did not measure anti-hla antibodies; therefore, exposure to 10 transfusions was a dichotomous categorical point chosen based on historical research regarding a continuously higher risk of nonengraftment and alloimmunization with increasing transfusion exposure [4,5,43,44]. While these historical data straddle the adoption of universal blood product leukoreduction, concerns persist for the risk of rejection in multiply transfused patients . While we acknowledge this as a limitation of our study, increasing
6 1294 McGuinn et al. exposure to transfusions may encompass additional high risk factors such as prolonged time to transplantation, increased risk of infection or even potentially iron load, which was not determined in our study. As our medical center is an ethnically diverse referral center, the interval between diagnosis and HLA results allowing for identification and mobilization of well-matched donor may account for the limited prior IST therapy, despite the majority of patients with >10 transfusion in our series. This highlights the importance of incorporating CB stem cell sources balancing the competing risks of time to transplantation against challenges suitable donor identification . In summary, our data suggest that an upfront risk-adapted cyclophosphamide intensity approach to AlloHSCT from related and alternative unrelated CB donor sources is a feasible strategy for treatment of pediatric and adolescent patients newly diagnosed with SAA. However, the increased risk of graft rejection in the lower dose arm warrants additional research regarding the optimal intensity of cyclophosphamide-based conditioning regimen to reduce toxicity without increasing graft failure. Given the increased risk of prior IST therapy and prolonged duration from diagnosis to transplant, additional prospective trials incorporating unrelated CB donor sources into the upfront treatment of acquired SAA should be explored in patients lacking an HLA matched related donor. ACKNOWLEDGMENTS We would like to thank the patients and their families for participating in this clinical research and the nurses for their assistance in the excellent care of these patients. We would also like to thank Kristen Petrillo, RN, for her assistance with data collection. M.B.B. is Director of Oncology Global Clinical Research, Research and Development, Bristol-Myers Squibb. 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