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Horse antithymocyte globulin as salvage therapy after rabbit antithymocyte globulin for severe aplastic anemia Phillip Scheinberg,1,2* Danielle Townsley,1 Bogdan Dumitriu,1 Priscila Scheinberg,1 Barbara Weinstein,1 Olga Rios,1 Colin O. Wu,3 and Neal S. Young1 The effectiveness of salvage therapy for aplastic anemia patients unresponsive to initial rabbit antithymocyte globulin (r-ATG) or cyclophosphamide is not known. We investigated the administration of standard horse ATG (h-ATG) plus cyclosporine (CsA) in patients who were refractory to initial r-ATG/CsA (n 5 19) or cyclophosphamide/CsA (n 5 6) (registered at clinicaltrials.gov as NCT00944749). The primary endpoint was hematologic response at 3 months and was defined as no longer meeting the criteria for severe aplastic anemia. Of the 19 patients who received r-ATG as initial therapy, 4 (21%) achieved a hematologic response by 3 months, and of the 6 patients who received cyclophosphamide, only 1 (17%) responded by 6 months. Among the responders there were no cases of relapse, and in nonresponders 2 patients evolved to monosomy 7. The overall survival for the cohort at 3 years was 68% (95% CI, 50–91%). These results suggest that only a minority can be successfully salvaged after receiving as first therapy either r-ATG or cyclophosphamide. Although h-ATG may be utilized in the salvage setting, the overall response rate probably will be lower than when h-ATG is used as initial treatment. † C Published 2014. Am. J. Hematol. 00:000–000, 2014. V

䊏 Introduction Initial therapy with horse antithymocyte globulin (h-ATG) plus cyclosporine (CsA) is the standard immunosuppressive therapy regimen in severe aplastic anemia (SAA) patients who are not candidates for a matched sibling hematopoietic stem cell transplantation (HSCT). Hematologic response with this regimen is achieved in about 2/3 of cases and the long-term outcome in this group is excellent [1–5]. Multiple efforts to improve outcomes beyond h-ATG/CsA have been disappointing. Addition of mycophenolate mofetil or sirolimus, although mechanistically rational, did not improve hematologic responses or decrease the relapse and clonal evolution rates. Also, the use of more lymphocytotoxic agents such as rabbit ATG (r-ATG), alemtuzumab, or cyclophosphamide led to worse outcomes than with h-ATG/CsA in randomized studies, due to a lower response rate and/or excess toxicities [6–9]. Particularly notable, and unexpected, were the poor clinical results associated with r-ATG as first therapy in SAA [10]. From 2005 to 2010, we investigated r-ATG as first therapy in SAA with an observed response rate of only 30–40% [10]. Nonresponders with a histocompatible donor underwent a related or unrelated HSCT, whereas the remaining patients received alternative immunosuppressants. From 2010 to 2012, we investigated moderate dose cyclophosphamide (120 mg/kg) as initial therapy in order to confirm reports on the better tolerability, response, and low evolution rates associated with this regimen compared with higher dose (200 mg/kg) [11]. The activity of a repeat course of immunosuppression in primary r-ATG or cyclophosphamide failures is unknown; the response rate to alemtuzumab in this setting appears low [8]. Therefore, we developed a protocol using standard h-ATG/CsA as salvage therapy in patients who were unresponsive to initial therapy with rATG/CsA or cyclophosphamide. The primary objective was to evaluate the effectiveness of a second course of immunosuppression with h-ATG/ CsA in subjects refractory to an initial course of r-ATG/CsA or cyclophosphamide.

䊏 Methods Patients Patients were enrolled into two treatment protocols registered at clinicaltrials.gov as NCT00944749 and NCT00260689. Two patients received salvage h-ATG as part of a crossover in a study that randomized between h-ATG and r-ATG as first therapy (NCT00260689), whereas the remaining patients (n 5 23) received salvage h-ATG on an open-label, single-arm phase II study (NCT00944749). Nineteen patients received r-ATG as first-line therapy and six patients received cyclophosphamide as their first treatment (cyclophosphamide-treated patients were later included in the eligibility criteria after protocol initiation). All patients (or their legal guardians) signed informed consent according to protocols approved by the Institutional Review Board of the National, Heart, Lung, and Blood Institute. All patients were treated at the Clinical Center of the National

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Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland; 2Hematology Service, Oncology Center, Hospital S~ao Jose, Benefic^encia Portuguesa, S~ao Paulo, Brazil; 3Office of Biostatistics Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Conflict of Interest: The authors have no conflicts to disclose. *Correspondence to: P. Scheinberg, Rua Martiniano de Carvalho, 951, Bela Vista, S~ao Paulo – SP, Brazil 01321-001. E-mail: [email protected] Received for publication: 7 October 2013; Revised: 5 January 2014; Accepted: 8 January 2014 Am. J. Hematol. 00:00–00, 2014. Published online: 11 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ajh.23669 C Published 2014. †This article is a U.S. Government work and is in the public domain in the USA V

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TABLE I. Overall Response to Salvage Horse ATG Initial therapy

N

Response (3 mo) N (%)

Response (6 mo) N (%)

R-ATG Cy

19 6

4 (21) 0 (0)

4 (21) 1 (17)

r-ATG, rabbit antithymocyte globulin; Cy, cyclophosphamide Institutes of Health (NIH) in Bethesda, MD. ATG administration and landmark visits for evaluation (at 3, 6, and 12 months, and then each year thereafter) were conducted at NIH.

Eligibility and endpoints All patients 2 years old or over with SAA who had failed initial immunosuppression with r-ATG/CsA or cyclophosphamide and were not candidates for a histocompatible HSCT were considered for enrolment. Patients with a nonrobust (suboptimal) response to initial r-ATG, defined as both platelet and reticulocyte counts < 503109/L at 3 months post-treatment, were also considered for enrolment, given the known poor long-term outcome in this group of patients [8]. The primary endpoint was hematologic response at 3 months, defined as no longer meeting criteria for SAA. Secondary endpoints included robustness of hematologic recovery, relapse, response rate at 6 months, clonal evolution, and overall survival. For protocol entry purposes, SAA was defined as bone marrow cellularity of less than 30% and severe pancytopenia with at least two of the following peripheral blood count criteria: (i) absolute neutrophil count (ANC) less than 0.53109/L; (ii) absolute reticulocyte count less than 603109/L; and (iii) platelet count less than 203109/L. Exclusion criteria were a diagnosis of Fanconi anemia, evidence of a clonal disorder on cytogenetics, infection not adequately responding to therapy, HIV seropositivity, active cancer chemotherapy, serum creatinine > 2.5 mg/dL, pregnancy, inability to provide written informed consent, or moribund status and/or significant comorbidities that would preclude the patient’s ability to tolerate protocol therapy or that death was imminent. In subjects with a nonrobust hematologic response at 3 months, peripheral blood parameters (improvement in one or more of the listed parameters (a, b, c) were recorded as a response: (a) ANC—if baseline ANC was below 0.53109/L, increase in ANC by 0.33109/L; if baseline ANC above 0.53109/L, any increase in ANC by 0.53109/L of blood; (b) platelets—if baseline platelet count was 503109/L, any increase in platelet count by >203109/L of blood; and (c) hemoglobin—any increase in hemoglobin by 1.5 g/dL of blood in transfusionindependent patients and in absolute reticulocyte count to 603109/L of blood in transfusion-dependent patients. A complete response was defined as ANC above 1.03109/L, Hgb > 10 g/dL, and platelet count > 1003109/L and a partial response defined as a hematologic response that was not sufficient for a complete response.

Immunosuppressive therapy All subjects underwent h-ATG skin testing. Horse ATG (ATGAM, Pfizer) was administered at a dose of 40 mg/kg/day for 4 consecutive days as previously described [12]. For subjects  12 years of age, CsA was started on day 1 at 3 mg/ kg/dose by mouth administration every 12 hours (total daily dose of 6 mg/kg/day). For subjects  12 years of age, CsA was started on day 1 at 6 mg/kg/dose by mouth administration every 12 hours (total daily dose of 12 mg/kg/day). CsA dosing was adjusted to obtain a therapeutic trough level between 200 and 400 ng/mL. Serum sickness prophylaxis was with oral prednisone at 1 mg/kg/day started prior to the first dose of h-ATG and continued for a total of 10 days and then tapered over the subsequent 7 days. For Pneumocystis jiroveci prophylaxis, aerosolized pentamidine was administered at 300 mg every 4 weeks by inhalation beginning the first month of therapy and continued for at least 6 months.

Statistical methods We hypothesized that the actual response probability using this treatment would reach 30% or more, based on published data for rescue of patients who had failed h-ATG and were retreated with r-ATG or alemtuzumab, and a response probability of 10% or less would warrant terminating the treatment on this patient population. The total sample size of 25 patients was calculated using the Two-Stage Minimax Design at 5% significance level and 80% power. Following this design, 15 patients were accrued in the first stage and the null hypothesis would be accepted if no more than 1 patient responded to the treatment within 3 months, and the additional 10 patients were accrued if 2 or more subjects responded to the treatment within 3 months at the first stage. The null hypothesis of P  10% would be accepted if the total number of responders within 3 months were 5 or less. The response probabilities were estimated using the sample proportions, and their inferences including the nominal confidence intervals and hypotheses were evaluated

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Figure 1. Overall survival. Horse ATG salvage in patients who failed initial therapy with rabbit ATG or cyclophosphamide (N 5 25). Day 0 in this survival curve is the first day of horse ATG salvage. Dotted lines represent 95% confidence intervals (CI). using binomial distributions. Survival analysis included the Kaplan–Meier estimates and the Cox proportional hazard regression based on the survival days from the start of the salvage h-ATG therapy. Numerical results were computed using the SPLUS 8.0 software package (TIBCO).

䊏 Results Demographics In total, 25 patients received salvage h-ATG after failing an initial course of r-ATG or cyclophosphamide from 2009 to 2012. The median age of this cohort was 27 (range 4–74) years of age. Eight (32%) patients were 18 years of age or younger. Sixteen (64%) patients were males. In only one case, aplastic anemia occurred after an episode of seronegative hepatitis. The median time from r-ATG to h-ATG was 217 days (range 124–584). The median follow-up for the cohort was 608 days (range 57–1395).

Hematologic response, relapse, and clonal evolution after h-ATG salvage Of the 19 patients who received r-ATG as initial therapy, 4 (21%) achieved a hematologic response by 3 months after h-ATG salvage. Of the 6 patients who received cyclophosphamide as first therapy, only 1 (17%) responded to h-ATG salvage at 6 months (Table I). All hematologic responses were partial at 3 and 6 months. Among responders, all achieved transfusion independence. Of the 5 responders, none has relapsed or evolved to date. Two instances of clonal evolution to monosomy 7 occurred in nonresponders 179 and 524 days from salvage h-ATG. Among the 20 nonresponders, 5 underwent HSCT: 3 from a matched unrelated donor, 1 from a matched sibling donor, and 1 underwent an umbilical cord HSCT. All but one patient who received a graft from a histocompatible unrelated donor are alive. Nine patients unresponsive to salvage h-ATG received eltrombopag on a research protocol of which three responded. Four had received cyclophosphamide as first therapy (one responded) and five received r-ATG as initial therapy (two responded).

Overall survival In total 7 patients died. Five patients who died had received initial therapy with r-ATG and two cyclophosphamide as first therapy. Causes of death were central nervous system hemorrhage (1), evolution to myelodysplasia (1), complications of pancytopenia (2), infection (1), transplantation related (1), and unknown (1). Of the two

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patients who evolved to monosomy 7, one died and the other received an umbilical cord graft and is alive. The overall survival for the cohort at 3 years was 68% (95% CI, 50–91%; Fig. 1).

䊏 Discussion Our results show that about one in five patients who were unresponsive to initial r-ATG/CsA were salvaged with standard h-ATG/ CsA immunosuppression. These data appear similar to the salvage rate of about 30% when r-ATG/CsA was administered after h-ATG/ CsA failure [13]. The percentages of patients who were salvaged following initial cyclophosphamide therapy appeared low, at about 15– 20%. However, the efficacy of h-ATG/CsA in this setting is lower than what is observed with this regimen as first therapy, in which a response rate of 60–80% is routine [12]. Therefore, it cannot be assumed that a comparable response rate will be observed with hATG/CsA when this regimen is given as salvage therapy after initial alternative immunosuppressants such as r-ATG, cyclophosphamide, and alemtuzumab [8]. Our data suggest that for patients who remain unresponsive to initial alternative lymphocytotoxic regimens, salvage with standard hATG/CsA is possible but only in a minority of patients. With an initial hematologic response rate to initial h-ATG/CsA of 60–80% and salvage rate in refractory patients of about 30–40% with r-ATG or alemtuzumab [13,14], overall hematologic recovery of 70–90% can be expected after 1 or 2 courses of immunosuppression. Unfortunately, this response rate does not appear achievable when patients receive alternative immunosuppressants as first therapy. With a response rate of 30–40% to initial r-ATG [10,15–17] and about a 20% successful salvage rate with h-ATG (current study), approximately 50% of patients are expected to achieve hematologic recovery after 1 or 2 courses when ATGs are given in this sequence [8,10,13]. Thus, our data further emphasize the importance of using h-ATG as first-line therapy, as only a minority are likely to be benefited when alternative immunosuppressive regimens are used initially. The best opportunity for hematologic recovery in SAA with immunosuppressive therapy is with standard h-ATG/CsA as first treatment. The explanation for this difference is not clear; however, distinct kinetics of lymphocyte (and subsets) depletion have been reportedly consistently between these two agents, which might contribute in hematologic recovery [10,18,19]. Given the low salvage rate of h-ATG after r-ATG failure, it is reasonable to consider alternative approaches such as HSCT from a matched related donor in older patients or an unrelated donor HSCT in a younger patient. In cases where a histocompatible donor (related or unrelated) is not available, a repeat course of immunosup-

䊏 References 1. Young NS, Calado RT, Scheinberg P. Current concepts in the pathophysiology and treatment of aplastic anemia. Blood 2006;108:2509–2519. 2. Bacigalupo A, Bruno B, Saracco P, et al. Antilymphocyte globulin, cyclosporine, prednisolone, and granulocyte colony-stimulating factor for severe aplastic anemia: an update of the GITMO/EBMT study on 100 patients. European Group for Blood and Marrow Transplantation (EBMT) Working Party on Severe Aplastic Anemia and the Gruppo Italiano Trapianti di Midolio Osseo (GITMO). Blood 2000;95:1931–1934. 3. Kojima S, Hibi S, Kosaka Y, et al. Immunosuppressive therapy using antithymocyte globulin, cyclosporine, and danazol with or without human granulocyte colony-stimulating factor in children with acquired aplastic anemia. Blood 2000;96:2049–2054. 4. Tichelli A, Schrezenmeier H, Socie G, et al. A randomized controlled study in patients with newly diagnosed severe aplastic anemia receiv-

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5.

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pression with h-ATG is reasonable after failure of initial r-ATG prior to undertaking a higher risk transplant from a mismatched unrelated, umbilical cord, or haploidentical donor. Thus, search for a histocompatible unrelated donor is warranted in younger patients along with the initial course of immunosuppression. The current prospective study was done in a small number of patients and of course limited by comparison to historical controls. However, our historical dataset comprises a relatively large number of patients, uniformly treated in a single institution and with periodic and long-term evaluations. The definitions for clinical outcomes have remained consistent at our institution since the 1980s. A larger cohort would have increased the precision of the results; however, the enrolment to the current study slowed as the use of r-ATG upfront declined in the United States since the reporting of its inferior outcome compared to h-ATG as first therapy [10]. In conclusion, salvage h-ATG/CsA in patients who failed initial rATG/CsA is possible but in a minority of patients. The success rate with alemtuzumab administered in a similar setting (r-ATG failures) was also associated with a low response rate [8]. Thus, our results suggest that the best chance for hematologic recovery in SAA patients is with standard h-ATG-based immunosuppression as first-line therapy, as a minority of patients are likely to be salvaged with immunosuppression when alternative lymphocytotoxic immunosuppressants such as r-ATG or cyclophosphamide is given initially.

䊏 Acknowledgment This research was supported by the Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute.

䊏 Author Contribution P. Scheinberg was the Principal Investigator for the protocols and conceptualized, wrote, and conducted the clinical trials, analyzed the data, interpreted the results, and drafted the manuscript. O. Rios and B. Weinstein did the patient screening, data collection, and attended to all patients’ needs. Pr. Scheinberg attended to all the regulatory protocol requirements including data collection. D. Townsley and D. Bogdan attended to patient care and were involved with data collection and analysis. C.O. Wu was involved in the conceptualization, statistics, and writing of the protocols, and did the statistical analysis of the manuscript. N.S. Young was involved in the conceptualization, implementation, and writing of the protocols and their conduct, interim discussions, data analysis, interpretation of results, and writing of the manuscript.

ing antithymocyte globulin (ATG), cyclosporine, with or without G-CSF: a study of the SAA Working Party of the European Group for Blood and Marrow Transplantation. Blood 2011;117:4434–4441. Rosenfeld S, Follmann D, Nunez O, et al. Antithymocyte globulin and cyclosporine for severe aplastic anemia: association between hematologic response and long-term outcome. JAMA 2003;289:1130–1135. Scheinberg P, Nunez O, Wu C, et al. Treatment of severe aplastic anaemia with combined immunosuppression: anti-thymocyte globulin, cyclosporine and mycophenolate mofetil. Br J Haematol 2006;133:606–611. Scheinberg P, Wu CO, Nunez O, et al. Treatment of severe aplastic anemia with a combination of horse antithymocyte globulin and cyclosporine, with or without sirolimus: a prospective randomized study. Haematologica 2009; 94:348–354. Scheinberg P, Nunez O, Weinstein B, et al. Activity of alemtuzumab monotherapy in

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treatment-naive, relapsed, and refractory severe acquired aplastic anemia. Blood 2012;119: 345–354. Tisdale JF, Dunn DE, Geller N, et al. Highdose cyclophosphamide in severe aplastic anaemia: a randomised trial. Lancet 2000;356:1554– 1559. Scheinberg P, Nunez O, Weinstein B, et al. Horse versus rabbit antithymocyte globulin in acquired aplastic anemia. N Engl J Med 2011; 365:430–438. Zhang F, Zhang L, Jing L, et al. High-dose cyclophosphamide compared with antithymocyte globulin for treatment of acquired severe aplastic anemia. Exp Hematol 2013;41: 328–334. Scheinberg P, Young NS. How I treat acquired aplastic anemia. Blood 2012;120:1185–1196. Scheinberg P, Nunez O, Young NS. Retreatment with rabbit anti-thymocyte globulin and cyclosporine for patients with relapsed or refractory severe aplastic anaemia. Br J Haematol 2006; 133:622–627.

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14. Di Bona E, Rodeghiero F, Bruno B, et al. Rabbit antithymocyte globulin (r-ATG) plus cyclosporine and granulocyte colony stimulating factor is an effective treatment for aplastic anaemia patients unresponsive to a first course of intensive immunosuppressive therapy. Gruppo Italiano Trapianto di Midollo Osseo (GITMO). Br J Haematol 1999;107:330–334. 15. Marsh JC, Bacigalupo A, Schrezenmeier H, et al. Prospective study of rabbit antithymocyte globulin and cyclosporine for aplastic anemia from the EBMT Severe Aplastic Anemia Working Party. Blood 2012;119:5391–5396.

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16. Atta EH, Dias DS, Marra VL, et al. Comparison between horse and rabbit antithymocyte globulin as first-line treatment for patients with severe aplastic anemia: a single-center retrospective study. Ann Hematol 2010;89: 851–859. 17. Bekassy AN, Locasciulli A, Marsh JC, et al. Immunosuppression with ALG and CsA is first line treatment in children with SAA lacking an HLA identical sibling. Pediatr Blood Cancer 2005;45:359–360; author reply 361–352. 18. Scheinberg P, Fischer SH, Li L, et al. Distinct EBV and CMV reactivation patterns following

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antibody-based immunosuppressive regimens in patients with severe aplastic anemia. Blood 2007;109:3219–3224. 19. Thomas FT, Griesedieck C, Thomas J, et al. Differential effects of horse ATG and rabbit ATG on T cell and T cell subset levels measured by monoclonal antibodies. Transplant Proc 1984; 16:1561–1563.

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Horse antithymocyte globulin as salvage therapy after rabbit antithymocyte globulin for severe aplastic anemia.

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