Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Scheinberg P, Nunez O, Weinstein B, et al. Horse versus rabbit antithymocyte globulin in acquired aplastic anemia. N Engl J Med 2011;365:430-8.
Supplementary Appendix Supplemental Figure 1. Schematic representation of the original study design. Schematic representation of the original study design. The original design included randomization among horse anti-thymocyte globulin (ATG) plus cyclosporine, rabbit ATG plus cyclosporine, and alemtuzumab. Horse ATG was administered at 40 mg/kg/day for 4 days, rabbit ATG at 3.5 mg/kg/day for 5 days, and alemtuzumab 10 mg/day for 10 days. After 16 patients were randomized to alemtuzumab, accrual to this arm was discontinued at the recommendation of the Data Safety and Monitoring Board due to low response rate and early deaths in this group. The study continued as 1:1 randomization between horse and rabbit ATG. Details of treatment-naïve patients who received alemtuzumab as well as the efficacy of alemtuzumab in relapsed and refractory severe aplastic anemia will be reported separately. The primary endpoint was hematologic response assessed at 6 months. Responders in all groups were evaluated long-term for secondary endpoints. Non-responders in the horse ATG arm went off study. In the rabbit ATG arm, non-responders were allowed to cross-over to the alemtuzumab arm, while non-responders in the initial alemtuzumab arm allowed to cross-over to the rabbit ATG group. 1
Supplemental Figure 2. Enrollment, randomization, and treatment through 6 months. Enrollment, randomization, and treatment through 6 months. All consecutive patients who met eligibility criteria enrolled into the study. There was an early evolution to leukemia in one patient treated with horse ATG prior to the 3 months and the patient underwent a matched sibling stem cell transplantation. In the rabbit ATG arm, 3 patients died prior to the 3-month evaluation time point: 2 from infectious complications (polymicrobial sepsis (1) and necrotizing fasciitis (1)) and one from intracranial hemorrhage shortly after initiating rabbit ATG administration. Between 3 and 6 months, one patient in the horse ATG arm and 6 in the rabbit ATG arm went off study due to rapid clinical deterioration, recurrent life-threatening systemic infections not adequately controlled with antimicrobials, and progressive pancytopenia. In the horse ATG group, the patient received second line immunosuppression, and in the rabbit ATG group, 2 received second line immunosuppression and 4 underwent emergent stem cell transplantation (3 from a histocompatible sibling and one from a matched unrelated donor). In total, 58 patients in the horse ATG and 51 in the rabbit ATG arm were evaluable for hematologic response at 6 months (primary endpoint). Analysis was intention-to-treat and patients who died or went off study prior to 6 months were counted as non-responders for the primary endpoint (see Methods, Statistical Analysis). 2
Supplemental Figure 3. Disposition among non-responders at 6 months to immunosuppression. Disposition among non-responders at 6 months to immunosuppression. Among the 17 nonresponders to horse ATG, 5 underwent stem cell transplantation, 4 from a histocompatible sibling and one from a matched unrelated donor. Eight patients underwent second line immunosuppression (rabbit ATG plus cyclosporine or alemtuzumab). Of the 29 non-responders in the rabbit ATG group, 23 received second line immunosuppression (horse ATG plus cyclosporine or alemtuzumab). Four patients underwent stem cell transplantation, 3 from a histocompatible sibling and one from an unrelated donor. Six patients who did not respond to second line immunosuppression went on to receive a stem cell transplantation, one from a histocompatible sibling, 4 from an unrelated donor, and one received a haploidentical/umbilical cord transplant. 3
Supplemental Figure 4. Late events after immunosuppressive therapy. Late events after immunosuppressive therapy. (A) Cumulative incidence of hematologic relapse (among responders). The effectiveness of continued cyclosporine cannot be determined in the current study due to the relative short follow-up among the responders to horse ATG. (B) Cumulative incidence of clonal evolution (all patients). Numbers at the bottom of each graph indicate patients at risk for each time point. 4
Supplemental Methods Study patients For protocol entry purposes, severe aplastic anemia was defined as bone marrow cellularity less than 30% and severe pancytopenia by at least two of the following peripheral blood count criteria: 1) absolute neutrophil count < 500 /µl; 2) absolute reticulocyte count < 60,000 /µl; and 3) platelet count < 20,000 /µl. 1 For children and young adults (< 40 years of age) chromosomes were assayed after in vitro exposure of lymphocytes to diepoxybutane and to mitomycin C to exclude Fanconi anemia. Patients with a clonal hematologic disorder as inferred from bone marrow cytogenetics or characteristic morphologic changes on bone marrow were excluded. All patients were tested for paroxysmal nocturnal hemoglobinuria by flow cytometry. 2 Immunosuppressive regimens Among responders in the horse ATG arm, cyclosporine was tapered starting at 6 months over subsequent 18 months and in the rabbit ATG arm, cyclosporine was discontinued at 6 months. (At our institution it is standard practice to discontinue cyclosporine at 6 months, but since 2003, all responders to horse ATG have had the cyclosporine dose tapered to determine its effectiveness in preventing relapse.) Prophylaxis for Pneumocystis carinii was with aerosolized pentamidine monthly and for Herpes simplex, valacyclovir daily. 5
Flow cytometric analysis After thawing, cells were washed, counted, and resuspended in phosphate buffered saline (PBS). Viability staining was performed for 30 min in presence of LIVE/DEAD Aqua fixable viability dye (Life Sciences, Carlsbad, CA) followed by washing in staining buffer (PBS supplemented with 1% normal mouse serum, 1% goat serum and 0.02% sodium azide; Gemini Bioproducts, West Sacramento, CA). Cell membranes were then stained at room temperature for 30 minutes with the following directly conjugated monoclonal antibodies: anti-cd45 quantum dot (QD) 800 (Life Sciences), anti-cd3 APC-H7 (BD Biosciences), anti-cd4 v450 (BD Biosciences), anti-cd8 QD 605 (Life Sciences), anti-cd 127 Alexa 647 (BD Biosciences), and anti-cd25 PE-Cy7 (BD Biosciences) was performed for 30 min in the dark at room temperature. Cells were then washed three times with FACS staining buffer and were acquired the same day. Data were acquired on a Becton Dickinson LSR II equipped with four lasers (407 nm, 488 nm, 532 nm, and 633 nm wavelengths) with 18 PMT detectors, optimized as previously described. 3 Data were acquired using DIVA 6.1.2 software (BD, San Jose, CA) and the analysis was performed using Flowjo 9.3 (Treestar Inc., San Carlos, CA). For the analysis, lymphocytes were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of an aqua viability dye. T cells were identified by expression of CD3 and CD45 among previously selected viable lymphocytes. For this analysis, regulatory T cells were identified as CD4 + CD25 + CD127 - as previously reported. 4 6
Statistical Methods The response rates between the two treatment arms were calculated and compared using the two-sample T-test for proportions. Patient characteristics were described using summary statistics including means, proportions, standard errors and 95% confidence intervals. P-values for comparing these patient characteristics between the two treatment groups were calculated using T-tests. Kaplan-Meier and the Cox proportional hazard models were used to compare overall survival (all patients), time-to-relapse (among responders) and time-to-evolution (all patients) between the two therapies. Log-rank p- values based on the Cox proportional hazard models were used to compare the survival and cumulative event curves between the two treatments. For response analysis, patients who were not evaluable at 6 months due to death or the need to pursue alternative therapies secondary to significant clinical deterioration were counted as non-responders. For the analyses of time-to-relapse and time-to-evolution distributions, patients who died or underwent stem cell transplant before these events were counted as censored. Based on the O Brien-Fleming spending function with one interim analysis, a P-value < 0.047 was considered statistically significant for the final analysis of the primary endpoint. A P- value of < 0.05 was considered statistically significant for all secondary endpoints. Analysis was intention to treat and conducted after the last patient became evaluable for the primary endpoint in January 10, 2011. The numerical results were computed with S- PLUS 8.0 statistical package (TIBCO Software Inc, Palo Alto, CA). For the flow cytometric data, a paired t-test was used to determine significance of differences between populations at each time point (GraphPad Prism version 5, San Diego, CA). 7
Supplemental Table 1. Summary of serious adverse events Related to immunosuppression Event Horse ATG Rabbit ATG Serum sickness 2 5 Chest pain 1 0 Hemolysis a 0 1 SVT 0 1 Azotemia 1 0 PRES 0 1 Hemorrhage CNS 0 1 Vitreous 0 1 Hematemesis 0 1 Epistaxis 1 1 Menorrhagia 2 0 Hemoptysis 1 0 Infection Neutropenic fever, negative cultures 23 16 Neutropenic fever, positive cultures 17 14 Polymicrobial b 0 6 Pneumonia c 1 3 Necrotizing fasciitis d 0 1 Clostridium difficile 2 2 Retropharyngeal abscess 0 1 Peri-rectal abscess 0 1 Peri-orbital cellulitis 1 0 Tonsillitis/pharyngitis 2 1 Apendicitis/typhlitis 0 4 Otitis media 1 0 Epididymitis 0 1 Upper respiratory infection 5 e 1 Other 9 11 Serious adverse events depicted are those that resulted in prolonged hospitalization, hospital admission or death. Events shown are those that occurred after the first cycle of immunosuppression. Repeated hospitalizations in the 8
same subject were counted as separate events. PRES, posterior reversible encephalopathy syndrome; SVT, supraventricular tachycardia; CNS, central nervous system. a Hemolysis occurred after ATG infusion in a patient with a large underlying paroxysmal nocturnal hemoglobinuria clone b Patients with multiple microbial isolates and/or different sites of infection are categorized as having polymicrobial infection. The organisms recovered and sites for each of the patients were: 1) Escherichia coli, Staphylococcus epidermidis, Enterococcus faecium, Acinetobacter baumanii (all in blood); Enterococcus sp., Staphylococcus sp, and mold (left turbinate biopsy); 2) Pseudomonas sp, Klebsiella sp, Staphylococcus epidermidis, Streptococcus viridans, Staphylococcus hemolyticus, Enterococcus faecium, and Stenotrophomonas maltophilia (all in blood); 3) Enterobacter cloacae, Pseudomonas aeruginosa (respiratory cultures); Acinetobacter baumanii, Mycobacterium gordonae, Fusarium sp, Acinetobacter baumanii, Enterococcus faecalis (sinus culture), Corynebacterium sp (blood), Proteus mirabilis (urine); 4) Propionibacterium acnes, Staphylococcus aureus, Candida albicans (throat); 5) Streptococcus sp (blood), Trichosporon asahii (sinus), Candida albicans (skin); 6) Enterococcus faecium, Staphylococcus epidermidis (blood), Klebsiella pneumoniae (urine). c One case of fungal pneumonia in each group d Caused by Clostridium sp e In 3 subjects viral infection was confirmed by nasopharyngeal wash. References for Supplemental Appendix 1. Rosenfeld S, Follmann D, Nunez O, Young NS. Antithymocyte globulin and cyclosporine for severe aplastic anemia: association between hematologic response and long- term outcome. JAMA 2003;289:1130-5. 2. Scheinberg P, Marte M, Nunez O, Young NS. Paroxysmal nocturnal hemoglobinuria clones in severe aplastic anemia patients treated with horse anti- thymocyte globulin plus cyclosporine. Haematologica 2010;95:1075-80. 3. Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay P, Roederer M. Quality assurance for polychromatic flow cytometry. Nat Protoc 2006;1:1522-30. 4. Banham AH. Cell- surface IL- 7 receptor expression facilitates the purification of FOXP3(+) regulatory T cells. Trends Immunol 2006;27:541-4. 9