Prospective Phase II Pilot Study of Rabbit Antithymocyte Globulin (ATG, Thymoglobuline) with Ciclosporin for Patients with Acquired Aplastic Anemia and Matched Pair Analysis with Patients Treated with Horse ATG (Lymphoglobuline) and Ciclosporin: A Study From the EBMT Severe Aplastic Anemia Working Party (RATGAA07)

Previously, horse antithymocyte globulin (hATG) with ciclosporin (CSA) was the standard immunosuppressive therapy (IST) for patients with aplastic anemia (AA) ineligible for HSCT, with response rates of 60–75% within 3–4 months. Rabbit ATG (rATG, Thymoglobuline, Genzyme) was reserved for non-response or relapse after a course of hATG. Following the withdrawal of hATG (Lymphoglobuline) from the market, the use of rATG as first line IST has been evaluated prospectively with hATG (ATGAM, Pfizer) in a randomised study from NIH (Scheinberg et al, NEJM, 2011), reporting significantly lower response rate and worse outcome after rATG. We report here the results of a multicenter, EBMT, phase II pilot study of 35 patients with AA enrolled between August 2008 and August 2010 at 10 centres (EUdraCT number 2007–000902–55). Patients enrolled were ineligible for matched sibling HSCT, had not received prior ATG or CSA and included both severe AA (SAA) and transfusion dependent non-severe AA (NSAA). The median age was 36 years (range 17–75). Six (17%) patients had very severe AA, 20 (57%) SAA and 9 (26%) had NSAA. Primary endpoint was response at 6 months. Dose of rATG was 3.75mg/kg daily for 5 days. CSA dose was 5mg/kg daily from day +1. For prevention of serum sickness, prednisolone was given at 1–2mg/kg daily from day 1–5, then the dose halved every 5 days. Results were compared in a 3: 1 ratio with 105 historical patients from the EBMT registry, matched for categorised age and disease severity, and who had been treated with hATG (Lymphoglobuline, Genzyme) and CSA. The study was designed to demonstrate a 25% difference in total response to rATG, with 80% power at a 5% level of significance. The median follow up for all patients treated with rATG is 285 days (6–781).

At 3 months, no patients had achieved a complete response (CR). Partial response (PR) occurred in 11 (34%). Two patients died, on day +6 and +9, and one was lost to follow up. At 6 months, CR rate was 7% (n=2) and PR rate 32% (n=11). One patient relapsed at 59 days. Five patients underwent unrelated donor HSCT for non-response to rATG, at day +153, 168, 321, 322 and 460 post rATG. Infections occurred in 22 (63%), elevated liver function in 10 (29%), rash in 8 (23%), haemorrhage in 7 (20%), hypertension in 6 (17%), abnormal renal function in 6 (17%), arthralgia in 5 (14%), avascular bone necrosis in one. There were 10 deaths after rATG (28.5%) and one following subsequent HSCT. Infections were the main cause of death in 9/10 patients. For rATG, overall survival at 2 yr was 56%, compared to 78% for hATG, p=0.001. Overall survival after censoring HSCT patients was 39% for rATG and 72% for hATG, p=0.0005. On multivariate analysis, rATG (RR = 3.4, p=0.003) and age > 37 years (RR = 3.8, p=0.002), were independent adverse risk factors for survival. Disease severity and time from diagnosis to treatment were not significant risk factors. CR at a given time was 11% for rATG and 44% for hATG, and PR for rATG was 43% and 23% for hATG. There were 19/105 (18%) deaths after hATG.

Changes in CD4+ T-cells were analysed in a subset of 5 patients from a single center, prior to rATG (n=5) and post rATG (n=8). The relevant (preliminary) findings are:pre-ATG therapy, number of Tregs was significantly lower in AA compared to healthy age-matched controls (p=0.009), while Th17 (p=0.014), Th1 cells (p=0.014) and Th2/Tregs ratio (p=0.025) were higher in AA patients. Following treatment with ATG, the frequency of Th1 (p=0.002) and B-cells (p=0.007) was reduced but there was no significant change in the frequency of Tregs, Th17/Tregs or T-helpers/Tregs ratios. However, the frequency of cytokine secreting non-Tregs (CD4⁺ CD25^high CD45 RA⁻ Foxp3^low) studied in 5 patients was significantly increased in post treatment samples (p=0.02) and there was a trend for a higher frequency in non-responders compared to responders.

We report significantly inferior response and survival with rATG (Thymoglobuline) compared to hATG (Lymphoglobuline), and a high incidence of serious infections with rATG, in association with significant changes in CD4+ T-cell subsets. These results have major implications for future treatment algorithms for AA and improve further our understanding of the mechanism of action of rATG in AA.

Marsh:Genzyme: Consultancy, Research Funding. Off Label Use: ATG used in treatment of aplastic anemia. Schrezenmeier:Genzyme: Research Funding. Risitano:Genzyme: Honoraria. Dufour:Pfizer: Research Funding. Elebute:Alexion: Honoraria. Mufti:Celgene: Consultancy, Research Funding; Genzyme: Research Funding. Bacigalupo:Genzyme: Speakers Bureau; Pfizer: Research Funding.