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Horse Antithymocyte Globulin as Salvage Therapy after Rabbit

Antithymocyte Globulin for Severe Aplastic Anemia

Phillip Scheinberg1,3, Danielle Townsley1, Bogdan Dumitriu1, Priscila Scheinberg1,

Barbara Weinstein1, Olga Rios1, Colin O. Wu2, and Neal S. Young1

1Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health,

Bethesda, MD, USA

2Office of Biostatistics Research, National Heart, Lung and Blood Institute, National Institutes of

Health, Bethesda, MD, USA

3Hematology Service, Oncology Center, Hospital São Jose, Beneficência Portuguesa, São Paulo,

Brazil

Abstract

The effectiveness of salvage therapy for aplastic anemia patients unresponsive to initial rabbit

antithymocyte globulin (r-ATG) or cyclophosphamide is not known. We investigated standard

horse ATG (h-ATG) plus cyclosporine (CsA) in patients who were refractory to initial r-

ATG/CsA (n=19) or cyclophosphamide/CsA (n=6) (registered at clinicaltrials.gov as

NCT00944749). The primary endpoint was hematologic response at 3 months and was defined as

no longer meeting 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 at 6 months. Among responders there have

been no cases of relapse, and in nonresponders 2 patients evolved to monosomy 7. The overallsurvival 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. While h-ATG may be utilized in the salvage setting, the overall response rate

likely will be lower than when h-ATG is used as initial treatment.

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).

*Corresponding author at [email protected], 10 Center Drive, Building 10 CRC, Rm 3E-5140, MSC 1202, Bethesda MD20892-1202 or Rua Martiniano de Carvalho, 951, Bela Vista, Sao Paulo – SP, Brazil 01321-001, [email protected]: P Scheinberg was the Principal Investigator for the protocols, 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 collectionand attended to all patient's needs. Pr Scheinberg attended to all the regulatory protocol requirements including data collection. DTownsley and D Bogdan attended to patient care and were involved with data collection and analysis. CO Wu was involved in theconceptualization, statistics, and writing of the protocols, and did the statistical analysis of the manuscript. NS Young was involved inthe conceptualization, implementation and writing of the protocols, their conduct, interim discussions, data analysis, interpretation of results, and writing of the manuscript.

Disclosure of Conflicts: The authors have no conflicts to disclose.

NIH Public AccessAuthor Manuscript Am J Hematol. Author manuscript; available in PMC 2015 May 01.

Published in final edited form as:

Am J Hematol. 2014 May ; 89(5): 467–469. doi:10.1002/ajh.23669.

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Hematologic response with this regimen is achieved in 60-80% of cases and the long-term

outcome in this group is excellent [1-5]. Multiple efforts to improve outcomes beyond horse

ATG/CsA have been disappointing. Addition of mycophenolate mofetil or sirolimus, while

mechanistically rational, did not improve hematologic responses or decrease the relapse and

clonal evolution rates, and 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]. Particularlynotable, and unexpected, were the poor clinical results associated with r-ATG as first

therapy in SAA [10].

From 2005-2010, we investigated r-ATG as first therapy in SAA with an observed response

rate of only 30-40% [10]. Non-responders with a histocompatible donor underwent a related

or unrelated HSCT, while the remaining patients received alternative immunosuppressants.

From 2010-2012 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 to 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, wedeveloped a protocol using standard h-ATG/CsA as salvage therapy in patients who were

unresponsive to initial therapy with r-ATG/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.

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 cross-

over in a study that randomized between horse and rabbit ATG as first therapy(NCT00260689) while the remaining patients (n=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 had 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 Institutes of

Health (NIH) in Bethesda, MD. ATG administration and landmark visits for evaluation (at

3, 6, and 12 months, and then yearly 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 non-robust (suboptimal) response to initial r-ATG,

defined as both platelet and reticulocyte counts < 50×109 /L at 3 months post-treatment, were

also considered for enrolment, given the known poor long-term outcome in this group of

Scheinberg et al. Page 2

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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 less than 0.5×109 /L; (ii) absolute reticulocyte count less than

60×109 /L; and (iii) platelet count less than 20×109 /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 non-robust hematologic response at 3 months, peripheral blood parameters

(improvement in one or more of the listed parameter (a,b,c) were recorded as a response: a)

ANC - if baseline ANC below 0.5×109 /L, increase in ANC by > 0.3×109 /L, if baseline ANC

above 0.5×109 /L, any increase in ANC by > 0.5×109 /L of blood; (b) platelets - if baseline

platelet count < 50×109 /L, any increase in platelet count by > 20×109 /L of blood; c)

hemoglobin - any increase in hemoglobin by 1.5 g/dl of blood in transfusion-independent

patients and in absolute reticulocyte count to > 60×109 /L of blood in transfusion-dependent

patients. A complete response was defined as ANC above 1.0×109 /L, Hgb > 10 g/dL, and

platelet count > 100×109 /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 administered every12 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 administered every 12 hours (total daily dose of 12

mg/kg/day). Cyclosporine 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/d

started prior to the first dose of h-ATG and continued for 10 days total and then tapered over

subsequent 7 days. For Pneumocytsis 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



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hypothesis would be accepted if no more than 1 patient responds 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 using Binomial distributions.

Survival analysis included the Kaplan-Meier estimates and the Cox proportional hazardregression based on the survival days from the start of the salvage h-ATG therapy.

Numerical results were computed using the S-PLUS 8.0 software package (TIBCO).

Results

Demographics

In total, 25 patients received salvage h-ATG after failing an initial course of r-ATG or

cyclophosphamide from 2009-2012. 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). Themedian follow-up for the cohort was 608 days (range, 57-1395).

Hematologic response, relapse, and clonal evolution after horse 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 1). All hematologic responses were partial at 3 and 6 months. Among responders, all

achieved transfusion-independence.

Of the 5 responders, none have 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 non-responders, 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 3 responded. Four had received cyclophosphamide as first therapy (1 responded) and

5 received r-ATG as initial therapy (2 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 2 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%; Figure 1).



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Discussion

Our results show that about one in five patients who were unresponsive to initial r-

ATG/CsA are salvaged with standard h-ATG/CsA immunosuppression. These data appear

similar to the salvage rate of about 30% when r-ATG/CsA is administered after h-ATG/CsA

failure [13]. The percentages of patients who are salvaged following initial

cyclophosphamide therapy appear 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 h-ATG/CsA when this regimen is given as

salvage therapy after initial alternative immunosuppressants such as r-ATG,

cyclophosphamide and alemtuzumab [8].

Our data suggests that for patients who remain unresponsive to initial alternative

lymphocytotoxic regimens, salvage with standard h-ATG/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 order [8, 10, 13]. Thus, our data

further emphasize the importance of using h-ATG as first line therapy, since only a minority

are likely to be benefited when alternative immunosuppresive regimens are used initially.

The best opportunity for hematologic recovery in SAA with immunosuppressive therapy is

with standard horse ATG/CsA upfront 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

immunosuppression 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 that we report here is in a small number of patients and of

course limited by comparison to historical controls. However, our historical data set

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 US since the reporting of its inferior outcome compared to h-

ATG as first therapy [10].



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In conclusion, salvage h-ATG/CsA in patients who failed initial r-ATG/CsA is possible but

in a minority of patients. The success rate with alemtuzumab administered in a similar

setting (r-ATG failures) 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 since a minority of patients are likely to

be salvaged with immunosuppression when alternative lymphocytotoxic

immunosuppressants such as r-ATG or cyclophosphamide are given initially.

Acknowledgments

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

Institute

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Figure 1.



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Table 1

Overall response to salvage horse ATG

Initial therapy N Response (3 mo) N (%) Response (6 mo) N (%)

R-ATG 19 4 (21) 4 (21)

Cy 6 0 (0) 1 (17)

R-ATG, rabbit antithymocyte globulin

Cy, cyclophosphamide


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