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Published in final edited form as:

Am J Hematol. 2014 May ; 89(5): 467469. doi:10.1002/ajh.23669.

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 So Jose, Beneficncia Portuguesa, So Paulo,

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Brazil

Abstract

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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 rATG/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 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. 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 scheinbp@mail.nih.gov, 10 Center Drive, Building 10 CRC, Rm 3E-5140, MSC 1202, Bethesda MD
20892-1202 or Rua Martiniano de Carvalho, 951, Bela Vista, Sao Paulo SP, Brazil 01321-001, scheinbp@gmail.com.
Authorship: 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 collection
and attended to all patient's 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. CO Wu was involved in the
conceptualization, statistics, and writing of the protocols, and did the statistical analysis of the manuscript. NS Young was involved in
the 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.

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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 (rATG), 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].

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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, we
developed 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 hATG/CsA in subjects refractory to an initial course of r-ATG/CsA or cyclophosphamide.

Methods
Patients

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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 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 rATG/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 < 50109/L at 3 months post-treatment, were
also considered for enrolment, given the known poor long-term outcome in this group of

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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 0.5109/L; (ii) absolute reticulocyte count less than
60109/L; and (iii) platelet count less than 20109/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.

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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 0.5109/L, increase in ANC by > 0.3109/L, if baseline ANC
above 0.5109/L, any increase in ANC by > 0.5109/L of blood; (b) platelets - if baseline
platelet count < 50109/L, any increase in platelet count by > 20109/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 > 60109/L of blood in transfusion-dependent
patients. A complete response was defined as ANC above 1.0109/L, Hgb > 10 g/dL, and
platelet count > 100109/L and a partial response defined as a hematologic response that
was not sufficient for a complete response.
Immunosuppressive therapy

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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 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 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 p10% 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 hazard
regression 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

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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). The
median 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.

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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 hATG. 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
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Our results show that about one in five patients who were unresponsive to initial rATG/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 hATG/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].

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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 hATG 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 hATG-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

References

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Figure 1.

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Table 1

Overall response to salvage horse ATG

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Initial therapy

Response (3 mo) N (%)

Response (6 mo) N (%)

R-ATG

19

4 (21)

4 (21)

Cy

0 (0)

1 (17)

R-ATG, rabbit antithymocyte globulin

Cy, cyclophosphamide

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