Abstract
Objective
Immunosuppressant therapy (IST) with antithymocyte globulin (ATG) and cyclosporine is an established treatment option for patients with aplastic anemia (AA), who are not eligible for allogeneic stem cell transplantation. However, data on the dose of ATG and its efficacy from the developing countries is minimal.
Methods
We performed a retrospective analysis of all AA patients (age >12 years), treated with equine ATG and cyclosporine from a single center in India. Patients who received or were eligible for stem cell transplantation were excluded. The overall response rate (ORR) to IST was calculated at 3 and 6 months. We also determined the influence of using a lower dose of Atgam ATG (25 mg/kg/day × 4 days) and compared its efficacy against the standard dose of locally manufactured Thymogam ATG (40 mg/kg/day × 4 days). Factors influencing the ORR were analyzed using Fisher's exact test with a significant P < 0.05.
Results
Thirty-nine patients with AA treated with ATG and cyclosporine were studied. Median age was 31 years with a male:female ratio of 0.85:1. The ORR was 58% at 3 months, 77% at 6 months and was similar with lower dose Atgam and standard dose Thymogam. On multivariate analysis of ORR at 6 months, the interval between the onset of symptoms to the initiation of therapy was close to attaining statistical significance (odds ratio 23.53, P value 0.053) while the other variables did not attain significance.
Conclusions
IST with equine ATG in a lower dose (25 mg/kg/day × 4 days) and cyclosporine is a feasible and effective treatment option for AA in resource-constrained settings.
Introduction
Aplastic anemia (AA) is a hematological disorder characterized by pancytopenia with a hypocellular bone marrow in the absence of any abnormal infiltrate or increase in marrow reticulin. Several congenital and acquired causes can predispose to this condition, the commonest etiology being idiopathic or immunomediated suppression of hematopoiesis.Citation1–Citation2 Although this disorder is seen in most parts of the world, there are geographic differences in its incidence with more number of cases being reported from the eastern countries compared with the west.Citation3 This difference is thought to be due to variations in socioeconomic conditions, genetic factors, and the degree of exposure to various factors associated with AA.Citation4 While supportive care is helpful in dealing with the complications of this disorder,Citation5–Citation7 treatments aimed at disease pathogenesis would provide long-term benefits with freedom from transfusions, infections, and potentially curing this condition. To date, allogeneic stem cell transplantation from an HLA-matched sibling donor is the definitive therapy for this disorder resulting in a long-term survival of >80% in these patients.Citation8 However, the patient's candidacy for transplant is often limited by lack of appropriate donor and factors like patient's age and co-morbidities. Immunosuppressant therapy (IST) with antithymocyte globulin (ATG) and cyclosporine is another promising approach for those patients in whom transplant is not an appropriate option.Citation9
While studies from the developed countries indicate a better survival with allogeneic transplant and IST in patients with AA, the treatment outcomes in developing countries is still uncertain. Key factors like the cost of therapy, availability of resources for stem cell transplantation, and the patient's willingness to undergo therapy influence the outcomes of this disorder in developing countries like India.Citation10 Similarly, the dose and the type of ATG used are also important factors that are considered to influence the overall outcome. While the ideal dose of ATG has not been well established, it is commonly used in the dose of 40 mg/kg/day over 4 days. However, a lower dose of ATG could be cost saving if it can produce similar overall response rates (ORRs). In this study, we have retrospectively analyzed the outcomes of IST and the factors associated with treatment response in a cohort of patients with AA from India. Moreover, we aimed to determine the influence of using a lower dose of Atgam ATG and compared its efficacy against the standard dose of another equine ATG preparation called Thymogam, which is commonly used in resource-constrained settings.
Materials and methods
Patients
Patients with newly diagnosed AA who were treated with IST as the first-line therapy were studied. These patients received treatment at the Postgraduate Institute of Medical Education and Research (PGMER, Chandigarh, India) from the years 2003 through 2010. Our center is located in the northwestern part of India and is a tertiary care referral institution. The patients included were of age >12 years at diagnosis, and received IST with ATG and cyclosporine. Although most patients had severe or very severe AA requiring IST, patients with non-severe AA who had declining trends in their blood counts with a high transfusion requirement requiring IST were also included. We excluded patients who had either received or planned for stem cell transplantation, hypersensitivity to ATG or cyclosporine, pre-existing renal failure, and constitutional AA. The study was approved by the Institutional Ethics Committee. Informed consent was obtained from all adult subjects and from the parents of patients <18 years of age, to review and utilize the medical records for the study.
Diagnostic criteria and disease severity
To be diagnosed with AA, patients had to satisfy two of the following three criteriaCitation11: (1) neutrophil count of less than 0.5 × 109/l, (2) platelet count of less than 20 × 109/l, and (3) anemia with a reticulocyte count of less than 1%. Bone marrow examination should demonstrate a cellularity of <25% (or <30% of the residual hematopoietic cells) along with the absence of abnormal infiltrates or any increase in reticulin.
AA was classified on the basis of disease severity according to the Camitta criteriaCitation12 as follows: Severe AA with a bone marrow cellularity of less than 25% (or 25–50% if <30% residual hematopoietic cells) and at least two of the following: (1) peripheral blood neutrophil count <0.5 × 109/l, (2) peripheral blood platelet count <20 × 109/l, and (3)peripheral blood reticulocyte count <20 × 109/l or corrected reticulocyte count <1%. Very severe AA has features similar to severe AA except that the peripheral blood neutrophil count is <0.2 × 109/l. Non-severe AA has hypocellular marrow with cytopenias but does not fulfill the criteria for either severe or very severe AA.
Therapy
Equine ATG was used in the treatment of all included patients. Two formulations of equine ATG available in India were used in this study – the Atgam (Pfizer Inc. NY, USA) and Thymogam (Bharat Serums and Vaccines, India). A total dose of 160 mg/kg (for Thymogam – standard dose) or 100 mg/kg (for Atgam – divided as 25 mg/kg/day × 4 days – low dose) was administered as an intravenous infusion over 6–8 hours. The choice of the ATG was based on patient's affordability, those who had better affordability received lower doses of Atgam and those who had poor affordability were treated with Thymogam. However, none of the patients could afford for the full dose Atgam ATG. Along with ATG, methyl prednisolone (2 mg/kg/day × 4 days followed by a taper in 2 weeks) was used for prophylaxis of serum sickness. Cyclosporine was given at 5 mg/kg/day in two divided doses starting from day 1 of ATG therapy and continued as per response, with periodic monitoring of cyclosporine levels and renal function. Supportive care with blood product transfusion was used as indicated.
Criteria for response and relapse
Complete response (CR) is defined by an achievement of absolute neutrophil count (ANC) >1500/μl, hemoglobin >11 g/dl, and a platelet count of >100 000/μl. Partial response (PR) is defined by an achievement of ANC >500/μl along with transfusion independence. Non-response (NR) is defined as not fulfilling the criteria for either CR or PR and has ongoing transfusion dependence. Relapse is defined by a decline in the peripheral blood cell count from CR to PR or PR to NR level and/or requirement of blood transfusions.
Statistical method
Descriptive statistics like mean, median, standard deviation, and proportions were used to describe the baseline characteristics which included age, sex, hemoglobin level, total leukocyte count, ANC, absolute lymphocyte count (ALC), reticulocyte percentage, platelet cousnt, lag from the diagnosis to treatment, period from the onset of symptoms to treatment complications. Owing to their wide distribution, quantitative variables like ANC, ALC, and platelet count were log arithmetically transformed (log[x] + 1). Age was analyzed in three groups – 12–39, 40–60, and >60 years. Response at 3 and 6 months was described using proportions. Overall response was defined by an achievement of either CR or PR status. Multivariate logistic regression analysis was performed to find out the determinants of response to IST using the following four variables: ANC, ALC, age, severity, and interval from symptoms to treatment. Overall survival (OS) was estimated by the Kaplan–Meier method. OS was defined as the time from beginning of IST to death due to any cause. Fisher's exact test was used to compare the response rates between the two groups of ATG (Atgam versus Thymogam). A P value of <0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS version 16 (IBM Inc. USA).
Results
Baseline characteristics
Thirty-nine patients with AA who received IST with ATG and cyclosporine were eligible for the analysis. Median age of the cohort was 31 years (range 13–67 years). There were 18 men and 21 women (M:F 0.85:1). Twenty-three patients received Atgam and 16 patients received Thymogam ATG. Baseline characteristics are summarized in . The median follow-up was 24 months. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones was assessed by flow cytometry and three patients had PNH clones (median cone size 59.49%, range 44.4–94.7%). Patients were evaluated for the response serially at 3 and 6 months.
Table 1. Baseline characteristics of the study group
Outcomes following immunosuppressive therapy
Response at 3 months
Thirty-three patients were analyzed at 3 months. Six patients could not be analyzed because of early treatment-related mortality (n = 5) and loss to follow-up (n = 1). There were 2 patients (6%) with CR, 17 patients (52%) with PR, and 14 patients (42%) with NR. The ORR was 58%.
Response at 6 months
Thirty patients were eligible for the analysis at 6 months after exclusion due to death (n = 1) and loss to follow-up (n = 2). There were 22 patients (74%) with PR, 1 patient (3%) with CR, and 7 patients (23%) with NR. Compared with response at 3 months, one patient had loss of response from CR to PR, one patient had loss of response from PR to NR, and five patients had improvement in response from NR to PR. The ORR was 77% ().
Table 2. Response to immunosuppressant therapy
Multivariate analysis of response at 6 months: As summarized in , there was a trend for interval from diagnosis to treatment to be predictive for response at 6 months, as this was not strictly significant (odds ratio 23.533, P = 0.053). Other factors like age, ANC, ALC, and disease severity did not attain statistical significance in this analysis.
Table 3. Multivariate analysis of response to IST at 6 months
Relapse
Of the seven patients who had relapse in the study, two patients relapsed at 6 months (decline from CR to PR in one patient and PR to NR in one patient), three patients had relapse at 1 year and 2 patients at 2 years. The significance individual variables could not be further evaluated due to the small sample size.
Additional therapy
Among the 18 patients who received additional therapy due to NR or loss of response to IST, 5 patients received danazol, 9 patients received oral methylprednisolone, 3 patients received both danazol and methylprednisolone, and 1 patient received mycophenolate mofetil (MMF). Among these, nine patients showed an improvement in response status, nine patients showed no change in response, one patient died, and two patients were lost to follow-up.
Mortality
There were seven patients (17.9%) who died within 6 months of receiving IST. Among these, three patients died within 1 month of IST. We observed a mean survival time of 51.7 ± 5.2 months. The predicted 5-year survival rate was 70.9 ± 0.07%. Five patients were lost to follow-up ().
Figure 1. Overall survival of patients treated with IST.
Type of ATG and response to IST
Based on the two types of ATG preparations used (low-dose Atgam versus standard-dose Thymogam), the response rate was analyzed during the follow-up. As shown in , there was no statistically significant difference in the ORR between the two groups on follow-up.
Table 4. Type of ATG and the overall response rate
Discussion
AA is a rare disease which is usually fatal if left untreated. The most common mechanism of this disorder is immune-mediated damage to the hematopoietic cells.Citation1,Citation4 The principal effectors mediating this bone marrow damage possibly originate from the CD4+ T-cell compartment.Citation13 In addition, regulatory T cells (TREG) that are thought to protect against autoimmunity are deranged in AA with various abnormalities like decreased frequency in peripheral blood and bone marrow, defective migration, and inability to suppress the effector T cells in vitro.Citation14 Initial clinical studies supported the benefit of ATG in the treatment of AACitation15 and subsequently, it was found that addition cyclosporine to ATG produces better outcomes compared with ATG monotherapy.Citation16 While ATG acts by destroying the T-lymphocytes through complement-mediated lyses, apoptosis and induction of natural killer cells,Citation17–Citation19 cyclosporine causes inhibition of IL-2 production from the activated T cells and prevents cytotoxic T-cell expansion.Citation20
In this study, we have demonstrated the effects of IST in a cohort of patients with AA treated at our center. Although the current standard first-line treatment for young patients with AA is allogeneic bone marrow transplantation from an HLA-matched sibling donor, it is quite challenging in a developing country like India to perform stem cell transplantation. The difficulties encountered in this context include resource constraints, cost of therapy, patient's willingness to undergo therapy, and population's perception or willingness to donate stem cells. Even in developed countries, it is presumed that only approximately 30% of the patients who are eligible for stem cell transplant actually get transplantation done due to lack of appropriate donor, patient's age, and comorbidities etc.Citation21 Hence, these facts strengthen the importance of IST in the treatment of AA. In general, studies from developed countries indicate good clinical response of 50–60% and an OS of 60% at 1 year with IST.Citation22 However, data from developing countries like India are lacking in this regard, highlighting the importance of the current study.
In our study, we found an ORR of 77% after 6 months of IST in patients with AA. One of the recent studies from India by Nair et al.Citation23 demonstrated that IST was able to produce an ORR of 85.8% at 6 months and OS of 83.4% in a cohort of 120 patients with AA. Although these two single-center studies from India reflect an outcome comparable to those from the developed countries, some of the prior studies showed a lower response rate to IST.Citation24,Citation25 In the pediatric population, a recent study from India by Gupta et al.Citation26 demonstrated an ORR of 33% at 6 months of IST with ATG and cyclosporine. Some of the prior studies in pediatric AA patients from India have also demonstrated a variably inferior outcome with IST as compared with the outcomes from developed countries,Citation27 even though children are conventionally thought to have a better response to IST as compared with adults. This may be due to the heterogeneity in treatment protocol and some proportion of undiagnosed Fanconi's anemia in pediatric AA patients.
Generally, two different formulations of equine ATG were used in the prior studies from the United States (Atgam 40 mg/kg/day for 4 days)Citation28,Citation29 and Europe (lymphoglobulin 15 mg/kg/day for 5 days).Citation30,Citation31 Even though head-to-head comparisons were not done, similar responses had been reported with the use of both these formulations. Owing to resource constraints, we had used a lower dose of equine ATG in this study (25 mg/kg/day versus 40 mg/kg/day dose in developed countries) and it appears that the ORR is comparable to those from the developed countries. However, a previous study using a lower dose of horse ATG showed inferior response rates in a cohort of elderly AA patients.Citation32 We were also able to demonstrate that the response rates are comparable with the two common ATG formulations available in India. Hence, treatment protocols adapted to the resource availability and patient-related factors would enhance the compliance to therapy and improve the outcome.
One of the difficulties with IST lies in predicting those patients who would respond favorably and survive with this therapy. In a study by the European Group for Blood and Marrow Transplantation group, a greater lag from the diagnosis to the initiation of IST (>23 days) was associated with a shorter survival period.Citation31 In a large retrospective study from the US NIH, Scheinberg et al. showed that baseline ANC >200 cells/cu mm, higher ARC, baseline ALC >1000 cells/cu.mm had a higher probability of predicting a favorable response at 6 months.Citation33 PNH clones can be detected in patients with AA and one of our previous study with 90 AA patients showed that the PNH clone was detected in 8 patients (8.9%) during diagnosis and in 28 patients (31.1%) during the follow-up.Citation34 However, its role in treatment response prediction is variable.Citation33,Citation35 While none of the above-mentioned predictive factors attained statistical significance in this study, an interval of less than 10 months from the onset of symptoms to the initiation of treatment had the highest odds ratio and was close to statistical significance. This is probably the factor which could potentially predict the treatment response in this study given the small sample size. This highlights the need for larger prospective studies from India to delineate the determinants of treatment response to IST.
Seven patients in this study had relapse. Studies from developed countries indicate a probability of relapse in about 30% of patients who initially respond to IST.Citation36 Although the standard protocol is to give a second course of IST in these circumstances, our patients could not receive a second course of ATG due to financial constraints. Alternative therapies with steroids, danazol, and MMF in the setting of relapse were able to improve the response status in a proportion of patients from our study.
The limitations of this study include the retrospective methodology, small sample size, and inclusion of patients from a single tertiary care referral center. Hence, these results could not be generalized to the population. Since our follow-up period was short, we could not estimate the incidence of clonal evolution to leukemia or myelodysplasia in these patients. However, considering the lack of data from developing countries such as India, this study helps to highlight the fact that IST is a feasible option to treat AA patients even in a resource-constrained setting.
Conclusion
Immunosupression with ATG and cyclosporine is a suitable treatment option for AA in the Indian setting. This single-center study demonstrates an ORR similar to those achieved in the developed countries, despite using a lower dose of Atgam ATG. ORR is comparable while using lower dose Atgam and standard dose Thymogam ATG. Future prospective studies from India with a larger patient population would give more information about the actual response rates, survival with IST, and factors predicting the treatment response and survival of AA patients.
Disclaimer statements
Contributors PM supervised, treated the patients and wrote the paper. VB and AKD collected the data. GSGM treated, analysed and wrote the paper. NV and SV helped in diagnosis, treatment and collecting the data. All authors have read the paper and agree with the content of this paper.
Funding None.
Conflicts of interest None.
Ethics approval The study was approved by Institute Ethics Committee.
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