Response to Immunosuppressive Therapy in Acquired Aplastic Anaemia: Experience of a Tertiary Care Centre from Eastern India

Abstract

The current study was conducted to assess response to immunosuppressive therapy (IST) in acquired aplastic anaemia (AA). It was a retrospective and prospective observational study. Patients were diagnosed as per standard international guidelines and IST was started as per standard protocol. Patients were followed up at 3 months and 6 months for assessment of response as per published standard guidelines. Total 76 cases were included in the study. The median age of the study population was 36 years with a range of 6–66 years with a male to female ratio of 2.04:1. Most common clinical presentation was pallor followed by bleeding. Commonest type of disease in the study group was severe AA. Among total 76 patients, 32 patients received Atgam and 44 patients received Thymogam. Within 3 months of ATG administration, 4 patients died and 1 patient was lost to follow up. At 3 months, 2 (2.63%) patients were on complete response (CR), 32 (42.10%) patients were in partial response (PR) and 37 (48.68%) patients were on no response (NR). Overall response (OR) at 3 months was 44.73%. At 6 months 5 (6.57%) patients were in CR, 43 (56.57%) patients in PR and 23 (30.26%) patients in NR; the OR was 63.14%. Overall response at 3 months was 44.73% and overall response at 6 months was 63.14%. The study revealed better overall survival for patients with ATGAM treatment than THYMOGAM treatment arm.

Introduction

Acquired aplastic anaemia (AA) is characterized pathologically by an “empty” bone marrow, in which pancytopenia results from replacement of haematopoietic precursor cells by fat [1]. Immunosuppressive therapy (IST) is an effective alternative treatment for patients who are not candidates for BM transplantation. Current standard first line IST is the combination of horse antithymocyte globulin (ATG) and cyclosporine (CSA) [2, 3]. The current study was conducted to assess response to IST for aplastic anaemia.

Materials and Methods

It was a prospective and retrospective observational study conducted in a tertiary care hospital in the eastern region of the country catering all the districts of West Bengal and adjoining states of Jharkhand, Bihar and Odhisa. Study from July 2013 to June 2018. Diagnosis of acquired AA was made as per standard international guidelines [2].

In this study, total 76 patients diagnosed with AA were included. Patients included in the study were of severe AA (SAA) and very severe AA (VSAA) in the absence of an HLA-matched sibling and/or > 35 years of age. Non-severe aplastic anaemia (NSAA) patients who were transfusion dependent, bleeding, encountering infections and required treatment for maintaining day to day activities were also included. Stress cytogenetics was done for all patients < 50 years of age to exclude cases of constitutional aplastic anaemia. Cases of secondary aplastic anaemia and active infection with Hepatitis B and Hepatitis C virus, HIV infection, renal and hepatic failure were excluded from the study. Following standard protocol, horse ATG (hATG) was administered for 4 days followed by Cyclosporin at a dose of 5 mg/kg/day in two divided doses starting from day 15 of ATG administration. All patients received prophylaxis for serum sickness in the form of oral prednisolone from day 5 to day 14, with dose tapering over next 2 weeks. Response was assessed at 3 months and 6 months of ATG administration. For statistical analysis data were entered into a Microsoft excel spreadsheet and analyzed by SPSS (version 24.0; SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 5.

Results

Median duration of follow up in the study was 12 months (range 6–60). Median age of the study population is 36 years (range 6–66). Among 76 cases, 25 (32.9%) patients were female and 51 (67.1%) patients were male; male and female ratio was 2.04:1. Pallor was the commonest (92.10%) presenting feature followed by bleeding (73.68%).

In the study population, 62 (81.6%) patients had severe AA, 5 (6.6%) patients had very severe AA and 9 (11.8%) patients had non-severe disease.

Only 4 patients in the study population had a PNH clone. All of them have a small (< 10%) population of glycosyl phosphatidylinositol–anchored proteins (GPI-APs) deficient neutrophils.

Among 76 patients, 32 patients received Atgam (Pharmacia and Upjohn Company, Division of Pfizer Inc., New York) and 44 patients received Thymogam (Bharat Serums And Vaccines Limited, India). Table 1 describes the baseline parameters at presentation of patients receiving Atgam and Thymogam. The median interval from diagnosis to therapy is 90 days (range 10–1825). Median duration of hospital stay was 25 days (range 14–43 days). Within 3 months of ATG therapy 4 patients died and 1 patient was lost to follow up. At 3 months of follow up 2 (2.63%) patients were on CR, 32 (42.10%) patients were in PR and 37 (48.68%) patients were in NR. Therefore overall response at 3 months was 44.73%. Figure 2 showing the response at 3 months and 6 months. In the Atgam group, 2 (6.25%) patients achieved CR, 15 (46.87%) patients achieved PR and 15 (46.87%) were in NR, OR was 53.12%. In the Thymogam group, no patient was in CR at the end of 3 months, 17 (38.63%) achieved PR and 22 (50%) patients were in NR. Overall response (OR) was 37.77%. Association of ATG preparation with response at 3 months was not statistically significant (p = 0.2483). At 6 months 5 (6.57%) patients were in CR, 43 (56.57%) patients were in PR and 23 (30.26%) patients were in NR. Therefore overall response at 6 months was 63.14%. In the Atgam group, 5 (15.62%) patients achieved CR, 15 (46.87%) patients achieved PR and 12 (37.5%) patients were in NR. OR was 62.5%. In the Thymogam group, no patient achieved CR, 28 (63.63%) patients achieved PR and 11 (25%) patients were in NR. OR was 63.63%. Association of ATG preparation with response at 6 months was statistically significant (p = 0.0153). Overall survival for patients treated with ATGAM and THYMOGAM was compared and it was found that the better overall survival (OS) for patients with ATGAM treatment than THYMOGAM treatment (Log-rank statistics—039; p = 0.843). Mean OS was 48.58 (± 14.783) months. This finding is depicted in Fig. 2. Total 4 patients experienced serum sickness, 2 patients had ATG induced posterior reversible encephalopathy syndrome. Commonly encountered infections are blood stream infection with Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and fungal pneumonia. Within 3 months of ATG therapy 4 patients expired, the cause of death was sepsis including probable invasive fungal pneumonia in two cases, acute myocardial infarction in one case, in one case the cause was not known as the patient died at some other hospital and record could not be tracked.

Table 1.

Baseline features of patients receiving Atgam and Thymogam

Parameters	Atgam	Thymogam
Total no of patients	32	44
Female/total	13/32	12/44
M:F	1.46:1	2.66:1
Median age in years (range)	35 (6–66)	31.5 (8–60)
Median Hb (g/L, range)	57.5 (41–78)	68 (53–104)
Median ANC (× 109/L) (range)	1.203 (0.457–1950)	0.600 (0.100–2.720)
Median platelet (× 109/L) (range)	17 (1–78)	10 (2–91)

Fig. 2.

Kaplan-Meier survival curve showing overall survival for patients with ATGAM and THYMOGAM treatment (Log-rank statistics—039; p = 0.843). The study revealed better overall survival for patients with ATGAM treatment than THYMOGAM treatment arm. Mean OS was 48.58 ± 14.783 months (Green line: Thymogam, Blue line: Atgam) (colour figure online)

Both univariate and multivariate analysis were done with following parameters to determine the predictors of IST:

1. Young age (< 18 years).

2. Less severe disease.

3. Absolute reticulocyte count > 25 × 10⁶/L.

4. Absolute lymphocyte count > 1.0 × 10⁶/L.

5. Absolute neutrophil count > 1.5 × 10⁶/L.

6. Median interval between diagnosis and therapy.

7. The presence of a PNH clone is predictive of response.

However, no statistically significant association was detected in any of the parameter.

Total duration of follow up was 5 years, 3 patients had a relapse and all of them succumbed to their disease. None had a clonal evolution within that period.

Discussion

Median age of the study population was 36 years, ranging from 6 to 66 years. There is a paucity of precise epidemiological data from this part of the continent. In one study from AIIMS, New Delhi by Mahapatra et al. [4] conducted in 1501 patients from North India found the median age at presentation 25 years (range 2–83). In another study from Western part of India conducted by Nair et al. [5] the median age at presentation was 36.5 years (19–77). AA is a disease of the young. Studies from western countries found that most patients present between 15 and 25 years of age or older than 60 years of age [6]. Unlike western countries, most of the studies from India show a younger age at presentation which may be related to some different pathophysiology or exposure to different kind of environmental toxins.

In the study population, 25 (32.9%) patients were female and 51 (67.1%) patients were male. Male and female ratio was 2.04:1. In the study by Mahapatra et al. [4] they also found male (70%) preponderance. In the study by Nair et al. [5], male to female ratio was 1.11:1. Although there is no major sex difference in the occurrence of the disease as per western published literatures [6], the population in question in the present study probably reflects the prevailed socio-economic status showing more accessibility of the male patients to health facility in comparison to their female counterpart.

In the present study, 70 (92.10%) patients presented with pallor, 56 (73.68%) patients with bleeding and 4 (5.26%) patients presented with fever. Therefore, pallor was the commonest presenting feature followed by bleeding. Mahapatra et al. [5] also got similar type of results; they found pallor (97%), bleeding manifestations (69.6%) and fever (54%) as common clinical manifestations. The present study also corroborated with the results of Killick et al. [2].

In this study, 62 (81.6%) patients had severe AA, 5 (6.6%) patients had very severe AA and 9 (11.8%) patients had non-severe disease. In the study by Mahapatra et al. [4] 75% of patients were classified into severe aplastic anaemia, another 15% were of very severe and only 10% of our patients were of non-severe variety. In the similar study, Nair et al. [5] found that 80% patients had SAA, 14.2% had VSAA and 5.8% patients had NSAA. Shah et al. [7] stated that, VSAA was 33%, SAA was 62.6% and NSAA was 4.4% in their study. In the current study also, SAA was the commonest variety of the disease. In the present study, the median interval from diagnosis to therapy is 90 days (range 10–1825). Mahapatra et al. [4] showed, the median interval from diagnosis to treatment was 120 days (range 30–2160). This difference in turnover could be due to the availability of resources and number of patients. There is not much comparative data available in the western literature because of the difference in socio-economic condition. In the current study, the median duration of hospital stay during ATG therapy was 25 days (range 14–43 days) in contrast to 14 days (range 7–30) as shown in the study by Mahapatra et al. [4]. Table 2 depicts the comparison of baseline haematological values between present study and similar previous studies. In this study, within 3 months of ATG therapy 4 patients expired, the cause of death was sepsis including probable invasive fungal pneumonia in two cases, acute myocardial infarction in one case, in one case the cause was not known as the patient died at some other hospital and record could not be tracked. One patient was lost to follow up. At 3 months of follow up 2 (2.63%) patients were on CR, 32 (42.10%) patients were on PR and 37 (48.68%) patients were on NR. Therefore overall response at 3 months was 44.73%. At 6 months 5 (6.57%) patients were in CR, 43 (56.57%) patients were in PR and 23 (30.26%) patients were in NR. Therefore overall response at 6 months were 63.14%. Among the total 76 patients 32 patients received Atgam and 44 patients received Thymogam. As shown in Fig. 1, at 3 months of IST: In the Atgam group, 2 (6.25%) patients achieved CR, 15 (46.87%) patients achieved PR and 15 (46.87%) were in NR. OR was 53.12% and in the Thymogam group, no patient was in CR at the end of 3 months, 17 (38.63%) achieved PR and 22 (50%) patients were in NR. OR was 37.77%. At 6 months of IST: In the Atgam group, 5 (15.62%) patients achieved CR, 15 (46.87%) patients achieved PR and 12 (37.5%) patients were in NR and OR was 62.5% and in the Thymogam group, no patient achieved CR, 28 (63.63%) patients achieved PR and 11 (25%) patients were in NR and OR was 63.63%. Association of ATG preparation with response at 6 months was statistically significant (p = 0.0153). Overall survival for patients treated with ATGAM and THYMOGAM was compared and it was found that the better overall survival (OS) for patients with ATGAM treatment than THYMOGAM treatment (Log-rank statistics—039; p = 0.843). Mean OS was 48.58 (± 14.783) months. This finding is depicted in Fig. 2.

Table 2.

Comparison of baseline haematological values between present study and similar previous studies

Haematological parameters	Mahapatra et al. [4]	Nair et al. [5]	Present study
	Median (range)	Median (range)	Median (range)
Hb (g/L)	59 (45–72)	58 (22–99)	67.5 (3–13.8)
ARC (× 109/L)	Not mentioned	16 (7–24)	15 (10–30)
TLC (× 109/L)	2.7 (2.1–3.5)	1.1 (0.4–4.7)	2.595 (0.5–7.0)
ANC (× 109/L)	0.380 (0.120–0.880)	0.36 (0.1–0.9)	0.590 (0.1–2.72)
Platelet (× 109/L)	10 (8–17)	22 (2.0–50.0)	2.09 (0.4–3.3)

Fig. 1.

Response at 3 months and 6 months in Atgam and Thymogam groups

Table 3 depicts the comparison among different studies in India and also many others from outside India. At the end of 3 months, Mahapatra et al. [4] found 5.82% CR, 33.98% PR and 54.36% NR and at 6th month of therapy 9.70% CR, 45.63% PR, 38.83% NR. The overall response was 58.7%. Nair et al. [5] reported their response at 6th month of IST; 9.2% achieved CR, 76.7% demonstrated PR and 14.16% patients did not respond to IST. Overall response (response ≥ PR) to IST was observed in 85.8% patients at 6 months. Shah et al. [7] showed, at 3 months, 2.2% CR, 39.6% PR and at 6 months, 8.8% CR and 39.6% PR. Patel et al. [8] also showed an overall response of 43.8%, although their study population is smaller. Bacigalupo [9] also mentioned in his recently published review article that after first-line IST with ATG and CSA, haematological recovery was seen in 50–70% of cases. Also in the 1980s and 1990s, most former studies on the efficacy of ATG therapy in patients with SAA conducted in Europe, Japan, and the United States used horse ATG, with haematologic responses observed in about two-thirds of cases [10–12].

Table 3.

Comparison between response to IST in different studies conducted with both Atgam and Thymogam (hATG^A: Atgam; hATG^B:Thymogam)

Study	IST used	Design	Total no patients (receiving IST)	Age group (years)	Response (%)
Studies from other countries
Scheinberg [13]	hATGA + CSA	Prospective	77 (42)	4–78	At 3 months: 50%
					At 6 months: 62%
Teramura [14]	hATG + CSA	Prospective	101 (50)	18–75	At 3 months: 51%
					At 6 months: 57%
Rosenfeld [15]	hATGA + CSA	Prospective	122	Not specified	At 3 months: 60%
					At 6 months: 61%
Indian studies
Agarwal [16]	hATGB + CSA	Prospective	30	9–58	At 3 months: 40%
					At 6 months: 50%
Shah [7]	hATGB + CSA	Retrospective	91	2–67	At 3 months: 41.8%
					At 6 months: 48.4%
Nair et al. [5]	hATG + CSA	Retrospective	120	> 18	At 6 months: 85.8%
Patel [8]	hATG + CSA	Retrospective	18	7–58	At 6 months: 43.8%
Current study	hATGA + CSA	Prospective and retrospective	32	6–66	At 3 months: 53.12%
					At 6 months: 62.5%
	hATGB + CSA		44	8–60	At 3 months: 37.77%
					At 6 months: 63.63%

In the present study, at 3 months, there was no association between age distribution and response, p = 0.1021. At 6 months, association between mean age and 6 months response was statistically significant (p = 0.0230), showing that CR and PR was more in the age group > 18 years.

In the study conducted by Mahapatra et al. [4] showed no statistical difference in response rate between paediatric (< 18 years) and adult (> 18 years) group (p = 0.08).

Schienberg et al. [13] showed an inverse relationship between response and age, younger patients having a higher probability of response compared to older patients (p = 0.01).

These differences in findings in different studies might be explained by the difference in the sample size, age distribution of the study population as well as use of different therapeutic protocols.

In the study under discussion, the association between disease severity and response at 3 months and 6 months was not statistically significant.

Mahapatra et al. [4] stated that the patients with severe aplastic anaemia (SAA) responded better than very severe AA (p = 0.02) in their study.

Vaht et al. [17] showed that VSAA patients had only a 19% probability of response. In a multivariable analysis, age and VSAA at the time of treatment were the independent factors for inferior survival.

Association between PNH clone and response at 3 months and 6 months was not statistically significant in the current study.

In several studies, investigators have attempted to reveal the clinical significance of such PNH clones in patients with bone marrow failure. However, the reliability of results obtained from minor PNH populations remains controversial.

Several studies conducted on adults and/or children with AA reported that the presence of minor PNH clones was associated with a favourable response to IST [18]. Kulagin et al. [19] in their prospective study confirmed the favourable prognostic value of presence of PNH clone in the setting of IST for AA.

In contrast, a retrospective National Institutes of Health (NIH) study done by Schienberg et al. [13] did not find differences between AA patients who did or did not respond to IST.

In the study under consideration, value of baseline haematological parameters like Hb, TLC, ANC, ALC, ARC, platelet and median interval between diagnosis and treatment were not associated with response at 3 months and 6 months.

In the study by Scheinberg et al. [13], in multivariate analysis, higher baseline absolute reticulocyte count (ARC), and absolute lymphocyte count (ALC) were highly predictive of response at 6 months. Patients with baseline ARC ≥ 25,000/cmm and ALC ≥ 1000/cmm had a much greater probability of response at 6 months following IST compared to those with lower ARC and ALC (83% vs. 41%, respectively; p < 0.001). The number of cases was high in comparison to the current study (n = 316) and they used different immunosuppressive protocols (h-ATG/CsA, h-ATG/CsA/mycophenolate mofetil (MMF) and h-ATG/CsA/sirolimus). These two factors can influence the result of the analysis.

Conclusion

In the present study, an overall response at 3 months was 44.73% and at 6 months was 63.14%. OR was 62.5% and 63.63% in the Atgam and Thymogam group, no patient achieved CR in the Thymogam group. The study revealed better overall survival for patients with ATGAM treatment than THYMOGAM treatment arm. None of the factors discussed above could predict the response after IST. Therefore, small sample size and short follow up period were the main limitation of the present study. In future, large sample size and long term follow up will be required to predict the outcome of the patient of aplastic anaemia on IST therapy.

Compliance with Ethical Standards

Conflict of interest

The authors have declared no competing interests.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional ethical committee (IEC).