Idiopathic Aplastic anemia: Indian Perspective

Abstract

Aplastic anemia (AA) is a rare immunologically mediated bone marrow failure syndrome, characterized by progressive loss of hematopoietic stem cells resulting in peripheral pancytopenia. Elaborative investigation including molecular tests is required to exclude inherited bone marrow failure syndrome (IMBFS) as the treatment and prognosis vary dramatically between them. Haematopoietic stem cell transplant with a fully matched sibling donor (MSD-HSCT) is still the only curative treatment. Management of AA is a real-time challenge in India, because of the delay in the diagnosis, lack of proper supportive care, limited availability of the expertise centre, and the patient’s affordability. Recently, results with intensified immunosuppressive therapy that includes anti-thymocyte globulin with cyclosporine-A (CsA) and eltrombopag, are enough encouraging to consider it as treatment of choice in patients lacking MSD or who are not fit for HSCT. However, limitations in resource constraints settings including the cost of therapy limit its full utilization. Relapse of the disease or evolution to myelodysplasia or paroxysmal nocturnal haemoglobinuria (PNH) in a proportion of patients is another challenge with immunosuppressants. The majority of the AA patients still receive CsA with or without androgens in India, mostly because of increased cost and limited availability of HSCT and ATG. The use of the unrelated or alternative donor is still upcoming in India, with unavailable data in terms of response and survival. Therefore, there is an utmost need for novel agents for the better management of AA having a balanced efficacy and toxicity profile to improve the survival and quality of life.

Introduction

Aplastic anemia (AA) is a rare hematological disorder with heterogeneous clinical presentation. It may be life-threatening and if untreated is usually associated with a high mortality rate. AA is a misnomer and is characterized by bi- or pancytopenia (not just anemia), hypocellular bone marrow with no features of dysplasia, fibrosis or infiltration, and absence of splenomegaly. The presentation may be challenging in terms of both diagnosis and management. The only definitive cure is still the allogeneic hematopoietic stem cell transplant, although new approaches have recently emerged expanding the possible treatment options for transplant-ineligible patients. Apart from the definitive treatment, managing the complexities including infectious risks and long-term transfusion support are an integral part of the management of AA patients [1]. This review provides an overview of idiopathic aplastic anemia (AA) epidemiology, pathogenesis, clinical presentation diagnosis, and management from an India perspective.

Epidemiology

The incidence of AA varies considerably worldwide and so also varies geographically in India. There is a lack of epidemiological studies in Asia as well as in India. The incidence in Asia reported is as high as 2–3 times (upto 8.8 per million individuals per year) than the western world [2–7]. Few studies from India reported that upto 20–30% cases of pancytopenia are later diagnosed as aplastic anemia[8]. The study from prestigious institute in New Delhi (2015), enrolled 1501 patients diagnosed with aplastic anemia over seven and a half years among 20 different states of India [9]. The incidence of AA in the pediatric population was found 6.8 per million in a study from Lucknow [10]. The incidence of AA [severe and non-severe] was reported as high as 33.33% and 57.14% in northern districts of West Bengal [11]. An observational study on 300 patients found that the overall survival (OS) for AA at 1, 2, and 3- years were 82%, 46%, and 32%, respectively. When evaluated effect of age on OS, there was statistically differences [1‐year survival was 90.0% and 42.10% in the < 21 and ≥ 21 age groups, respectively] [14].

The median age at the presentation from western countries found bimodal age distribution, 15–25 years, or ≥ 60 years [3]. Unlike western countries, most of the studies from India found a younger age at presentation, the median age at presentation 25 years (range 2–83 years) [9] and 36.5 years (range 19–77 years) [12]. This difference may be related to some different pathophysiology or exposure to different kinds of environmental toxins [13, 17].

The gender variation in the incidence of AA is heterogeneous with studies from developed countries have nearly equal sex ratio [15], while preponderance among male patients in countries like India [9, 12] due to social bias [16].

Pathophysiology

The main cause of idiopathic aplastic anemia is the antigen-driven and auto-immune like dysregulated cytotoxic T-lymphocyte mediated destruction of CD34 + hematopoietic stem cells (HPSCs). The etiology implicated leading to an antigenic alteration in the HPSCs includes infections, medications, exposure to chemicals, such as benzene, and pregnancy [17]. The immune dysregulation served as the basis for treatment with immunosuppressive therapy, predominantly anti-thymocyte globulin (ATG) combined with cyclosporine-A [18]. Recent studies have shown the role of dysregulation of interferon-γ (IFN- γ) and its effect on bone marrow niche leading to suppression of hematopoiesis [19] and hamper the interaction of thrombopoietin (TPO) and c-MPL due to TPO‐IFN‐γ heterodimerization leading to disruption in its downstream signaling pathway and thereby reduced the self-renewal property of the stem cells [20]. Eltrombopag (EPAG), a TPO-receptor agonist, has shown significant effectiveness in the treatment of aplastic anemia with IST therapy [21]. Figure 1 illustrates the mechanisms contributing to antigenic modulation and immune destruction of hematopoiesis in idiopathic aplastic anemia.

Fig. 1.

Pathogenesis of idiopathic aplastic anemia. Immune-mediated destruction of hematopoietic stem cells (HSC) or progenitor cells. Direct killing by a noxious agent (e.g. cytotoxic T cells) or functional inhibition by interferon-γ leads to depletion of the HSC pool, with consequent impaired production of hematopoietic progenitor cells (HPCs) and of mature blood cells, resulting in pancytopenia. IFN- γ: Interferon- γ, TNF-α: Tumor necrosis factor- α

Clinical presentation

The initial presentation varies considerably with age and severity of AA, and can present as bicytopenia or multilineage cytopenia. Symptoms varies as per degree of anemia and thrombocytopenia, with most presenting in clinics as requirement of frequent blood transfusion support. Bleeding manifestations are more alarming and significant reason for hospital visits [9]. The presence of constitutional symptoms including weight loss, persistent fever, or night sweats points towards alternative diagnosis. A family history of inherited bone marrow failure syndrome (IBMFS), cryptogenic cirrhosis, pulmonary fibrosis, or the presence of congenital malformations (skeletal deformities, nail dystrophy, skin pigmentation disorders, gastrointestinal, genitourinary malformations, or endocrine dysfunction) or the presence of splenomegaly or lymphadenopathy, rules out the acquired AA [6, 10]. A high index of suspicion is required in patients with cytopenia and subtle phenotypical findings and hence both clinical examinations and investigations play a vital role in the confirmation of the definitive diagnosis [6, 10, 17].

Severity is defined as per the modified Camittas criteria as defined below [22].

Diagnosis

Patient with cytopenia required to undergo multiple investigations:

• I.To confirm the suspected diagnosis.
• II.To exclude other causes of pancytopenia and a hypocellular bone marrow like hypoplastic myelodysplasia (MDS).
• III.To exclude IBMFSs like Fanconi anemia.
• IV.To screen for an underlying cause like a viral infection.
• V.To document co-existing abnormal cytogenetic and paroxysmal nocturnal haemoglobinuria (PNH) clones.

Figure 2a and 2b provides a diagnostic workflow and interpretation of pancytopenia.

Fig. 2a.

Diagnostic approach in patients with pancytopenia. AML: Acute myeloid leukemia, ANA: Antinuclear antibody test, BMF: Bone marrow failure, CMV: Cytomegalovirus, EBV: Epstein–Barr virus, FISH: Fluorescence In-situ hybridization, HCV: Hepatitis C virus, Hepatitis AA: Hepatitis associated aplastic anemia, HIV: Human immunodeficiency virus, MDS: Myelodysplastic syndrome PNH: Paroxysmal nocturnal haemoglobinuria, SLE: Systemic lupus erythematosus, Viral AA: Viral associated aplastic anemia, Vit B12: Vitamin B12. *Chromosomal aberrations characteristic of MDS includes Deletion of chromosome 5, del(7q), monosomy 7, del 20q, del 5q

Fig. 2b.

Differential diagnosis of pancytopenia and differentiation of Aplastic anemia from hypocellular MDS. AA: Aplastic anemia, MDS: Myelodysplastic syndrome, PNH: Paroxysmal nocturnal haemoglobinuria.

Classification

As per the International agranulocytosis and aplastic anemia study (IAAS), AA was defined as Hb < 100 g/L, platelet < 50 × 10⁹/L and total leucocyte count < 3.5 × 10⁹/L or absolute neutrophil count < 1.5 × 10⁹/L. Later the diagnostic and the severity criteria were defined by the modified Camittas criteria [22].

Diagnostic Criteria

Bone marrow must be hypocellular with cellularity of < 25% of normal age appropriate cellularity or ≥ 25 to ≤ 50% of normal age appropriate cellularity with < 30% of residual cells are hematopoietic with at least one of the following.

1. Platelet count < 20,000/ µL.

2. Absolute reticulocyte count < 20,000/µL.

And

Severity Criteria

depends on absolute neutrophil count (ANC) as.

• Non-severe aplastic anemia (NSAA): ANC > 500/µL.

• Severe aplastic anemia (SAA): > 200 – ≤ 500/µL.

• Very severe aplastic anemia (VSAA): ≤ 200 /µL.

With the availability of newer automated cell counters, the reticulocyte count criterion has now been modified from manual < 20 × 10⁹/L to absolute reticulocyte levels < 60 × 10⁹/L as assessed by automated technologies [23].

The severity of the disease is directly related to the overall survival (OS) and treatment decisions, with VSAA & SAA having a worse prognosis requiring urgent treatment [24].

Management

The optimal management of AA includes early diagnosis and timely initiation of treatment (immunosuppression or HSCT) as per the age and performance status of the patient [25, 26]. In the last few decades, the survival of the AA has been improved substantially due to the availability of better supportive care and definitive management options in both children and adults [27]. Supportive care includes safe and ideal blood product transfusion strategy, prophylaxis, and early management of infections, along with treatment of the iron overload. However, in developing countries like India, the prognosis is still not improved as compared to the western countries because of the limited availability of well-equipped centre, the heterogeneity of health care (private and public sector), and lack of proper supportive care due to the cost and affordability by the patient [12].

The main limitation in the management of AA is the delay in the diagnosis and the definitive management like bone marrow transplant. Meanwhile supportive care needed in AA patients to maintain quality of life to prevent complications, and early mortality are also lacking in developing countries. The major life-threatening complications include bleeding manifestations and severe bacterial or invasive fungal infections (IFI) due to severe neutropenia [27]. The practice of supportive care varies from centre to centre and depends on the clinical presentation and its severity. There is a lack of availability of randomized controlled trials (RCT) based recommendations for supportive care in AA [25].

Infection

There is no consensus on the routinely prophylactic use of antiviral, antifungal, antibacterial, or against Pneumocystis jirovecii pneumonia (PJP) as primary prophylaxis [22]. However, early recognition of signs and symptoms of infection and implementation of the therapeutic intervention including antimicrobial can significantly reduce infection-related mortality [28]. Isolation of the microorganism and the use of specific anti-microbial is the mainstay of the management. In cases where isolation of the organism is not possible empirical therapy should be implemented. Previous studies have reported a mortality rate of upto 70–80% in severely neutropenic patients due to IFI including aspergillosis [12]. With the emerging armamentarium to deal with IFI, the survival of VSAA has been improved in the last few years. Secondary prophylaxis is generally recommended in patients with VSAA for IFI to prevent relapse [22]. Routine prophylaxis against viral and PJP infections is recommended to those who undergo HSCT [29]. There is insufficient evidence to recommend the use of G-CSF or erythropoiesis-stimulating agents (ESA) or granulocyte infusion except in some special circumstances [25, 27]. However, small interventions like hand washing, sitz bath and use of mouthwash can make huge difference in reduction of infection rates in AA patients.

Blood Transfusion

Most of the patients require a frequent blood transfusion to relieve symptomatic anemia or bleeding manifestations, however, it should be judiciously used to prevent both short- and long-term complications related to the transfusion. As long-term transfusion support is required, an extended RBC phenotype (or genotype) before their initial transfusion should be preferably be determined. The leukoreduced blood products should be preferred to prevent the alloimmunization [30], however, it’s a major limitation in India due to lack of the facility and cost [31]. The threshold for PRBC transfusion (Hb ≤ 6 g/dl), varies with the patient profile including age and comorbidities like cardio-pulmonary dysfunction (Hb ≥ 8-10 g/dl) [22]. A restrictive transfusion should always be preferred in comparison to the liberal transfusion policy, in order to reduce the risk of blood transmitted infections, iron overload and risk of alloimmunization [25]. The guidelines recommend transfusing platelets if active bleed or platelet count < 10,000/µL or fever with platelets < 20,000/ µL. The routine use of irradiated blood products should be discouraged in AA patients and should be considered only with HLA-selected platelets, transfusion of granulocytes, donations from first- or second-degree relatives, or planned relevant treatment (e.g.ATG, alemtuzumab, HSCT) and are based on as per international guidelines [27]. In countries like India with limited availability of blood products, there is a significant role of adjuvant therapy like antifibrinolytic agents, to reduce bleeding due to thrombocytopenia, although there are mixed results concerning its utility and efficacy [32].

Definitive treatment

In India, AA patients attend a range of medical and other health professionals before being referred to the specialist centre, leading to the acquisition of infections, increasing the relative risk of treatment-related mortality [24]. It is crucial to develop awareness regarding the significance of early referral to a specialist centre to improve the overall survival of AA.

The primary modalities of treatment include Allogenic-Hematopoietic stem cell transplant (HSCT) and immunosuppressive therapy (IST) including anti-thymocyte globulin with Cyclosporine-A. Both IST and HSCT have the potential to restore the haematopoiesis but only HSCT has the curative potential. However, no RCT till now directly compared the outcome of HSCT and IST in AA in India.

The factors to consider before deciding the definitive therapy include age, disease severity, comorbidities, HLA-MSD availability, and patient affordability. Figure 3 illustrates an algorithm to select the definitive therapy in AA.

Fig. 3.

a) Proposed treatment approach in severe and very severe acquired aplastic anemia in India. Patients are stratified according to affordability, age, and transplant eligibility. In young patients (< 50 years) fully matched related donor allogeneic HSCT is standard of care. Patients lacking a matched donor or older than 50 years, or unfit for HSCT, intensified immunosuppressive therapy including hATG with cyclosporine-A with or without eltrombopag is the treatment of choice b) Proposed treatment approach in Refractory aplastic anemia (non-responder to IST): Patients not responding to initial IST have several choices, depending on the performance status, the availability of an HLA-matched unrelated donor (UD), alternative donor HSCT, and patient preference (affordability). In selected patients with very severe disease, an unrelated matched donor (UD) graft may be considered first-line therapy. Elderly patients (older than 60 years of age), non-transplant therapy should be considered. Allo SCT: Allogeneic stem cell transplant, EPAG, eltrombopag, hATG: Horse (equine) anti-thymocyte globulin, HSCT: Haematopoietic stem cell transplant, IST: Immunosuppressive therapy, MSD: Matched sibling donor, MUD: Matched unrelated donor, SAA: Severe aplastic anemia, UD: unrelated donor, UD BMT, unrelated donor HSCT, VSAA: Very severe aplastic anemia

Immunosuppressive therapy (IST)

The efficacy of IST in AA is well established since the 1970s, with several large prospective trials in the United States, Europe, and Japan showing consistent results [33–35]. As per the British Society for Standards in Haematology guidelines (BCSH-2015), IST is the standard of treatment for SAA and VSAA patients who lack a suitably matched sibling donor (MSD), age > 40years, medically unfit, and as bridging therapy while waiting for the HSCT [22].

The standard effective first-line IST regimen includes a combination of ATG with CSA ± EPAG. The preparation of ATG includes equine-ATG (hATG) and Rabbit-ATG (rATG).

The meta-analysis including 13 western studies compared to the efficacy of hATG versus rATG for SAA, found a higher response rate (RR) and modestly lower early mortality with hATG as compared to rATG [36].

Various large prospective studies have proven a consistent efficacy of the hATG-based IST (hATG & CsA) in the developed countries in the last 4 decades with hematologic responses (graded by BCSH) of upto 60–75% over 3 to 12 months [37–41]. IST is usually associated with excellent long-term survival of > 80% in responders [40, 42]. However, only upto 20–25% of patients sustain hematologic recovery after IST.

Recently, prospective studies have shown a good therapeutic effect with triple-drug therapy (hATG, CsA, and EPAG) with upto an overall response of 90% (CR of 50%) [21, 43, 44]. The inclusion of eltrombopag (TPO agonist) offers a favorable balance between outcomes and toxicity in adults with SAA [21].

The inferior response rates to ATG in Indian settings is seen particularly involving children, which is lower even in the western world compared to their adult counterparts, ranging from 35 to 70% (CR 10–30%) especially in the paediatric population. Table 1 illustrates various studies on hATG based IST conducted in India.

Table 1.

Response rate of IST (hATG and CsA) in various studies conducted in India

S No	Author (Year) [Ref]	Study design	Age group (Median age)	Sample size (n)	hATG Type*	ORR (CR%, PR%) [Time- Period]
1.	Ramzan M (2014) [45]	Retrospective	Children (9 years)	20	Thymogam	70% (10%, 60%) [15-months]
2.	Gupta V (2012 [46]	Retrospective	Children (9 years)	30	Thymogam	33.3% (11.1%, 22.2%) [6-months]
3.	Agarwal MB (2015) [47]	Prospective	Adults (30 years)	30	Thymogam	50% (6.6%, 43.4%) [6-months]
4.	Shah S (2019) [48]	Retrospective	Total	91	Thymogam	68.2% (27.5%, 40.7%) [24-month]
			Children (10 years)	23		69.5% (39.1%, 30.4%) [24-month]
			Adults (29 years)	68		67.6% (23.5%, 44.1%) [24-month]
7.	Mahapatra M (2015) [9]	Retrospective	Mixed cohort (27 years)	26	Thymogam	50% (3.8%, 46.2%) [6-months]
8.	Amalnath DS (2019) [49]	Retrospective	Adults (39 years)	60	Thymogam	68.3% (3.3%, 65%) [6-months]
9.	Dutta B (2019) [50]	Prospective and Retrospective	Mixed cohort (31.5 years)	44	Thymogam	63.63% (0%0.63.63%) [6-months]
10.	Dutta B (2019) [50]	Prospective and Retrospective	Mixed cohort (35 years)	32	ATGAM	62.42% (15.62%,46.8%) [6-months]
11.	Mahapatra M (2015) [9]	Retrospective	Mixed cohort (27 years)	97	ATGAM	58.7% (10.3%, 48.4%) [6-months]
12.	George B (2015) [51]	Retrospective	Total	530	ATGAM/ Lymphoglobulin	58.4% (23.3%, 35.1%) [6-months]
			Adults	410		65% (24.1%, 40.9%) [6-months]
			Children	120		35.8% (20.8%, 15%) [6-months]
15.	Nair V (2012) [52]	Prospective	Children (14 years)	33	ATGAM	87.9% (24.2%, 63.7%) (6-months)
16.	Nair V (2013) [12]	Retrospective	Adults (36.5 years)	120	ATGAM	85.8% (9.2%, 76.6%) [6-months]
17.	Malhotra P (2015) [53]	Retrospective	Adults (31 years)	30	ATGAM (Low dose 25 mg/kg) or Thymogam (Std dose)	77% (3%, 74%) [6-months]
18.	Patel AB (2015) [54]	Retrospective	Total	16	Not mentioned	50% (31.25%, 18.75%) [12-months]
			Children (9 years)	6		33.3% (16.6%, 16.7%) [12-months]
			Adults (35 years)	10		50% (30%, 20%) [12-months]
21.	Sharma R (2012) [55]	Retrospective	Children (10 years)	28	ATGAM/ Thymogam/ Lymphoglobulin	50% (14.3%, 35.7%) [12-months]
22.	Jain R (2019) [56]	Retrospective	Children (10 years)	43	ATGAM/ Thymogam/	53.5% (21%, 32.5%) [12-months]
23.	Chandra J (2008) [57]	Prospective	Children (8 years)	20	ATGAM/ Thymogam/ Lymphoglobulin	40% (10%, 30%) [6-months]

*hATG includes ATGAM (manufactured by Pfizer Inc., USA) and Thymogam (manufactured by Bharat Serums and Vaccines Ltd., India). Dose- hATG = 40 mg/kg/day for 4 days, CsA: Cyclosporine A = 5 mg/kg/day in two divided doses

CR: Complete response, hATG: Horse Antithymocyte globulin, IST: Immunosuppressive therapy, ORR: Overall response rate, PR: Partial response

The hATG available in India includes ATGAM (manufactured by Pfizer Inc., USA) and Thymogam (manufactured by Bharat Serums and Vaccines Ltd., India). The response rate of Thymogam ranges from 35 to 70% and of ATGAM 50–85% in the Indian population. However, a study conducted in 2019 in the Eastern part of India found similar RR of Thymogam and ATGAM in the adult Indian population [50].

The preference of the hATG and its brand is dependent on several factors like clinician preference, product availability, and the cost of therapy (most significant factor in India). Many Indian medical institutes are being supported by government or non-government organizations (NGOs) directed financial aid to bear the cost of therapy, although a still significant number of patients are unable to avail the treatment due to cost limitation (many not even CsA single-agent IST). The cost of ATGAM is upto two times than the indigenous hATG Thymogam [47, 49].

The biomarkers which can predict the response to IST includes [50, 58]:

• Young age (< 18 years).

• Less severe disease.

• Absolute reticulocyte count ≥ 25 × 10⁹/L.

• Absolute lymphocyte count ≥ 1 × 10⁹/L.

• The median interval between diagnosis and therapy.

• Presence of mutations including PIGA, BCOR, and BCORL1.

• Presence of a PNH clone (usually minor clone and is seen in upto 50% AA patients).

• Telomere length (TL).

The combination of detection of PNH clones and TL may represent a promising tool for future personalized IST in AA [58].

Administration of ATG requires central venous catheter insertion and premedication with methylprednisolone to prevent infusion reactions including anaphylaxis and serum sickness. The incidence of serum sickness reported varies from nil to 25.9% [9, 47–50].

CsA is given at a dose of 5–6 mg/kg/day in two divided doses and subsequently titrated to maintain a trough drug level between 100 and 250 ng/mL. The duration of CSA varies among various centres, some with a minimum of 12 months while others taper the dose after 6 months of stable response [40, 59].

Follow up

The late consequences of IST include disease relapse and the most significant, the clonal evolution including progression to the MDS or acute myeloid leukemia (AML). Clonal evolution reported in upto 15% of patients over the decade following the initial IST. Most often manifesting as cytopenia associated with cytogenetic abnormality usually loss of all or part of chromosome including chromosome 7 aneuploidy which has a poor prognosis and triggers efforts to transplant strategy [59–61]. Therefore morphologic, and cytogenetic evaluation of marrow cells should be considered for SAA patients managed with IST at a yearly interval [61].

Immunosuppressive therapy (IST)

Upfront MSD-HSCT is the curative treatment of choice for young (≤ 50yrs) fit SAA/VSAA patients [59, 62], while matched unrelated donor (MUD) only after failure of one course of IST (refractory AA). Alternative donor-HSCT including haploidentical or umbilical-cord blood may be considered in patients with refractory AA who lacks MSD or suitable MUD [22]. The 10-years overall survival after MSD-HSCT in the decade 2001–2010 as per EBMT varies with age as 86% (1-20years), 76% (20-40years), and 55% (> 40years) [63].

Studies comparing HSCT and immunosuppression in AA are lacking, however, two meta-analyses with 7955 and 302 patients respectively, found insufficient and biased data to clarify the comparative effectiveness of MSD-HSCT and IST in terms of OS and mortality [64, 65].

The successful outcome of HSCT pre-requisites includes patients age (strongest impact), selection of appropriate donor by HLA matching at high resolution, comorbidities assessment, lack of clonal evolution, absence of donor-directed antibodies, conditioning regimen used, source of the stem cells (PBSC or bone marrow) and the interval between diagnosis and transplant. There is an overall survival advantage of bone marrow over peripheral blood as a stem cell source in all the age groups [66]. However various studies have shown that with improved conditioning and immunosuppression, G-CSF mobilized peripheral blood stem cells could be a practical solution, compared to cumbersome bone-marrow source [74–81].

International as well as Indian data suggests that MSD-HSCT offers an excellent outcome in children. The OS after MSD-HSCT found in Indian transplant centres varies from 65 to 80%. High-risk transplant includes AA patients with either infection, > 20units transfusion, or previous exposure to IST. The survival advantage in high risk (65%) is found to be lesser than in low-risk transplant (95%) [67, 68].

Outcomes with HSCT in adults also continue to improve in the last 2 decades with the advancement in the conditioning regimens, infection prophylaxis and treatment (including fungal), immunosuppressive therapy (GVHD prevention), and other supportive care [69].

The reported incidence of acute GVHD varies from 9 to 39%, chronic GVHD from 11.5 to 47.5%, graft rejection from 5 to 32% as per various Indian transplant centres (Table 2).

Table 2.

Details of different HSCT studies conducted in India

S.no	Centre (Year) [Ref]	Donor (Stem cell source)	Sample size (n)	Median Age (Years)	Conditioning Regimen (Immuno-prophylaxis for GVHD)	Acute GVHD	Chronic GVHD	Graft Failure (%)	OS (Median follow up)
1.	CMC Vellore (2001) [73]	MRD (BM)	22 (24 transplant)	13	Cy & ATG (CsA & MTx)	17	-	31%	36% (5-years)
2.	AIIMS Delhi (2006) [74]	MRD (PB)	5	18	Flu, Cy &ATG (CsA & MTx)	-	-	-	100% (171 days)
3.	CMC Vellore (2007) [75]	MSD (BM-7 PB-28)	35	20	Flu, Cy ± ATG (CsA & MTx)	29%	32%	9.5%	82.8& (22months)
4.	CMC Vellore (2008) [76]	MRD (PB)	84 (90 transplant)	21	Flu- based (CsA & MTx)	-	-	-	-
5.	CMC Vellore (2010) [77]	MRD (BM-20 PB-10)	30	10.2 ± 3	Cy & ATG Flu & Cy ± ATG Flu & Bu ± ATG (CsA & MTx)	16.6%	16.6%	6.7%	70% (3-years)
6.	AIIMS Delhi (2012) [78]	MSD (PB)	41	22	Flu, Cy & ATG (CsA & MTx)	39%	25%	29.3%	75.6% (6.5-years)
7.	NCRI Kol (2012) [79]	MSD = 3 (PB) UCB = 2	5	19.6	Flu & Bu ± ATG (CsA & MTx)	9.1% (Gut)	18.18	32%	81.25% (4.6-years)
8.	Army hospital R and R (2013) [80]	MRD (BM-1 PB-15)	16	22 (12–46)	Flu & Bu ± ATG (CsA & MTx)	26.5%	11.5%	31.3%	68.8% (16months)
9.	Apollo Ahmedabad (2014) [81]	MRD (PBSC)	3	21	Cy & ATG (CsA & MTx)	-	-	-	66.67% (22months)
10.	AIIMS Delhi (2015) [9]	MSD (BM-5 PB-64)	69 (72 transplant)	22	Flu & Cy ± ATG (CsA & MTx)	25%	17%	24.6%	75.3% (32months)
11.	CMC Vellore (2015) [82]	MRD (BM-36 PB-178)	214	22	Cy & ATG Flu & Bu ± ATG Flu & Cy ± ATG (CsA & MTx)	38.4%	47.5%	8.8%	64.8 ± 3.3% (5-years)
12.	ISCTR (2015) [82]	MSD (BM-124 PB-510)	634	21	Cy & ATG Cy & TBI Flu & Cy ± ATG (CsA & MTx)	14.4%	41%	12.9%	66.3 + 2% (5-years)
13.	CMC Vellore (2018) [83]	MSD (PB)	30	29	Flu, Cy & TBI (CsA & MTx)	22%	22.7%	6.6%	65.9 ± 8.8% (3-years)
14.	CMC Vellore (2020) [96]	MRD (PB)	212	23	Flu, Cy (CsA & MTx PTCy in few)	27.9%	41.6%	9.4%	75.3 ± 3.0% (5-years)
15.	BLK Delhi (2020) [84]	MRD (76)	111 (BM-19 PB-89 Mixed-3)	17	Flu, Cy & ATG (CsA & MTx)	9%	21%	5%	79% (2-years)
		Haplo (35)			Flu, Cy & TBI (PTCy &Tac/MMF)		6%	21%	42% (2-years)
17.	BLK Delhi (2014) [85]	Haplo	2	8	Flu, Cy & TBI (PTCy & Tac/MMF)	-	-	-	100% (18months post-transplant)

Centres: AIIMS: All India Institute of Medical Science, New Delhi; Army Hospital R & R: Army Hospital Research and Referral, New Delhi; BLK: Dr. B L Kapur Hospital, Delhi; CMC: Christian Medical College, Vellore; ISCTR: The Indian Stem

Cell Transplant Registry, NCRI: Netaji Subhas Chandra Bose Cancer Hospital, Kolkata;

Conditioning regimens: (1) Cyclophosphamide (Cy; 200 mg/kg over 4 days) with horse anti-lymphocyte globulin ( hATG; 40 mg/kg over 3 days); (2) Fludarabine (Flu; 180 mg/m² over 6 days) with busulfan (Bu; 8 mg/kg over 2 days) and hATG (hATG; 40 mg/kg over4 days); (3) Fludarabine (Flu; 180 mg/ m² over 6 days) with cyclophosphamide (Cy; 120 mg/kg over 2 days) ± ATG (hATG; 40 mg/kg over 4 days)

Immuno-prophylaxis for GVHD: CsA + MTx: Cyclosporine (3 to 5 mg/kg twice daily) and methotrexate (10 mg/m² on days 1& 3); PTCy + Tac/MMF: post-transplant cyclophosphamide (50 mg/kg on days + 3 & +4) and tacrolimus/mycophenolate mofetil (day + 5 onward)

(ATG: Anti-thymocyte globulin, BM: bone marrow source of stem cells, GVHD: Graft versus host disease, Haplo: Haploidentical stem cell transplant HSCT: Haematopoietic stem cell transplant, MRD: matched related donor, MSD: Matched sibling donor, OS: Overall survival, PB: peripheral blood source of stem cells, TBI: Total body irradiation, UC: Umbilical cord)

Survival in MUD HSCT depends on donor factors including gender (male vs. female), HLA-matched (high resolution at the A, B, C, DRB1 loci), age ≤ 30 years, and cytomegalovirus status [70]. The survival after MUD-HSCT varies with age and found to be better (upto 85%) if performed at younger age (< 10years) [70]. There is a paucity of Indian data on MUD-HSCT.

The standard conditioning regimen for matched sibling transplants is cyclophosphamide 200 mg/kg (CY 200) and ATG [71]. The recommendations from the standard guidelines include adding FLU with CY and ATG (FCA) or alemtuzumab (FCC) for > 30years SAA patients receiving MSD-HSCT [22, 72, 75]. The standard GVHD prophylaxis includes methotrexate with CsA for MSD and high dose post-transplant cyclophosphamide (PTCy) for haplo-matched HSCT [86]. The conditioning regimen recommended for MUD-HSCT includes FCA and low-dose TBI (2-4 Gy) or FCC for haploidentical donor [61]. However due to high cost and more associated toxicities, alemtuzumab is still not being favoured in conditioning regimens in Indian centres.

Table 2 summarizes the outcome of HSCT in AA in India. There is a paucity of published Indian data on outcomes of alternative donor HSCT (MUD, Haploidentical, or Mismatch) in SAA.

HSCT is still inaccessible for the majority of eligible AA patients in a developing country like India, because of the lack of resources, expertise, and limitation of donor availability. The cost (MSD-HSCT range-15,000 to 20,000 USD) as well as time to receive HSCT (lack of donor registries), goes up significantly with consideration of unrelated donors and therefore, HSCT is not easily available to the majority of the patients in India [56, 79, 80].

Other Treatment Options

Cyclosporine (CsA) as single agent

In resource-limited countries like India, most of the AA patients are unable to receive HSCT or ATG due to both cost constraints and limited bed availability in the expertise centres. CsA as a single agent can be the treatment option for them and for those who are unfit for the HSCT/ATG therapy. Various studies conducted in India (Table 3), have shown an overall response rate of 19.6 to 56.2%, with minimal adverse effects [9, 86–89]. With the advantage of easy availability, low cost, safety profile, and monitored immunosuppressant (trough level 150-250ng/ml), CsA can be an option for SAA patients [9].

Table 3.

Response rate of cyclosporine and androgen as monotherapy and as combination in various studies of India

S No	Author (Year) [Ref]	Design	Median Age (years)	Sample size (n)	Major Adverse Effects	ORR (Follow up period)
Cyclosporine as Single-agent IST (Dose – 5 mg/kg/day in 2 divided doses)
1.	Verma S (1999) [86]	Prospective	21	15	Gum Hypertrophy	30.8% (3-months)
2.	Rai M (2001) [87]	Prospective Cyclosporine (6 mg/kg/day in divided doses)	19.4	12	Myalgia, Hypertrichosis, hyperbilirubinemia	41.6% (6-months)
3.	Mahapatra M* (2015) [9]	Prospective	25	62	Gastrointestinal upset	32.2% (6-months)
4.	Shetty M (2016) [88]	Prospective	27	16	Renal dysfunction, Gum Hypertrophy	56.2% (3-months)
5.	Mandal PK (2017) [89]	Prospective	37	57	Renal dysfunction, Deranged LFT, Gum Hypertrophy	19.6% (6-months)
Androgens as Single-agent therapy
1.	Marwaha RK (2004) [92]	Prospective (Stanozolol − 1 mg/kg/day in divided doses)	9	47	Virilization	20.4% (25 months)
Androgens and Cyclosporine as Combination Therapy
1.	Rai M (2001) [87]	Prospective Cyclosporine (6 mg/kg/day in divided doses) and Stanozolol (1 mg/kg/day in divided doses)	19.4	11	Hypertrichosis, hyperbilirubinemia	9.09% (6-months)
2. 2	Mahapatra M* (2015) [9]	Prospective Cyclosporine (5 mg/kg/day in divided doses) and Stanozolol (2 mg/kg/day in divided doses)	25	59	Gastrointestinal upset	45.6% (6-months)

* Study shows no statistical difference in response rate between cyclosporine monotherapy or combination with androgen. There was more GI upset with combination therapy

OR: Overall response rate

Androgens

The role of androgens in AA is limited, early studies failed to show any response in one trial [90] and while females in another RCT showed a superior response rate with comparable OS [91]. Synthetic androgens (anabolic steroids) namely, stanozolol (0.5 mg-2 mg/kg/day), oxymetholone (2 mg/kg/day), and danazol (600 mg-800 mg/day) increases endogenous erythropoietin production, thereby raises Hb levels [94]. A response rate of 20.4% was found with stanozolol (1 mg/kg/day) as single agent in AA [92]. When CsA was added to stanozolol (Table 3), response rate varies from 9.09 to 45.6% [87]. However. the combination therapy of androgens and CsA failed to show any statistically significant response and survival benefit when compared with single-agent CsA therapy in the Indian population [9].

Eltrombopag (EPAG)

EPAG is a non-peptide thrombopoietin receptor (TPO-R) agonist that is approved by the US Food and Drug Administration (FDA) as a single agent in refractory SAA (inadequate response to IST therapy), and as a component of triple IST in newly diagnosed SAA [93]. Its role in childhood AA is not well-defined.

EPAG acts as a bone marrow stimulating agent by acting on TPO-R expressed on hematopoietic stem cells, multipotent progenitor cells, and megakaryocytes [94]. EPAG as single-agent in moderate AA (both not previously treated or who failed prior IST), achieved a response rate of 50% [95], although there is a paucity of published Indian data on EPAG as a single agent in AA.

EPAG dose in AA is 150 mg/day orally empty stomach [93]. Because of population pharmacokinetics, a lower dose of EPAG (75 mg/day) is found to be effective in East or Southeast Asian individuals. It is generally well-tolerated with minimal adverse effects such as skin reactions or mild hepatic dysfunction [21].

Table 4 illustrates the comparison of the cost of different therapies available in India for AA.

Table 4.

Comparison of the cost between various therapies available in India

S No	Therapy	Approximate Cost in Indian Rupees (Adult weight 50 kg)	Comment
1.	MSD HSCT*	12–15 lacs	Limited availability of Transplant centre
2.	hATG (ATGAM)	7 lacs	Cost may vary from centre to centre
3.	hATG (Thymogam)	3.5 lacs
4.	rATG	6 lacs
5.	Triple IST** (hATG + CsA + EPAG)	11 lacs (ATGAM) 7.5 lacs (Thymogam)	Cost and limited expertise centre availability
6.	hATG + CsA**	7.5 lacs (ATGAM) 4 lacs (Thymogam)
7.	Cyclosporin A (Sandimmune Neoral)	7500/month	Limited response rate in SAA and VSAA
8.	Stanazolol (Neurobol)	4000/month	Not effective in SAA or VSAA
9.	Danazol (Danogen)	2000/month
10.	CsA + Androgens	10,000/month	Limited response
11.	Eltrombopag (Revolade)	60,000/month	Paucity of data

*Excluded the cost of post-transplant IST therapy

** Cost includes standard 6 months duration therapy of CsA & EPAG

CsA: Cyclosporine, EPAG: Eltrombopag; hATG: Horse anti-thymocyte globulin, rATG: Rabbit anti-thymocyte globulin, IST: Immunosuppressive therapy; MSD-HSCT: Matched sibling hematopoietic stem cell transplant, rATG: Rabbit anti-thymocyte globulin, SAA: Severe aplastic anemia. VSAA: Very severe aplastic anemia

Future Perspective

Newer agents like EPAG provides a favorable balance of outcomes and toxicity and therefore, the addition of IST or androgens and possibly other growth factors (G-CSF or erythropoietin) to EPAG may improve the marrow function and blood count recovery. In the coming years, more clinical research is required to learn and found real-world evidence to solidify the role of these promising non-transplant combination therapy in VSAA and SAA patients in India.

Summary

Acquired aplastic anemia is a rare life-threatening hematological disorder in India. The survival and outcome of SAA are still dismal, because of the limitations in the availability of the expertise centres and cost of the therapy. The standard of care includes early referral of the transplant eligible patient to an HSCT expertise centre or initiation of IST in transplant ineligible patient. Recently, triple-drug therapy-based IST is the treatment of choice for patients who are transplant-ineligible or who lacks a matched donor. The response of intensified IST is encouraging, although it is associated with relapse and clonal evolution to MDS or PNH. Single-agent CsA IST with or without androgens may be considered for patients who are unable to afford the expensive IST. The emerging non-transplant therapies may transform the management of AA in the future especially in the resource constraint countries like India.

Authors’ Contributions

Dr Tuphan Kanti Dolai- Concept, Literature search, write up, corrections.

Funding

Not Applicable.

Data Availability

Not applicable.

Compliance with Ethical Standards

Not applicable.