Introduction
Acquired aplastic anemia (AA) is an immune-mediated bone marrow failure caused by a cytotoxic T-cell attack on hematopoietic stem cells. Diagnosing AA requires ruling out congenital conditions and myelodysplasia. Treatment options include immunosuppressive therapy (IST) or allogeneic hematopoietic cell transplantation (alloHCT), chosen based on patient age, comorbidities, and donor availability. Since 2022, horse ATG (hATG) has been the preferred IST over rabbit ATG, improving treatment outcomes.
Despite advancements in IST with hATG and eltrombopag, some patients experience relapse or require delayed alloHCT. Transplantation techniques have improved, making haploidentical HCT with posttransplant cyclophosphamide a promising option. Upfront alloHCT from unrelated donors is also gaining traction. This paper explores the latest guidelines and studies on managing severe AA in pediatric and adult patients.
Pathophysiology and clonal evolution
Acquired aplastic anemia (AA) is an immune-mediated bone marrow failure where cytotoxic T-cells attack hematopoietic stem cells (HSCs). This process is driven by type I interferons, which initially activate T-helper 1 responses and later shift toward T-helper 17 and CD8+ T-cell responses in severe cases. Hepatitis-associated AA, a variant affecting 5% of patients, typically appears months after acute hepatitis and responds well to immunosuppressive therapy (IST) in those without a suitable donor. Bone marrow dysfunction, mesenchymal stromal cell dysregulation, and immune-driven gene alterations contribute to disease progression, with telomere shortening linked to poor outcomes and relapse risks.
Genetic mutations play a role in AA evolution. Paroxysmal nocturnal hemoglobinuria (PNH) clones, caused by PIGA mutations, are common in AA patients. Next-generation sequencing (NGS) has identified somatic mutations that influence disease progression. Mutations in BCOR/BCORL1 indicate a lower risk of transformation to myelodysplastic syndrome (MDS), whereas mutations in DNMT3A and ASXL1 signal a higher malignancy risk. While most AA patients have a normal bone marrow karyotype, abnormalities like trisomy 8 and del(13q) predict a better response to therapy, while monosomy 7 suggests a higher likelihood of MDS. Poor-risk mutations (ASXL1, TP53, RUNX1, DNMT3A) are associated with inferior survival, increased relapse risk, and reduced IST response.
Diagnosis
The diagnosis of AA requires differentiating it from other causes of pancytopenia and bone marrow failure (BMF) syndromes. Bone marrow (BM) histopathology should exclude conditions like hypoplastic MDS, leukemia, metastases, and fibrosis while assessing cellularity. A comprehensive workup includes cytogenetic analysis with FISH, immunophenotyping for PNH (as nearly half of AA patients have PNH clones), and screening for viral infections such as hepatitis viruses, parvovirus B19, CMV, and EBV. Exposure to radiation, chemotherapy, and toxic substances like benzene, along with certain medications, can also lead to BMF and should be ruled out.
Inherited BMF syndromes are more common in children and young adults and may result from mutations affecting DNA repair, telomere maintenance, or hematopoiesis regulation (e.g., Fanconi anemia, dyskeratosis congenita, GATA2 deficiency). NGS-based genomic screening is recommended, especially for atypical cases. Hypocellular MDS can mimic AA and complicate diagnosis, with genetic studies identifying inherited BMF syndromes in about 6.6% of clinically diagnosed AA cases. Diagnostic guidelines from EBMT and North American Pediatric Aplastic Anemia Consortium emphasize laboratory tests, BM karyotyping, FISH, PNH clone detection, and telomere length assessment. NGS panels for somatic mutations (PIGA, DNMT3A, ASXL1, TP53, etc.) are being explored for future clinical use. Given the increasing understanding of monogenic defects in cytopenias, genetic testing should be considered in suspected cases.
History of AA therapy
Initially, AA treatment relied on supportive care, steroids, and vitamins. In the 1960s, androgen therapy emerged but had significant side effects without improving mortality. Though less favored today, it remains an option for transplant-ineligible patients.
In the 1970s, alloHCT became the curative standard, especially for children with matched sibling donors (MSDs) and young adults. Matched unrelated donors (MUDs) are increasingly used, though the process takes 2-3 months.
The 1980s introduced immunosuppressive therapy (IST) after observing remission in patients with alloHCT rejection. Antithymocyte globulin (ATG), which depletes T-cells, became a key IST component. Since 2022, horse ATG (hATG) has been reintroduced in Europe, warranting updated immunosuppressive strategies.
Eltrombopag (ELT), a thrombopoietin receptor agonist, was later added to IST. It enhances blood counts in nearly half of IST-refractory patients by stimulating marrow progenitor cells. Studies showed ELT combined with hATG and CsA improved response rates to 74% at 3 months and 80% at 6 months.
Current SAA treatment guidelines are provided by EWOG-MDS/SAA (2023) for children and EBMT for adults, with additional UK national guidelines available.
Nontransplant therapeutic options in AA
Evaluation of pediatric recommendations
IST with hATG and CsA has improved 5-year OS in children under 16 from 50% (pre-1990) to ~80%. A Polish study reported 78% 10-year OS, while a German study found better CR (68% vs 45%) and survival (93% vs 81%) in vSAA vs SAA cases.
hATG was widely used but withdrawn in 2007, leading to rATG use, which showed lower efficacy (e.g., 3-year OS: 76% vs 96%). Meta-analyses confirmed better ORR with hATG. pATG showed similar results to rATG in some studies.
Factors improving IST response include disease severity, younger age, higher pretreatment counts, and male sex. G-CSF may reduce infections but has no survival benefits and potential long-term risks.
If hATG is unavailable, rATG is an alternative. Non-responders or relapsed cases may need alloHCT or second-line IST with serotherapy switch, but success rates vary (Fig 1)
Figure 1. Therapeutic algorithm in children with severe aplastic anemia.
Evaluation of adult recommendations
BMT Recommendations:Patients under 40 should undergo alloHCT from MSD. Those above 40 or without an MSD are candidates for IST. The best outcomes are achieved with hATG + CsA + ELT. The current treatment schedule for patients with SAA based on EBMT recommendations and data from the available literature is shown in Figure 2.
Figure 2. Therapeutic algorithm in adults with severe aplastic anemia.
hATG Dosage & Efficacy: The standard hATG dose is 160 mg/kg (40 mg/kg for 4 days). Studies show hATG + CsA leads to 60-65% response, with 10% achieving CR. hATG is superior to rATG, with a higher ORR and OS.
Safety & Adverse Events:Common side effects include skin reactions, fever, headache, and chills. Rare complications like anaphylaxis and cytokine release syndrome can occur. Preventive measures include steroids, antihistamines, and central line infusion.
Response Evaluation: Best response is assessed 4-6 months after treatment. CR is defined as ANC >1 × 10⁹/L, Hb >10 g/dL, and Platelets >100 × 10⁹/L. Most patients achieve PR, gaining transfusion independence.
Relapse & Second-line Treatment:Relapse occurs in 10-30% of cases. Options include alloHCT or repeat IST, using an alternative ATG, alemtuzumab, or TPO-RA, though stem cell reserve limitations may impact efficacy.
Thrombopoietin-mimetic therapy and immunosuppression
ELT in Adult SAA Patients:
ELT is effective in AA patients refractory to IST, with Desmond et al. reporting 40% long-lasting responses. The RACE trial (EBMT-SAA-WP) compared hATG + CsA vs. hATG + CsA + ELT in 197 patients (median age 53). ELT (150 mg daily) significantly improved CR at 3 months (22% vs 10%) and ORR at 6 months (68% vs 41%). ELT accelerated responses without added toxicity, and 2-year OS was comparable in both groups.
ELT in Pediatric SAA Patients:
ELT’s role in pediatric SAA remains controversial. In the Groarke et al. analysis, ELT showed no significant ORR/CR improvement. However, Fang et al. reported higher CR (50% vs 17.9%) and ORR (94.4% vs 69.2%) at 6 months. Goronkova et al. found a higher CR rate (31% vs 12%) with ELT but similar ORR. ELT was beneficial in SAA (ORR 89% vs 57%) but not vSAA (52% vs 50%). Due to insufficient data, EWOG does not recommend ELT for pediatric SAA.
TPO-RA in SAA Treatment:
Other TPO-RA, like Romiplostim, are under investigation. A phase 2 trial in IST-refractory SAA showed 30% platelet response at 2-3 years with a 10 μg/kg weekly dose. A phase 2/3 study titrating 10-20 μg/kg weekly found 84% response at 27 weeks. Avatrombopag, an oral second-gen TPO-RA, is being tested in SAA patients (treatment-naïve or refractory) at 60 mg/day for 180 days with or without IST in an ongoing phase 2 trial.
Other nontransplant options in AA
Androgens have been used in AA for decades. Their mechanism of action in BMF is complex. Androgens induce erythropoiesis by increasing erythropoietin secretion and response to erythropoietin; they also stimulate the expansion of hematopoietic progenitors. Androgens also act by ameliorating telomerase activity in HSCs, resulting the in prevention of telomere attrition both in acquired SAA and in constitutional BMF syndromes. Some androgens have additional immunosuppressive mechanisms. For example, danazol inhibits interleukin-1 and TNF-α secretion by human monocytes in a dose-dependent manner. Several studies, including one randomized trial, showed the benefit of adding androgens to IST. Combined therapy with an immunosuppressive agent, ELT, and androgens in refractory SAA was also reported, with an ORR of 42%. The recommended doses for androgens in SAA are 2.5 mg/kg/d for oxymetholone and methenolone and 1 mg/kg/d for methandrostenolone and norethandrolone.
Alemtuzumab (a humanized monoclonal antibody directed against CD52 protein) is one of the lymphocytotoxic serotherapeutic options for the treatment of autoimmune diseases. However, in SAA the best results for alemtuzumab were obtained in the relapse and refractory settings, with an ORR of 56% and a 3-year OS of 86%
Hematopoietic cell transplantation
Challenges in pediatric HCT
AlloHCT in AA:
AlloHCT in AA has faced challenges, notably graft rejection due to T-lymphocytes and high HLA sensitization in heavily transfused patients. Nonmyeloablative immunosuppressive protocols with hATG and cyclophosphamide improved outcomes, particularly with MSD transplants. Similarly, rATG-based therapy with cyclophosphamide showed high engraftment rates but risk of fungal infections. Advances in donor typing, less toxic conditioning regimens, and improved supportive care have reduced mortality.
Pediatric AA and MUD-HCT:
In pediatric SAA, MUD-HCT as upfront therapy is controversial. Some studies suggest better OS with upfront MUD-HCT over IST, with similar outcomes to MSD-HCT. A randomized trial by the North American Pediatric Aplastic Anemia Consortium and Pediatric Transplantation and Cellular Therapy Consortium showed promising results for MUD-HCT as an upfront therapy with 18-month follow-up.
Challenges in adult HCT
Transplantation Options in Adult AA Patients:
In adult AA, alloHCT can be either upfront or delayed after failed IST. Over the last decade, alloHCT outcomes have improved with OS rates of 70%-90%. However, some patients still face relapse, clonal evolution, or aplastic relapse. Chromosomal aberrations, such as chromosome 7 defects, and the risk of MDS or AML progression persist, especially 3-6 months post-IST. The decision to proceed with alloHCT should weigh the benefits of IST vs. alloHCT.
Factors Influencing IST Response & Biomarkers:
Baseline absolute lymphocyte count, reticulocyte count, and mutations like PIGA, BCOR, and BCORL help predict IST response. Negative markers include undetectable PNH clones, short telomere length, and adverse mutations (ASXL1, DNMT3A, RUNX1, TP53), which also increase clonal evolution and relapse risk.
Risk Factors & Treatment Pathway:
The choice between IST and alloHCT depends on age, comorbidities, donor availability, AA severity, and access to agents like hATG and ELT. Risk factors for alloHCT include PB as the cell source, age >20 years, delayed transplant (>6 months), and CMV mismatch. Fludarabine-based regimens showed better outcomes for patients under 20 but are less effective for older patients, especially those with comorbidities or a high HCT-CI score.
Age & Response to IST:
For patients <40 years without comorbidities, MSD-alloHCT is optimal, while older patients should generally opt for IST due to higher mortality. However, Flu-based regimens show no significant OS difference across age groups, though the risk of GVHD is higher in the elderly. Upfront alloHCT may be suitable for SAA or vSAA patients with poor IST response or high clonal evolution risk.MUD-HCT Outcomes:
MUD-alloHCT has shown improved outcomes in the past decade, attributed to better donor typing, supportive care, and conditioning protocols. MUD-HCT may be considered for young patients (<20) with urgent indications, while older patients (>40 years) may face lower OS and higher transplant-related mortality.
Pretransplant Management
Before alloHCT, patients should undergo an HCT-CI score assessment, review of past and current infections (including tuberculosis, viral, and fungal), and laboratory screening for latent infections. Blood products must be irradiated and leukodepleted to prevent alloimmunization. For patients with transfusion burden, serum ferritin levels should be monitored, and iron chelation therapy may be needed if ferritin >1000 ng/ml to reduce organ toxicity, infections, and graft failure.
Patients should discuss fertility preservation if a gonadotoxic conditioning regimen is planned. The risk of infertility depends on age and cumulative exposure to gonadotoxic agents. Irreversible infertility is highly likely with cyclophosphamide equivalent dose (CED) >4000 mg/m² for men or >6000 mg/m² for women.
Conditioning regimens
Over the last 40 years, the conditioning regimen for pediatric SAA has evolved, with irradiation omission improving outcomes. The addition of Flu to MUD-HCT regimens in the last two decades has also led to better results. Reducing conditioning intensity improves survival but GVHD-free/relapse-free survival (GRFS) may not exceed 80%. This is especially important in children due to long life expectancy and potential GVHD and steroid-related complications.
Flu-Cy-ATG greatly improved MUD-HCT outcomes for children. A study showed a 5-year overall survival (OS) of 82% for MRD-HCT, compared to 69% for MUD-HCT. In 2023, Flu/Cy chemotherapy became standard for pediatric SAA, with lower Cy dosing improving survival. Low-dose Cy (60 mg/kg) may increase graft rejection risk.
Studies show favorable outcomes with Flu-Cy-ATG conditioning, and acute GVHD rates are manageable. 5-year OS for children post-2017 has increased to 97%. Graft rejection remains a risk with certain protocols. Flu/Cy-based regimens are recommended for young adults and those at risk for cardiotoxicity. For haploidentical transplants, higher TBI doses (4-6 Gy) may help ensure engraftment.
Transplantation techniques
The choice of transplant material in alloHCT for SAA is debated, influenced by resource availability. Bone marrow (BM) is preferred for its higher survival rates and lower GVHD incidence, while peripheral blood stem cells (PBSC) have been associated with higher chronic GVHD. A study by Bacigalupo et al. found BM superior to PBSC across all age groups. However, PBSC is more popular for technical reasons and is considered a viable alternative in countries with limited resources. For GVHD prevention, ex-vivo T-cell depletion of PBSC shows promise but requires specialized facilities, while posttransplant Cy (PTCy) is emerging as an effective option for GVHD prophylaxis. In adults, BM is recommended for MSD and MUD settings, but PB may be used with alemtuzumab. In haploHCT, BM or G-CSF-mobilized PB is often preferred, though no clear recommendations exist. Syngeneic HCT offers excellent long-term outcomes, with PB as the preferred source.
Serotherapy in HCT
Serotherapy, including rATG, hATG, or alemtuzumab, is used in conditioning regimens for SAA and has been shown to offer a survival advantage. rATG, in particular, has been linked to a reduced risk of chronic graft-versus-host disease (cGVHD), though it also increases the risk of opportunistic infections. A multicenter study revealed that conditioning with Cy alone or Cy/ATG resulted in different outcomes for cGVHD and overall survival (OS). For instance, the cGVHD incidence was 21% with Cy alone and 32% with Cy/ATG, while OS was 74% and 80%, respectively.
In comparison, rATG showed a lower risk of acute GVHD (aGVHD) but no difference in cGVHD rates compared to hATG. Further studies found that rATG was protective against both aGVHD and cGVHD, although it resulted in a higher incidence of fungal infections and mixed chimerism. Regarding recommended doses, rATG for children undergoing HCT typically ranges from 40 to 60 mg/kg for Grafalon and 8 to 10 mg/kg for Thymoglobulin.
Alemtuzumab in SAA
To reduce GVHD, alemtuzumab is sometimes used as an alternative to ATG. A study examining the use of Flu, Cy, and alemtuzumab in SAA patients showed promising results with only 13.5% of patients developing aGVHD and a minimal incidence of cGVHD. The optimal dose of alemtuzumab ranges from 40 to 100 mg, administered over five consecutive days. Despite concerns about CD52 expression on lymphocytes in patients with paroxysmal nocturnal hemoglobinuria (PNH), there have been no significant reports of graft rejection or failure with alemtuzumab-containing regimens.
Haploidentical HCT
Over the past two decades, haploidentical HCT (haploHCT) has gained attention for its potential in SAA. The conditioning regimen, which includes cyclophosphamide (Cy), has been associated with cardiotoxicity risks, leading to alternative protocols for high-risk patients. Early reports of haploHCT showed promising results, with a 2-year OS rate of 78%. A reduced-dose PTCy approach was used in studies to reduce GVHD, resulting in a low incidence of aGVHD and cGVHD.
Several studies on haploidentical donors with reduced-intensity conditioning showed favorable outcomes, with a high OS and low incidence of GVHD. However, variations exist between protocols across different regions. For instance, Chinese protocols with more intensive chemotherapy resulted in excellent transplant outcomes in pediatric SAA patients. While haploidentical donors show comparable results to matched unrelated donors (MUDs), they are often considered when MUDs are unavailable or after the failure of immunosuppressive therapy (IST).
Cord Blood Transplantations
The role of cord blood (CB) in SAA treatment remains uncertain, with a decline in CB transplants in recent years. In a Chinese study, patients receiving haploidentical CB showed similar OS rates to those receiving haploidentical bone marrow (Haplo) HCT. However, CB was associated with inferior engraftment and a higher graft failure (GF) rate. In pediatric cases, some studies reported success with CB transplants, showing good survival rates but delayed engraftment. Though promising results were noted in some cases, the use of CB is limited by lower graft cellularity, and it is generally not recommended for adult patients due to slower engraftment times.
In adult studies, cord blood transplantations have demonstrated an OS of 88% with low incidences of aGVHD and cGVHD. Protocols like APCORD, which combine Flu, Cy, ATG, and low-dose TBI, have shown good results, indicating that CB might still be a viable option in specific cases, though it is often reserved for situations where other donor options are not available.
Posttransplant Management and Sequelae
The main advantages of MUD-HCT include quick neutrophil engraftment and high survival rates. However, they are offset by the delays in therapy due to the time required for donor searches and graft arrangements, which can take up to 4 months. During this waiting period, pharmacotherapy involving IST can be considered, sometimes combined with additional treatments like TPO-RA or androgens. Post-HCT complications include organ toxicities, infections, graft failure (GF), acute and chronic GVHD, infertility, and secondary cancers, necessitating careful evaluation before referring for HCT.
The greatest impact on quality of life post-transplant is often from GVHD, while the success of alloHCT is best measured through GFRS. A significant concern after alloHCT for AA is the risk of GF, which emphasizes the importance of personalized posttransplant IST to improve outcomes.
In children, GVHD prophylaxis typically includes MTX and CsA, as this combination has been shown to be superior to CsA alone. In adult patients, calcineurin inhibitors (CNI) along with short courses of MTX are standard in the MSD and MUD platforms, while protocols with alemtuzumab focus on CNI-based IST. In haploHCT, protocols involving PTCy ± rATG with MMF and tacrolimus, or the Chinese protocol, are used. Chimerism monitoring at various stages post-transplant is essential for tracking engraftment and predicting GF.
If graft failure occurs, second transplants can be highly successful, with excellent outcomes even after retransplanting from the original donor. The long-term survival rate for children post-HCT is very high, with probabilities of 96% at 5 years and 94% at 10 years. However, late effects such as gonadal dysfunction and growth disturbances become more apparent over time, especially with MUD-HCT.
The risk of subsequent malignancies is a concern, with an incidence of 1.1% among SAA patients. Improved diagnostics and genetic testing have reduced misdiagnosis of clonal disorders as SAA, which may help lower the incidence of myeloid malignancies post-HCT. Long-term follow-up is essential to detect malignancies, as many arise more than 5 years after transplantation.
Conclusions
Treatment outcomes for AA patients are often derived from retrospective studies. Key recommendations include testing for constitutional defects, pretransplant care such as transfusions and anti-infective screening, and upfront alloHCT for children and young adults. Elderly patients or those without an MSD should undergo IST without delay. In adults, adding ELT to IST is recommended. MUD-HCT can be considered if donor searches are completed within 2 months.
Bone marrow is the preferred source of stem cells, and conditioning regimens should be adapted for age and specific therapies. GVHD prophylaxis typically includes CNIs and MTX or CNI alone with alemtuzumab. HaploHCT and CBT are generally not first-line therapies unless as a salvage option after IST failure. Fertility preservation and long-term surveillance are crucial for patients undergoing alloHCT.
References
1. Young NS. Aplastic anemia. N Engl J Med. (2018) 379:1643–56.
2. Peslak SA, Olson T, Babushok DV. Diagnosis and treatment of aplastic anemia. Curr Treat Options Oncol. (2017) 18:70.
3. Camitta BM, Rappeport JM, Parkman R, Nathan DG. Selection of patients for bone marrow transplantation in severe aplastic anemia. Blood. (1975) 45:355–63.
4. Giudice V, Selleri C. Aplastic anemia: pathophysiology. Semin Hematol. (2022) 59:13–20.
5. Locasciulli A, Bacigalupo A, Bruno B, Montante B, Marsh J, Tichelli A, et al. Hepatitis-associated aplastic anaemia: epidemiology and treatment results obtained in Europe. A report of The EBMT aplastic anaemia working party. Br J Haematol. (2010) 149:890–5.
6. Osugi Y, Yagasaki H, Sako M, Kosaka Y, Taga T, Ito T, et al. Antithymocyte globulin and cyclosporine for treatment of 44 children with hepatitis associated aplastic anemia. Haematologica. (2007) 92:1687–90.
7. Rauff B, Idrees M, Shah SA, Butt S, Butt AM, Ali L, et al. Hepatitis associated aplastic anemia: a review. Virol J. (2011) 8:87.
8. Zhang X, Yang W, Yang D, Wei J, Zhang P, Feng S, et al. Comparison of hematopoietic stem cell transplantation and immunosuppressive therapy as the first-line treatment option for patients with severe hepatitis-associated aplastic anemia. Front Immunol.
9. Atmar K, Tulling AJ, Lankester AC, Bartels M, Smiers FJ, van der Burg M, et al. Functional and immune modulatory characteristics of bone marrow mesenchymal stromal cells in patients with aplastic anemia: A systematic review. Front Immunol. (2022) 13:859668.
10. Pagliuca S, Gurnari C, Hercus C, Hergalant S, Nadarajah N, Wahida A, et al. Molecular landscape of immune pressure and escape in aplastic anemia. Leukemia. (2023) 37:202–11.
11. Ball SE, Gibson FM, Rizzo S, Tooze JA, Marsh JC, Gordon-Smith EC. Progressive telomere shortening in aplastic anemia. Blood. (1998) 91:3582–92.
12. Barade A, Aboobacker F, Korula A, Lakshmi K, Devasia A, Abraham A, et al. Impact of donor telomere length on survival in patients undergoing matched sibling donor transplantation for aplastic anaemia. Br J Haematol. (2022) 196:724–34.
13. Brummendorf TH, Maciejewski JP, Mak J, Young NS, Lansdorp PM. Telomere length in leukocyte subpopulations of patients with aplastic anemia. Blood. (2001) 97:895–900.
14. Scheinberg P, Cooper JN, Sloand EM, Wu CO, Calado RT, Young NS. Association of telomere length of peripheral blood leukocytes with hematopoietic relapse, Malignant transformation, and survival in severe aplastic anemia. JAMA. (2010) 304:1358–64.
15. Calado RT, Cooper JN, Padilla-Nash HM, Sloand EM, Wu CO, Scheinberg P, et al. Short telomeres result in chromosomal instability in hematopoietic cells and precede Malignant evolution in human aplastic anemia. Leukemia. (2012) 26:700–7.
