Immune-mediated cytopenias (IMCs) after HSCT for pediatric non-malignant disorders: epidemiology, risk factors, pathogenesis, and treatment

Hematopoietic stem cell transplantation (HSCT) represents the main therapeutic option for several non-malignant disorders, including bone marrow failure syndromes, primary immunodeficiencies, and inborn errors of metabolism. The delicate immune balance characterizing immune reconstitution post-HSCT [1] could be disrupted by the development of immune-mediated cytopenias (IMCs) [2]. This is particularly evident in pediatric patients transplanted for non-malignant diseases, in whom IMCs represent an important cause of morbidity and mortality post-HSCT [3]. However, the increasing awareness, prompt recognition, and a broader range of therapeutic approaches have led to improving survival in this cohort of patients [3]. We reviewed current evidence in epidemiology, risk factors, pathogenesis, and available treatment options for these complications with the aim to exploit a better understanding of these potentially life-threatening complications for a more accurate clinical management and better outcomes.

IMCs occurring post-HSCT in patients affected by non-malignant diseases have a reported incidence that varies widely through literature: from a 1.5 to 22% of pediatric reports [4‐17]. IMCs may manifest as hemolytic anemia, thrombocytopenia, and neutropenia, alone or in combination (e.g., Evans syndrome, which results from the association of immune hemolytic anemia and thrombocytopenia or neutropenia). The median reported time to develop IMCs is approximately 2 to 40 months post-HSCT, regardless of the ongoing immunosuppression for prophylaxis or treatment of graft-versus-host disease (GvHD) [4‐10, 18, 19]. Hemolytic anemia remains the most frequent form of IMC [2, 6, 8‐10, 17, 18, 20, 21]. Immune thrombocytopenia has been reported in 0.5–2% of pediatric HSCT patients [3, 6, 14]. Immune neutropenia is rare, and no data about its incidence is available to date [22]. Risk factors related to IMCs post-HSCT might be classified in patient-specific, donor–recipient HLA matching-related, and transplant-associated [3] (see Table 1). For the first group, patient age and the underlying disease appeared to have the utmost influence on the development of IMCs [3]. Particularly, a greater incidence of IMCs has been highlighted in several reports among younger patients [2, 5‐10, 17‐19, 23] and in patients transplanted for non-malignant diseases (especially primary immunodeficiencies and inborn errors of metabolism) [4, 6, 9, 10, 20, 23, 24]. It could be stated that being transplanted for inherited disorders represents per se a risk factor for developing IMCs post-HSCT [23]. Interestingly, a recently published retrospective single-center study found pediatric patients transplanted for hemoglobinopathies as having an increased 1-year cumulative incidence of IMCs [25]. This suggests that previous alloimmunization consequent to multiple transfusions might somehow contribute to the development of these complications [26], even if it is difficult to demonstrate its pathogenicity, because of the multiple concurrent factors increasing the risk in this cohort of patients. Moreover, Szanto et al. demonstrated that chemo-naivety presented a stronger relationship (P = 0.004) than “non-malignant indication” (P = 0.02), highlighting that the chemo itself prevents the development of IMCs, perhaps because chemotherapy removes the immune cells involved in IMCs development, prior to HSCT [15]. From a donor–recipient compatibility standpoint, the choice of unrelated donors, particularly if mismatched, and haploidentical ones have been related to the development of these complications [4, 6, 13, 27]. Finally, the HSCT procedure itself and its following complications might increase the risk of immune cytopenias [2]. Indeed, the use of lymphodepleting agents to prevent GvHD during the conditioning regimen has been reported in several pediatric reports as a crucial risk factor (alemtuzumab > ATG) [2, 9, 13, 15, 26]. Besides, the conditioning choice may increase the risk for IMCs: this has been demonstrated in patients affected by severe aplastic anemia, in whom the use of reduced intensity conditioning was associated with the development of post-HSCT immune complications [14, 26]. Indeed, it has been postulated that the development of mixed chimerism (that often follows a reducedintensity conditioning regimen and is more tolerated, as well as found, after HSCT for a non-malignant indication) could further increase the risk of IMCs after HSCT, even if this point is still a matter of great debate (9,12,15). Some pediatric reports are identified as a risk factor even the source of stem cells (UCB > PBSC > BM), but multivariate analysis did not confirm the findings [6, 14, 15, 17‐19, 26, 28]. Likewise, the development of GvHD seemed to increase the risk of immune complications, but only a single-center retrospective study was able to classify aGvHD grades II–IV as an independent risk factor for IMCs in the pediatric population [15]. Interestingly, this study was also able to underline that pediatric patients treated with serotherapy, chemo-naïve prior to HSCT, and who developed aGVHD grades II–IV before IMC development had a 22% chance to develop immune cytopenias post-HSCT [15]. Furthermore, chronic GvHD has been reported as one of the key risk factors for developing IMCs, even for pediatric patients [2]. Finally, infectious complications, particularly viral reactivations, could act as a trigger for the development of IMC post-HSCT during pediatric age [2, 26]. This has been highlighted especially for CMV [9], which has been recognized as one of the primary risk factors.

The mechanisms behind the development of IMCs post-HSCT are not completely understood (see Fig. 1). This is because of the unique picture characterizing the post-transplantation period, in which a delicate balance between the reconstituting donor immune system and the depleted but still present recipient immune system is crucial for avoiding immune complications [1]. In this scenario, it is of paramount importance a prompt reconstitution and appropriate functioning of T regulatory cells (Tregs) which suppress autoreactive T and B cells, ultimately preventing their expansion which leads to consequent aberrant immune responses [2, 3]. Indeed, a paucity of Tregs is characteristic of younger patients in whom the thymus is still under development [2, 3, 7, 17] and, broadly, of the early post-HSCT setting (because the thymus is damaged by the conditioning regimen) or late post-HSCT setting (in which additional complications, such as GvHD and its related treatments, could further damage the thymus) or, finally, of the T-deplete haploidentical setting [29]. Similarly, patients with IMCs showed inadequate Tregs’ capabilities in controlling T helper cell type 2 (Th2) activation, which ultimately resulted in Th2-driven autoimmunity [9]. Besides Tregs/Th2 imbalance, pediatric patients presenting IMCs showed also aberrant immune reconstitution profiles, with a trend toward lower NK, CD3 + CD8 + cells, and higher values of IgM at the time of IMC onset [15]. Taken all together, these data confirm that both T cell and B cell dysfunction lay the foundation for the development of immune cytopenias, and it is quite challenging to distinguish auto- from allo-immunity in this context. Thus, the definition of “immune cytopenias” better fits these complications. Besides the proposed mechanisms, the donor could cause the transfer of autoantibodies and autoreactive B and T cells, or both, into the recipient, triggering the development of IMCs [22]. Furthermore, taking advantage of lessons learned from patients affected by inborn errors of immunity, in whom several immune mechanisms breaking immune tolerance have been proposed [30], we could also speculate that other pathomechanisms could be implied in autoreactive B and T cells expansion in patients with IMCs post-HSCT. Indeed, from a T cell standpoint, aberrant T cell receptor signaling could compromise central tolerance by affecting negative selection of autoreactive T cells, while defective lymphocyte apoptosis could lead to a failure in controlling lymphocyte expansion in the context of an adaptive immune response [30]. On the other hand, the central B cell tolerance, which is deployed within the bone marrow and is achieved through several mechanisms (e.g., anergy, clonal ignorance, clonal deletion, and receptor revision), could be incomplete leading to a considerable proportion of autoreactive B cells escaping the bone marrow [30]. Considering the limited information on these variables, further studies are needed for a thorough exploration of the interconnection of T cell- and B cell-mediated pathomechanisms.

In this context, it has to be considered that all the factors which influence the speed and quality of donor immune reconstitution may lead toward IMCs’ development: the underlying disease, the use of lymphodepleting agents (ATG or alemtuzumab), the source of the stem cells, the choice of a mismatched donor, the development of GvHD, and the administration of calcineurin inhibitors to prevent GvHD or infectious complications [1, 31]. This reflects the known reported data about the relationship between the cited variables and IMCs’ development.

Finally, we could speculate that all the cited factors are interrelated in children affected by non-malignant disorders as compared to those with hematological malignancies who undergo HSCT. As explained above, these patients are relatively younger (with a still immature and under development thymus), and they also have an underlying immune system that is presumably intact because of no prior exposure to chemotherapy. In some of them presenting with metabolic diseases, even lysosomal glycosaminoglycan accumulation could be implicated in innate and adaptive immune system dysfunction, while others with hemoglobinopathies (especially those affected by thalassemia) could present an alloimmunization consequent to multiple transfusions which could eventually hamper post-HSCT immune dysregulation.

IMCs could be related to several causes when they occur in the post-HSCT setting. Hence, important SCT-related complications which usually present with overlapping features should be ruled out before considering IMCs: graft-versus-host disease, hemolytic anemia owing to ABO blood group incompatibility, transplantation-associated thrombotic microangiopathy, graft failure, disease relapse, active infections, side effects of drugs, and disseminated intravascular coagulation.

A stepwise diagnostic approach is recommended [22], starting from a proper review of the transplantation procedure itself (evaluating patient-specific, donor–recipient HLA matching-related and transplant-associated risk factors).

First-line diagnostic tests include differential blood count with reticulocytes and a blood smear, chemistry including creatinine and hemolytic parameters (i.e., direct and indirect bilirubin, lactate dehydrogenase, and haptoglobin), and urinalysis. It is worth remarking that very young infants could falsely show decreased haptoglobin because they often have poor synthesis capabilities for this protein [3].

In case of hemolytic anemia, a direct antiglobulin test (DAT) is mandatory, and it is often positive in patients with post-HSCT autoimmune hemolytic anemia, even if a negative DAT does not rule out the condition [32]. DAT usually reveals complement (C3) and/or immunoglobulin (IgM or IgG) antibodies on the surface of red blood cells. Indirect Coombs testing may also yield the presence of serum antibodies.

In case of immune thrombocytopenia and neutropenia, anti-human platelet antigen or anti-human neutrophil antigen antibodies may be present on testing, but their presence does not exclude other causes of these cytopenias, and their absence does not exclude a diagnosis of an immune-mediated destructive process [15]. Indeed, the relatively low sensitivity of anti-platelet and anti-neutrophil antibody testing compared to that for immune hemolytic anemia adds another hurdle to diagnose multiple lineage cytopenias or isolated immune thrombocytopenia and neutropenia. For both platelet and neutrophil antibody testing, the earlier techniques, ELISA-based, have low specificity and sensitivity (60–70%) [23]. The more recent assays, often flow cytometric immunobead assays, are only marginally better, reaching a sensitivity and specificity of about 80% [33, 34]. For these reasons, the diagnostic criteria for immune thrombocytopenia and neutropenia could not be routinely and exclusively based on the presence of antibodies alone. Further, in case of thrombocytopenia, a pattern of transient response or refractoriness to platelet transfusions could help corroborate diagnosis, while a trial with granulocyte colony-stimulating factor (G-CSF) administration could help confirm immune neutropenia (poor or no response is expected) [22]. A bone marrow aspirate and biopsy should be considered to evaluate chimerism and confirm normal megakaryopoiesis in case of immune thrombocytopenia, while islands of neutrophil precursors and occasionally clear evidence of myeloid maturation arrest are seen in patients presenting immune neutropenia.

There are no guidelines for IMCs’ treatment post-HSCT; therefore, therapy is usually performed following the treatment of immune cytopenias presenting in non-transplanted patients. However, children undergoing HSCT who develop IMCs post-HSCT usually present refractory forms of cytopenias, and second- or third-line therapies are often required.

The greatest challenge is to make conclusions from currently available reports (see Table 2 for a summary of the most relevant cited studies and Fig. 2 for a compendium of therapeutic options). Indeed, most of the available treatments have been reported in single case reports or very small case series, so no evidence supports their administration in treating patients with IMCs post-HSCT. Further studies, possibly perspective or even better randomized controlled trials, are needed to address this question. Moreover, preclinical studies should be devoted to correctly assessing the pathogenesis of IMCs arising post-HSCT, with the aim of developing tailored approaches in this unique cohort of patients. The aim of clinicians should be to enroll refractory patients in designed clinical trials aiming to search for the best therapeutic options in a controlled setting.

Notwithstanding the difficulties in drawing conclusions from the available data, the authors suggest that some unique patient subgroups (e.g., those expected as refractory to standard therapies) could benefit from a prompt second- or even third-line treatment based on the newest drugs, even if their potential effectiveness deserves to be further assessed, so far. Indeed, patients presenting mixed chimerism should receive DLI or a second HSCT, either if affected by hemolytic anemia or immune thrombocytopenia. For young patients with hemolytic anemia after HCT and concomitant GvHD, a good choice could be to administer drugs that could indirectly help manage the GvHD itself: ibrutinib or bortezomib could be the best option, from this standpoint. Ultimately, for patients with immune thrombocytopenia and GvHD, low-dose IL2 or fostamatinib could represent a valid therapeutic approach. A careful risk/benefit balance should always guide the clinician’s decision.

IMCs arising in the post-HSCT setting represent a rare but potentially life-threatening complication. It is widely reported that younger age at transplantation carries a higher risk of IMCs post-HSCT, especially considering young patients transplanted for non-malignant disorders. Moreover, as compared to adults, children present unique risk factors which could be interpreted as the result of the complex relationship between the immaturity of their infantile immune system and all the perturbing agents and factors which characterize the post-HSCT period. Corticosteroids, intravenous immunoglobulin, and rituximab represent the undiscussed first-line therapeutic approach, whereas future efforts should be directed to establish the best option for refractory patients, for whom a standard and validated approach is not currently available. Notwithstanding this uncertain scenario, the last decade has witnessed an improvement in the management of these challenging immune diseases, but further studies are warranted to offer a better outcome for these fragile patients.