Although an increasing number of studies are reporting that unmanipulated haplo-HSCTs with PTCY are effective, the effects of conditioning regimen , stem cell source  and donor characteristics [5, 13] on the outcome remain unclear. An important point of contention is regarding the timing of CNI administration (pre- or post-CY) in terms of efficacy and complications [2, 5, 6]. Since factors such as conditioning regimen, stem cell source, donor gender, stem cell count, and duration of immunosuppression affect haplo-HSCTs, the haplo-HSCTs vary between patients, and more importantly, between centers, and thus, it is difficult to draw general conclusions. Our single-center study of 49 patients with leukemia will therefore be a useful standard to compare the results of large multi-center studies, such as the one by Ruggeri et al. , which included 509 patients with leukemia from 34 centers. Our results show that pre-CY CNI administration could potentially benefit cases of relapse in acute leukemias and a noninferiority to post-CY application in survival outcomes, similar to Ruggeri et al. In this study, we found that pre-CY application provides an advantage for patients with CRS.
Although post-CY setting had a positive effect on OS in the higher risk groups (CR3 or refractory), this effect is lost in EFS due to increased relapse frequency. The slightly negative effect on OS in the pre-CY group is possibly associated with increased infections. As the increased frequency of viremia in haplo-HSCTs is a known problem [5, 14], the increase in the pre-Cy group was higher than that in the post-CY group. The fact that early CNI initiation in the pre-CY group prevents T cell proliferation earlier and consequently for a longer time, possibly increases the non-relapse mortality.
CRS development is an expected complication in unmanipulated haplo-HSCTs [15, 16]. Although it mostly occurs in cases where the stem cell source is peripheral stem cells, it can also occur in transplants where the stem cell source is bone marrow. As CRS diminishes shortly after CY administration, proliferating lymphocytes may be responsible for this phenomenon, although this has not been proven yet. In our study, this argument is also supported by the fact that the frequency of CRS in the pre-CY group was much lower and less problematic, possibly because of the direct effect of CNIs in preventing rapid proliferation of alloreactive T-cells. In our study, almost all of the patients in the post-CY group developed CRS where immunosuppressives did not start early, and they required dexamethasone because of the refractory fever, but almost no patient required dexamethasone in the pre-CY group due to the fact that it only caused a slight non-refractory fever, suggesting that the CRS development was not related solely to haploidentical feature, but due to the alloreactive cells that emerged by infused stem cells which were not prevented by an immediate efficient immunosuppressive such as cyclophosphamide or methotrexate. The fact that CRS can develop not only in haploidentical HSCTs but also in HLA-identical HSCTs with post-CY application also supports this . In addition, as the rate of CRS was not related to the number of cells used, it may not only be associated with alloreactive cells, but also with organ damage developed after the conditioning regimen, as all patients with TBI developed CRS in our study.
Since almost all patients in the post-CY group developed CRS, we looked at the effect of CRS development on survival analysis in the pre-CY group and found that the development of CRS significantly reduced OS and EFS in the 2-year survival analysis, similar to recent studies [15, 17]. Hence, it is important to prevent CRS with drugs such as dexamethasone. Routine administration of corticosteroids before PTCY is generally avoided because preventing the proliferation of alloreactive T-cells that make them susceptible to CY could theoretically decrease the efficacy of CY in terms of GVHD. Nevertheless, severe and refractory courses of fever, despite antibiotics, in patients with deep neutropenia necessitates the use of short-term steroids. We used dexamethasone in about 33% of the patients, mostly in the post-CY group (Table 2), and almost always had a positive response within a day. The use of dexamethasone before CY did not increase the frequency of GVHD in our study but it did increase the tendency to relapse in the post-CY group; thus, it significantly decreased the probability of EFS in this group. Dexamethasone has an apoptotic effect on lymphocytes, while CNIs decrease the activity of lymphocytes without triggering apoptosis in them [18, 19]. Alloreactive cells in the pre-CY group may have eliminated the sparing effect of CY because of previously initiated CNIs; thus, it increased the effect of graft-versus-leukemia (GVL) and decreased the possibility of relapse (although it was not significant). However, this effect did not occur when dexamethasone was used before CY in the post-CY group suggesting that dexamethasone was effective in eliminating alloreactive cells completely, resulting in a decreased GVL effect. We recruited a small number of patients in our study; therefore, a larger prospective randomized study is needed to show whether dexamethasone has a negative effect on the development of GVHD or increases relapse when administered before PTCY.
Eltrombopag is a thrombopoietin receptor agonist primarily used in immune thrombocytopenic purpura. It has also been used in acquired aplastic anemia in recent years, because of its effects on trilineage hematopoiesis. Due to the similarities between the poor graft problem that develops after transplantation and acquired aplastic anemia, eltrombopag is increasingly being used in this context as well . The main causes of poor graft function in an haplo-HSCT setting are conditioning, GVHD, viral infections and donor specific antibodies [21, 22]. In vivo T cell depletion using PTCY overcomes T cell- and NK cell-mediated graft rejection and we observed that, in our study, pre-CY initiation of CNIs did not have a negative effect on this mechanism. Further, no effect of the timing of the initiation of CNIs in the development of poor graft function in haplo-HSCTs with PTCY was observed suggesting that poor graft development might be more related to antibody-mediated rejection rather than cell-mediated rejection.
Our study has a few limitations. First, our study population comprised a heterogeneous patient population with significant variation in disease properties, conditioning regimens, types of donors, sources of stem cells, and conditions of the recipient. Second, the lack of specific diagnostic methods for the diagnosis of CRS has resulted in a vague definition of CRS. However, it is obvious that fever is more common in post-CY patients in the days after stem cell infusion. Third, haplo-HSCTs seem to be affected by multifactorial effects, and the trend for increased relapses in patients treated with dexamethasone may not actually be directly related to dexamethasone use.
Although the dataset studied had some limitations, our study shows that the argument for starting CNI administration after CY is tenuous, and the rationale for not starting CNI before CY needs to be reconsidered. The timing of initiation of CNIs had no effect on survival analyses and GVHD frequency in our study; it supports the finding  that cyclophosphamide induces alloreactive T cell functional impairment rather than destroying them. In an unmanipulated haplo-HSCT setting, the CNI administration in a PTCY may be individualized to start before or after CY. It may be more logical to start CNIs before CY for patients with a high probability of relapse. Although dexamethasone may be useful for CRS patients, it should be avoided in high-risk patients with malignancies because of the possibility of increasing relapse.