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Long-term outcome of mixed chimerism after stem cell transplantation for thalassemia major conditioned with busulfan and cyclophosphamide

Abstract

Mixed chimerism (MC) occurs frequently after allogeneic hematopoietic stem cell transplantation (HSCT) for thalassemia major (TM) and may be associated with rejection. We report the outcome of MC in 132 TM patients conditioned with Busulphan/Cyclophosphamide, who had successful engraftment and had 1 year follow-up. Chimerism was first assessed at day +28, then every 3–9 months or more frequently if there was MC. If rejection was suspected, immunosuppression was stopped and donor-lymphocyte infusion (DLI) was given if there was no response. Among 132 patients, aged 7 years (range: 2–24), 46/132 (34.8%) had MC in the first year, 32/46 (69.6%) at day +28 and another 14 (30%) between day +28 and 1 year post HSCT. MC was quantified at level 1 (residual host chimerism (RHC) <10%) in 20 (43.5%), level II (RHC 10–25%) in 14 (30.4%) and level III (RHC >25%) in 12 (26.1%). On tapering immunosuppression, 15 (32.6%) developed acute GvHD and 8 (17.4%) had chronic GvHD with reversal to complete chimerism (CC). DLI was administered to 5/46 (10.9%), 1 evolved to CC but 4 rejected the graft. At median follow-up of 60 months (range: 16–172), 20/46 (43.5%) had CC, 18/46 (39.1%) had persistent MC with hemoglobin of 11.5 g/dL (range: 8.4–13.6), whereas 8 (17.4%) rejected the graft. Close monitoring and early intervention is needed with increasing recipient chimerism. Novel strategies are required for preventing graft rejection.

Introduction

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available curative treatment for β-thalassemia major (TM).1, 2 Graft rejection is a major complication of HSCT for TM and has been reported to occur in 5–30%.3 Different approaches have been taken to reduce graft rejection by either intensifying myeloablative conditioning or by enhancing immunosuppression.2 Residual host cells (RHCs) can also persist without graft rejection in a stable state referred to as mixed chimerism (MC), observed in 30–40% of patients after HSCT for hemoglobinopathies.1, 3 Intriguingly, many of these patients demonstrate stable and durable co-existence of donor and host cells associated with transfusion independence and the lack of continued clinical manifestations of their disease.1, 2, 3, 4, 5, 6, 7, 8

The reasons for such persistent MC are not well understood. Conditioning regimen, stem cell dose, immune status of recipient and post HSCT immunosuppression are some of the possible factors that can influence chimerism status.2, 5, 9 The long-term outcome of patients with persistent MC post HSCT depends on the time of occurrence and the extent of MC.7 There are limited data on the outcome of MC with long-term follow-up.

In this study, we evaluated the course of MC in TM patients who underwent HSCT using Busulphan/Cyclophosphamide (Bu/Cy)-based conditioning, to determine the clinical course, final outcome and possible predictors of such outcomes of MC.

Patients and methods

All patients who underwent HSCT at our center for TM between January 2000 and December 2010 using Bu/Cy-based conditioning and a HLA-identical related donor were included in the analysis. Consent for analyzing and reporting of data made anonymous is obtained from all patients who undergo transplant and has been approved by our Institutional Review Board. All data were collected from prospectively and systematically maintained structured hospital records.

Among 200 patients transplanted during this period, we selected 132 patients with successful engraftment at day +28 post HSCT and who had 1 year follow-up. Patients (n=68) excluded were as follows: 49 with early TRM, 2 with primary graft rejection and 17 who had <1 year follow-up. HLA identical donors were identified from the family. HLA typing of parents and extended family members was considered when siblings were not HLA identical. All patients had liver biopsy done pre-HSCT, to evaluate for fibrosis for categorizing according to Pesaro criteria.10 Eleven patients were class 1, 75 were class 2 and 46 were class 3. Bu was administered at a total dose of 14–16 mg/kg (4 divided doses orally for 4 days (day −9 to −6) and Cy 160–200 mg/kg (2 h IV infusion daily for 4 days (day −5 to −2) along with ATG 30 mg/kg/day for 3 days (day −8 to −6). Patients aged >15 years received lower doses.11, 12

The source of stem cell graft was bone marrow in 131 patients and peripheral blood in one. GvHD prophylaxis was with cyclosporine (5 mg/kg/day in two divided doses from day −4 with regular monitoring of drug levels) and methotrexate (10 mg/m2 on day +1 and 7 mg/m2 on days +3, +6 and +11 post HSCT). Myeloid engraftment (neutrophil recovery) was defined as an ANC 500 × 109/L for 3 consecutive days after nadir and platelet engraftment as a platelet count of >20 000 × 109/L independent of platelet transfusions for at least 7 consecutive days.5

Evaluation of chimerism

Presence of donor cells in the recipient peripheral blood (chimerism status) was assessed at day +28, 2 months, 3 months, 6 months and 1 year post HSCT for those with complete chimerism (CC) or more frequently and beyond 1 year if there was MC. Chimerism analysis was done on genomic DNA extracted from whole blood. Patients with MC underwent more frequent monitoring of chimerism status at the treating physician’s discretion based on blood counts. In general, for those with significant drop in blood counts, monitoring was done once in 1–4 weeks and intervention steps were initiated as mentioned below if there was >5% change in MC.

After initial screening of the informative STRs/variable number tandem repeats (VNTRs) (between patient/donor pairs), STR/VNTR PCR for the informative marker with pre-HSCT sample, donor and post-HSCT sample was done. Qualitative analysis of chimerism was done using agarose or PAGE. Quantitative analysis was done by capillary gel electrophoresis using ABI 3130 or 3500 genetic analyser with GeneScan 3.1 software (Applied Biosystems, Foster city, CA, USA).13, 14

Definitions of chimerism status

MC was defined as presence of >5% RHC at any time point post HSCT, whereas rejection as >90% RHC in peripheral blood with relapse of TM/red cell transfusion dependence.1, 5, 8 Transient MC is defined as MC, which subsequently revert to CC or rejection, whereas those who continued to have MC with stable graft function were called persistent MC (PMC).1, 8 The level of MC was classified based on the percentage of RHC in the recipient whole blood as Level 1 (RHC <10%), Level 2 (RHC 10–25%), and Level 3 (RHC >25%).5, 15

Therapeutic interventions

With a >5% change in MC towards rejection and a significant drop in blood counts, therapeutic interventions were made. These included taper/stopping of immunosuppression and donor-lymphocyte infusion (DLI) if there was no response to the former, unless GvHD had occurred already. DLI was administered only if there was at least 10% residual donor chimerism.

Statistical methods

Descriptive statistical methods were used to evaluate all variables. Differences in proportions were assessed using chi-square statistics or Fisher’s exact test. Differences in means were tested using Mann–Whitney U-test. For all tests, a two-sided P-value of 0.05 was considered statistically significant. SPSS 16.0 software (SPSS South Asia Private Limited, Bangalore, India) was used for the analysis.

Results

Frequency of MC

Among the 132 patients included in this analysis, 46 (34.8%) were observed to have MC at some time point in the post-HSCT period with a median follow-up of 60 months (range: 16–172) (Figure 1). At the first time point of chimerism evaluation (day +28 post HSCT), 32 (24.2%) patients were noted to have MC, whereas 100 (75.8%) remained in CC. Subsequently, 11 (11.0%) among the 100 with CC on day 28 developed MC between day 28 and 6 months, and 3 (3.4%) out of the remaining 89 patients with CC developed MC between 6 months and 1 year post HSCT. None of the remaining 86 patients with CC had a drop in blood counts to suggest the possibility of MC at any point beyond 1 year post HSCT. Therefore, in this cohort, there were 46 (34.8%) patients who had a MC status at some point post HSCT, whereas 86 (65.2%) maintained a stable CC status from day 28 till last follow-up. Comparison of the patients who maintained CC status with those who had MC at some time point (Table 1) showed no significant difference that could predict development of MC.

Figure 1
figure1

Frequency and outcome of MC based on time of onset of MC. MC in the early post-HSCT period (day +28), has high probability of graft rejection as compared with those who develop MC after the first month post HSCT (that is, 25% vs 0%; P= 0.040). CC=complete chimerism; HSCT=hematopoietic stem cell transplant; MC=mixed chimerism; PMC=persistent mixed chimerism; RHC=residual host cell.

Table 1 Comparison between complete and mixed chimerism groups a (N=132)

Outcome based on time of onset of MC

Among the 32 patients who had MC by day +28 post HSCT, 8 (25.0%) rejected the graft, whereas 15 (46.9%) evolved to CC and 9 (28.1%) had PMC (Figure 1). Out of the 11 patients who developed MC between day +28 and 6 months post HSCT, 4 (36.4%) evolved to CC, whereas 7 (63.6%) remained in PMC. Among the 3 patients who developed MC between 6 months and 1 year post HSCT, 1 (33.3%) evolved to CC and 2 (66.7%) had PMC. None among the 11 patients who developed MC between day +28 and 6 months or the 3 who developed MC between 6 months and 1 year post HSCT rejected the graft. Thus, in this cohort, rejection was associated only with very early (day +28) presence of MC (P=0.040).

Outcome based on level of MC

Most (20/46; 43.5%) of the patients had level 1 MC, 14/46 (30.4%) had level 2 and 12/46 (26.1%) had level 3 MC (Figure 2). With therapeutic interventions, that is, taper/immediate withdrawal of immunosuppression and/or DLI, 13/20 (65.0%) patients with level 1 MC evolved to CC and 7/20 (35.0%) continued with PMC. None of these rejected the graft. Among 14 patients with level 2 MC, 7 (50%) remained in PMC, 5 (35.7%) evolved to CC and 2 (14.3%) rejected the graft. Rejection of the graft occurred in 6/12 (50%) patients with level 3 MC, whereas 2 (16.7%) evolved to CC and 4 (33.3%) had PMC with no transfusion requirement. Thus, in this cohort, the risk of rejection of graft was observed to be significantly higher with increasing levels of MC, that is, 50% in level 3 MC vs 14.3% in level 2 and 0% in level 1 MC (P=0.001).

Figure 2
figure2

Outcome of MC based on initial levels of MC. Graft rejection is more likely in those with higher level MC (6/12 (50%) of level 3 MC) compared with those with level 1 (0%) and level 2 (14.3%) MC (P0.001). CC=complete chimerism; HSCT=hematopoietic stem cell transplant; MC=mixed chimerism; PMC=persistent mixed chimerism; RHC=residual host cell.

Therapeutic interventions and outcomes of MC

Immunosuppression was reduced and tapered in all patients with MC on follow-up, whereas only five received DLI (Table 2). With tapering of immunosuppression, the incidence of acute GvHD was 15/46 (32.6%), grade 1–2 in 14 (93.3%) and grade 4 in 1 patient. Chronic GvHD developed in seven of these patients, three had extensive and four had limited chronic GvHD. DLI was given to five patients; one infusion each with a median CD3+ cell dose of 4.2 × 108/kg (range: 1.2–7.6 × 108/kg) on post-HSCT days ranging 60–391 days. In these five patients, the RHC ranged from 17% to 74% (one had level 2 and four had level 3 MC) and had increased to 46–83% at the time of DLI. One child, a father to son graft, who received a high DLI dose of 6.2 × 108 CD3+ cells/kg, developed GvHD and subsequently evolved to CC, whereas the remaining four went on to reject the graft; all four had early onset MC (day +28). The one who evolved to CC post DLI had developed MC at 6 months post HSCT.

Table 2 Interventions and outcome of different levels of MC (N=46)

Evolution of MC

With a median follow-up of 60 months (range: 16–172), among the 46 patients with MC, 20 (43.5%) evolved to CC, 8 (17.4%) progressed to rejection (that is, total 28 (60.9%) had transient MC) and 18 (39.1%) remained in PMC throughout follow-up (Table 2). It was observed that among the 28 with transient MC, 15 (53.6%) evolved to CC by 6 months post HSCT, an additional 4 (14.3%) evolved to CC by 1 year post HSCT and 1 (3.5%) evolved to CC by 16 months post HSCT, whereas all the 8 (28.6%) patients who rejected the graft did so in the first year post HSCT. Graft rejection was noted only in those with MC at day 28 post HSCT and it happened by day 90 in 3/8 (37.5%), by day 100 in 1/8 (12.5%) and by 6 months post HSCT in the remaining 4/8 (50%), and these patients were transfusion dependent with RHC ranging from 89% to 100%. No graft rejection occurred beyond 6 months post HSCT.

All the 18 ex-thalassemic patients with PMC are transfusion independent with median hemoglobin of 11.5 g/dL (range: 9–13.6 g/dL) and RHC ranging from 6% to 63%. Five (10.6%) of these showed a PMC of level 3 (range: 31–63%). This finding suggests that although high numbers of RHC in a recipient early after HSCT leads to a high risk of rejection, the same proportion of RHC present 1 year from HSCT is associated with a state of tolerance between donor and recipient cells.

Discussion

This report describes a large series of patients with MC post HSCT for TM with long-term follow-up and close monitoring of MC, as well as therapeutic interventions based on those results. Although the overall incidence of MC in TM is similar to previous reports,6, 15 with about a third of patients showing MC in the first year, there are significant differences between the profile of these changes and the outcomes. In this cohort, all cases of MC occurred within the first year after HSCT unlike earlier reports15 where some occurred much later as well. Consistent with previous reports, the probability of rejection of the graft was related both to the number of RHC present in the recipient, as well as the time after HSCT when MC appeared.

We have observed that with early MC (day +28 post HSCT), the likelihood of graft rejection was more compared with those who developed MC after the first month post HSCT (that is, 24% vs 0%; P=0.043) (Figure 1). All 8 patients with graft rejection were found to have MC at day +28 post HSCT and in all of them rejection occurred within the first 6 months (3 within 3 months and 5 by 6 months). None of the patients who developed MC beyond day +28 rejected the graft. This is very different from several other reports where rejections have been reported to occur even with later occurrence of MC.2, 6, 15 The reasons for this difference are difficult to specify but the fact that we did closer monitoring of MC in such patients and combined it with modulation of immunosuppression given for prevention of GvHD to the point of stopping it completely in the case of rising MC could have had a role.

We also found that autologous recovery with thalassemic hematopoiesis is more likely in those with higher level MC (6/12 (50%) of level 3 MC) compared with those with level 1 (0/20) or level 2 (2/14) MC (P=0.008) (Figure 2). In contrast, if at the same period of observation the proportion of RHC is low, MC is more likely to evolve toward CC or PMC (that is 65.0% and 35.0% of patients with level 1 MC and 50.0% and 35.7% with level 2 MC evolved to CC and PMC, respectively). There was no rejection among those with level 1 MC but 14.3% of those with level 2 MC rejected the graft. Among those with level 3 MC, graft rejection was even higher at 50%, whereas 16.7% evolved to CC and 33.3% to PMC (Figure 2). This observation suggest that the presence of higher number of RHC with level 3 MC at day +28 post HSCT is associated with a very high rejection of the graft in 50% of patients15 and would therefore be candidates for early and more intensive interventions. The evolution to CC/PMC appears to be higher (83% vs 65%) and rejection rate lower (17% vs 35%) in our cohort of patients compared with that observed by Andreani et al.15 among all levels of MC (P=0.031). Although rapid tapering or stopping of immunosuppression with weekly monitoring of chimerism status would be the obvious first steps, followed by DLI if MC is rising, four out of five patients treated in this manner did not respond to DLI. In all these patients, DLI was given within a month of the detection of early MC. Whether higher doses of DLI or combining with immunosuppressive drugs such as Cy or fludarabine along with further infusion of hematopoietic stem cells could have given better results needs to be explored.

Although none of the patients with CC on day +28 rejected the graft, 14/100 (14.0%) of them developed MC (RHC ranging from 6% to 62%) within a year of HSCT. The resurgence of host cells could be related to inadequate cell killed by the conditioning regimen and the donor immune system, but the exact mechanisms involved in this phenomenon need further evaluation, particularly with regard to the pharmacokinetic and pharmacogenetic aspect of the drugs used for conditioning and the pattern of immunological reconstitution post transplant. We have previously reported an association between Bu and Cy pharmacokinetic parameters and graft rejection.16, 17 Patients with Bu Cmin1 <156 ng/mL had a higher rejection (30% vs 8%) compared with those with >156 ng/mL (RR=9.8; P=0.0001).16 Unfortunately, Bu pharmacokinetic data are not available for all the patients in this cohort to do a similar correlation. With modification of conditioning regimen, Anurathapan et al.18, 19 have reported a lesser incidence of MC in class three high-risk TM patients as well as in TM patients undergoing haplo-identical stem cell transplants. There have been a few recent reports that implicate T-regulatory and natural killer cells also in this phenomenon.4, 20 It has been shown that RHC of <50% in T cells is associated with a very low risk of rejection, but that higher levels of RHC in both T- and natural killer cells are associated with rejection in up to 90% of patients.21 We have also previously reported the correlation of rejection in patients with TM with the number of circulating natural killer cells on day 28 after HSCT.22

Among 18 patients with PMC, level 3 MC was observed in 5 (27.8%) (Table 2). Among them, the level of RHC ranged from 31% to 63% for durations ranging from 25 to 120 months. Although the level of PMC was variable in different patients, it was stable in each patient at a particular level. All these patients were independent of transfusion with median hemoglobin of 11.5 g/dL (range: 9–13.6). Specific assessment of erythroid chimerism would be useful to understand the basis for the adequacy of hemoglobin production in these patients. It is likely to be that the proportion of donor-derived erythroid cells is much higher than what would be predicted by the MC data from genomic DNA from unselected cells. In such cases, it has been shown that in patients with long-lasting stable or persistent mixed hematopoietic chimerism, there is a two- to fivefold enrichment of donor-derived mature erythrocytes in the peripheral blood than their unselected cell chimerism would suggest.1, 4 This observation from our study and other reports of significant level PMC with good hemoglobin levels in ex-thalassemics1, 4, 5, 15 suggests that enhanced erythroid chimerism is probably an important factor defining the hemoglobin level in such patients.

We attempted to evaluate potential patient, donor, graft characteristics or transplant variables that could contribute to the phenomenon of MC (Table 1). None of these could predict the development of MC.

In conclusion, our results show that MC occurs in about a third of patients with TM undergoing HSCT with Bu/Cy conditioning and about a sixth of these patients with MC go on to graft rejection in spite of current therapeutic intervention. The probability of graft rejection is almost 50% with early level 3 MC and 14% with early level 2 MC. It appears though that rejection can be restricted to those with early MC detected within one month after HSCT with frequent monitoring and modulation of immunosuppression. DLI at the doses administered was not found to reverse rejection in most patients in this cohort. Although MC did evolve in some patients beyond the first month after HSCT, none of these patients rejected the graft. No patient developed MC more than 1 year after HSCT. Patients with persistent MC maintain normal blood counts irrespective of the level of MC. The level of MC at around one month after HSCT with close quantitative monitoring currently seems to be the best guide for intervention until a more reliable predictor is available.

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Fouzia, N., Edison, E., Lakshmi, K. et al. Long-term outcome of mixed chimerism after stem cell transplantation for thalassemia major conditioned with busulfan and cyclophosphamide. Bone Marrow Transplant 53, 169–174 (2018). https://doi.org/10.1038/bmt.2017.231

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