Haploidentical bone marrow transplantation with post-grafting cyclophosphamide: multicenter experience with an alternative salvage strategy

Achieving a cure for relapsed leukemia or lymphoma remains a challenge. Allogeneic stem cell transplantation (aSCT) after myeloablative conditioning is a curative treatment option for younger patients. However, concerns regarding aSCT-related toxicity and questions regarding the overall benefit, limit its use for older patients and for heavily pre-treated patients with comorbidities. Another problem is donor availability, as a suitably human leukocyte antigen (HLA)-matched donor can be identified in a timely manner in only a subset of patients with relapsed or refractory disease.

Related HLA haplotype-mismatched (‘haploidentical’) bone marrow transplantation (BMT) is an alternative method to expand the potential pool of stem cell donors. The disadvantage of this strategy has been a high incidence of graft rejection and graft-versus-host disease (GvHD). Typically, investigators prefer to use T-cell-depleted or CD34+-selected grafts after intensive conditioning therapy. T-cell depletion of the graft significantly reduces the incidence and severity of GvHD, but at the expense of increased graft rejection, infection and relapse. Consequently, T-cell depletion of the graft has not been shown to improve outcomes after HLA-mismatched aSCT.1

High-dose cyclophosphamide (Cy), when administered in a narrow window after transplantation, depletes alloreactive T cells from the donor and the host, and can inhibit both GvHD and graft rejection.2, 3 It has been suggested that as a form of drug-induced immunological tolerance, the strategy of giving high-dose Cy after aSCT takes advantage of the higher cytotoxic sensitivity of proliferating, alloreactive T cells over non-alloreactive, resting T cells (which mediate graft-versus-leukemia (GvL) effects). This is supported by extensive studies in mice, in which it has been shown that aSCT induces the bidirectional activation and proliferation of alloreactive host-versus-graft and graft-versus-host reactive T cells, and that these recently activated T cells are more susceptible to being eliminated by high-dose Cy.3

Accordingly, clinical trials have been recently initiated by Luznik et al.4 in which reduced intensity conditioning and the infusion of bone marrow from HLA-mismatched relatives was followed by high-dose post-grafting pulsed Cy. This strategy was pioneered in patients with hematological malignancies for which no HLA-matched donor was available and who were ineligible for myeloablative high-dose conventional aSCT. High-dose Cy was given on day +3 or on days +3 and +4 after BM infusion, as pre-clinical models had suggested that earlier application of Cy might not lead to tolerance.5 At 24 h, after high-dose Cy application, GvHD prophylaxis was continued using mycophenolate mofetil and tacrolimus. Again, tacrolimus was not given before Cy, because the importance of this sequential strategy had been demonstrated in rodents.6 Surprisingly, in contrast to other clinical trials with haploidentical donor–recipient match situations in these patients, it has been shown that the rates of GvHD development and infection were comparable to the incidences after HLA-matched aSCT.4

Besides the disadvantage of a higher risk for GvHD, HLA-mismatched aSCT has the advantage of potentially enhanced GvL reactivity. One important mechanism is the efficient engagement of donor natural killer cells, which express inhibitory killer-cell immunoglobulin-like receptor(s) (KIR) for self-major histocompatibility complex class I ligand(s). Missing expression of the respective KIR ligands in the recipient has been shown to improve outcomes of HLA haploidentical transplantation in acute myeloid leukemia (AML) patients and potentially in other hematological diseases by controlling relapse without causing GvHD.7

In this study, we hypothesized that haploidentical BMT, using nonmyeloablative reduced-intensity conditioning, followed by high-dose post-transplantation Cy, could be utilized as a salvage protocol in patients with relapsed or refractory leukemia (in particular AML) or lymphoma. Patients for which no matched donor was available and who were not eligible for high-dose conditioning were included.

In a multi-center approach, 18 patients with myeloid or lymphoid leukemia or lymphoma, between April 2008 and June 2010, with a median age of 49 years were enrolled (Table 1). Nine patients suffered from high-risk AML. Patients with AML were either in cytopenia after repetitive salvage chemotherapy (patients 1–5), did not respond to salvage chemotherapy (patient 6), had experienced a molecular relapse (patient 7) or had suffered a failure of a cord-blood graft (patient 8) or a HLA-matched sibling graft (patient 9). Two patients suffered from high-risk acute lymphoblastic leukemia and were responsive to salvage chemotherapy (patients 10, 11). Patients 12 and 13 had multiple myeloma in very good partial remission after relapse and patient 14 had plasma cell leukemia in second complete remission. Finally, three more patients with treatment-refractory lymphomas were included, in particular T-cell-derived non-Hodgkin leukemia (patient 15), B-cell non-Hodgkin lymphoma (patient 16) and Hodgkin's disease (patient 17). All 17 patients above were pretreated with multiple high-dose polychemotherapy protocols, and suffered either from refractory or from high-risk disease (n=15) or allogeneic graft failure (n=2). Finally, another patient with secondary myelodysplastic syndromes (RAEB-1, 9% marrow blasts) after severe aplastic anemia and high risk of graft failure was included (patient 18).

Table 1 Patients, grafts, HLA-match, KIR-L mismatch (MM), CMV status and reactivation and engraftment

Most patients received conditioning therapy as published by Luznik et al.3 (n=11), in particular low-dose Cy (14.5 mg/kg at days −6 and −5), fludarabine (30 mg/m2 at days −6 to −2) and 200 cGy total body irradiation (on day −1) before the infusion of BM from HLA-mismatched related donors (Figure 1a). In seven patients, conditioning therapy was modified: six patients (8, 11, 12, 13, 17, 18) received busilvex (at a dose of 3.2 mg/kg at day −3) instead of 200 cGy total body irradiation (at day −1). One patient (15) with a progressive T-cell-derived non-Hodgkin leukemia received treosulfan (14 000 mg/m2/day, days −6 to −4) and etoposide (30 mg/kg at day −3) instead of low-dose Cy (at days −6 and −5).

Figure 1
figure1

(a) Schema of non-myeloablative, HLA-haploidentical bone marrow transplantation with high-dose post-transplantation cyclophosphamide. Cy, cyclophosphamide; BMT, bone marrow transplantation; TBI, total body irradiation; G-CSF, granulocyte-stimulating factor; MMF, mycophenolate mofetil, * alternatively Busulfan (at a dose of 3.2 mg/kg at day −3), ** alternatively only at day +3, *** alternatively MMF 3 × 15 mg/kg and Cyclosporine A (160–200 ng/ml). (b) Overall (OS) and progressive-free survival (PFS). The graphs depict Kaplan–Meier estimates of OS and PFS for all 18 patients included.

All patients received BM from HLA-mismatched related donors (Table 1). Nine patients had a complete haplotype mismatch (5/10), whereas 6/10 and 7/10 mismatches were documented in five and four cases, respectively. KIR ligand mismatching, according to the missing self-hypothesis,7 was present in 10 out of 17 evaluable patients (59%). Seven patients had one KIR-L-MM, two patients had two KIR-L-MM and one patient had three KIR-L-MM, respectively. The BM grafts infused contained a median number of 2.4 × 106/kg (range 0.94–4.76 × 106/kg) CD34+ cells. A gender mismatch with a higher risk for GvHD (female donor and male recipient) occurred in five patients (patients 4, 12, 14, 15, 18). In all patients with this gender mismatch, acute GvHD was observed, and four out of five of these patients also acquired chronic GvHD.

To suppress alloreactive donor T cells, high-dose (50 mg/kg) Cy was infused on days +3 and +4 (n=15) after transplantation. In three patients, high-dose Cy was only given at day +3 (n=3), with the intention to reduce toxicity and to increase GvL immune response with the potential higher risk of GvHD (patients 3, 6, 9). Previous studies have shown a higher risk for GvHD when high-dose Cy is only applied at day +3.3 Interestingly, all patients with a single dose of high-dose Cy developed no or only grade I acute GvHD, but no relapse. Starting 24 h, after high-dose Cy, GvHD prophylaxis was continued with mycophenolate mofetil (3 × 15 mg/kg) combined with tacrolimus (5–15 ng/ml target level, n=12) or cyclosporine (target levels 160–200 ng/ml, n=6).

To promote engraftment, granulocyte colony-stimulating factor was applied starting at 24 h after high-dose Cy in 13 patients (Lenograstim: 263 μg/day i.v., n=9; or filgrastim: 5 μg/kg/day s.c., n=6). Three patients did not receive granulocyte colony-stimulating factor (patients 4, 5, 10) and these patients also showed timely engraftment. In all patients, neutrophil and platelet engraftment was achieved with a median time of 19 and 30 days, respectively. Complete donor chimerism (>95% as measured by STR-PCR) was detectable after a mean time of 18 days. There was one case of grade 3–4 extramedullary toxicity: Patient 11 acquired veno-occlusive disease and died of this complication at day 15 after BMT.

Acute GvHD grade I occurred in 8 of 17 (47%) available patients, grade II acute GvHD was observed in 5 of 17 patients (29%) and grade III-IV GvHD occurred in 1 patient (8; Table 2). Limited chronic GvHD (requiring no steroid medication) was observed in 6 of 17 patients (35%). Extensive chronic GvHD has not occurred so far. Immunosuppression was discontinued in 6 of 17 patients between 60 and 340 days after transplantation (median 8.5 months). Cytomegalovirus reactivation occurred in 9 out of 14 sero-positive patients, out of which 6 patients required antiviral medication.

Table 2 Month post BMT, immunosuppression, acute and chronic GvHD, current status

After a median follow-up time of 12 months (range 4–24), 16 out of 18 patients (89%) were alive (Figure 1b). The probability of 12-month overall survival was 94% (95% confidence interval, 83–100%) and the probability of progressive-free-survival was 86% (95% confidence interval, 69–100%). One patient (patient 8) died of relapsed AML and one died from toxicity (patient 11). Of the 16 patients alive, three have relapsed: one patients with acute lymphoblastic leukemia experienced extramedullary relapse (patient 10), one patient with multiple myeloma (patient 11) acquired osteolysis with persistent complete chimerism and one patient with AML (patient 5) experienced hematological relapse and but achieved second hematological remission after donor lymphocyte infusion. However, donor lymphocyte infusion was associated with acute GvHD of the skin, gut and liver in this case, which responded to reinstitution of immunosuppression. Three patients with AML and one patient with multiple myeloma still have detectable levels of minimal residual disease.

Although the case number and the follow-up in this retrospective multi-center study are limited, we could confirm the feasibility of immunosuppressive conditioning followed by haploidentical BMT in heavily pretreated patients without excess morbidity and mortality. The strategy seems to be especially attractive in patients without hematopoietic recovery after intensive salvage therapy, or in cases in which a matched related or unrelated donor cannot be identified by the time of best response of high-risk hematological malignancies. In all patients, rapid engraftment was achieved after haploidentical BMT. No graft-rejection or graft failure has occurred, so far. Another major advantage of the current approach is the avoidance of laborious and expensive ex vivo manipulation of the graft. Given the therapy-refractory status of the patients in this study, the outcome argues for the effectiveness of GvL reactions with this approach. The GvL effector mechanism(s) might include T-effector cells that are not depleted by high-dose post-grafting Cy and natural killer cells that are activated by KIR-L mismatching. Because of the low toxicity, the strategy can be applied as a salvage maneuver in high-risk leukemia and lymphoma patients.

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Correspondence to M Bornhäuser.

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MF performed the determination of KIR-L mismatching. ST, JG, CS, JR, AK, DS, UP, GRM, MW, UH, PB, JMM, NS, JMC, AK, NG, JS, GE, MH, JLDM and MB were responsible for clinical care and out-patient follow-up. CT performed chimerism analyses. ST, JG, CS and MB wrote the paper and we have read and approved the final version of the manuscript.

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Tuve, S., Gayoso, J., Scheid, C. et al. Haploidentical bone marrow transplantation with post-grafting cyclophosphamide: multicenter experience with an alternative salvage strategy. Leukemia 25, 880–883 (2011). https://doi.org/10.1038/leu.2011.11

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