Pharmacokinetic (PK) Exposure of 4-Hydroxycyclophosphamide Associates with Clinical Benefits of Reduced-Dose Post-Transplantation Cyclophosphamide: Results of a Completed Phase I/II Trial to De-Escalate Post-Transplantation Cyclophosphamide for HLA-Haploidentical Myeloablative Bone Marrow Transplantation
Authors: Hyder Mustafa A. , Dimitrova Dimana , Sabina Ruby , DeVries Ashley , McCune Jeannine S. , McAdams Meredith J. , Flomerfelt Francis A. , McKeown Christi , Sadler Jennifer L. , Chai Amy , Hughes Thomas E. , Napier Scott , Stokes Anita , Sponaugle Jennifer , Rechache Kamil , Parta Mark , Cuellar-Rodriguez Jennifer , Figg William D. , Choo-Wosoba Hyoyoung , Steinberg Seth M. , Kanakry Jennifer A. , Kanakry Christopher G. 
Published: December 2024

Introduction
Standardly, post-transplantation cyclophosphamide (PTCy) dosing is administered at 50 mg/kg/day on days +3 and +4 (HD-PTCy). Yet, the optimal dosing has not been identified. Our MHC-haploidentical murine hematopoietic cell transplantation (HCT) studies showed that PTCy 25 mg/kg/day on days +3/+4 was more effective in preventing histopathologic and clinical graft-versus-host disease (GVHD) than higher or lower dosing and that the maximal efficacy of PTCy was on day +4.
Methods
Building on these two findings, at the NIH, we conducted a phase I/II clinical trial reducing PTCy exposure after myeloablative T-cell-replete HLA-haploidentical bone marrow HCT. Patients received myeloablative conditioning with targeted IV busulfan and fludarabine, followed by a fresh bone marrow graft on day 0. The PTCy dose levels were: PTCy 50 mg/kg on days +3/+4 (n=5), 25 mg/kg on days +3/+4, or 25 mg/kg on day +4 only. Additional GVHD prophylaxis included sirolimus from days +5 to +80, and mycophenolate mofetil from days +5 to +35. The primary endpoint used for PTCy dose de-escalation was grade III-IV acute GVHD (aGVHD) by day +60. Secondary endpoints included engraftment; grade II-IV and III-IV aGVHD at day +200; non-relapse mortality (NRM) at 100 days and 1 year; and chronic GVHD (cGVHD), relapse, overall survival (OS), and disease-free survival (DFS) at 1 year. Exploratory endpoints included immune reconstitution, weekly monitoring of BK virus and associated symptoms, and PKs of cyclophosphamide and 4-hydroxycyclophosphamide (4HCY). Patients underwent transplantation from July 2019 to January 2022.
Results
35 patients were treated across the three PTCy dose levels. 23 patients received the phase II dose, 25 mg/kg/day PTCy on days +3/+4 (ID-PTCy). For ID-PTCy recipients, median age was 34 years (range 18-57), 87% were racial/ethnic minorities, median HCT-CI was 3 (range: 0-7), 65% underwent HCT for acute leukemia, and 43% were high/very high risk by the revised Disease-Risk Index. Three patients were not evaluable for the primary endpoint: two had primary graft failure (one with primary myelofibrosis and the other with acute myeloid leukemia) and one patient had early relapse. Among evaluable ID-PTCy patients, the median time to engraftment was 14 days for neutrophils (vs. 19 days for HD-PTCy, p=0.0004), and 22 days for platelets (vs. 33 days for HD-PTCy, p=0.0097). The median number of units transfused for RBCs and platelets were 2 and 5 for ID-PTCy compared with 8 and 12 (p=0.015 and p=0.064, respectively) for HD-PTCy. Mixed T-cell chimerism was seen in 4 of 20 evaluable patients treated with ID-PTCy. Grade 3 mucositis was less severe and shorter in duration with ID-PTCy, with a median of 0 days for ID-PTCy and 11 days for HD-PTCy (p=0.0003). CD4+ and CD8+ T-cell reconstitution was markedly faster with ID-PTCy compared with HD-PTCy (median at day +14: CD4+ T cells 11.78 vs. 0.97 cells/µl, p=0.002; CD8+ T cells 4.02 vs. 0.09 cells/µl, p=0.003). BK-virus-associated cystitis symptoms were significantly shorter with ID-PTCy compared with HD-PTCy (hemorrhagic cystitis: median 7 vs 51 days, p=0.006) and this was associated with significantly lower BK viral shedding in blood (p=0.046).
No ID-PTCy patient developed grades II-IV acute GVHD and 2-year cumulative incidence of chronic GVHD requiring systemic therapy was 13%. With median follow-up of 2.4 years, 2-year OS, DFS, and GVHD-Free, Relapse-Free Survival (GRFS) were 61%, 61%, and 52%. CI of relapse at 2 years was 22%, and non-relapse mortality at 100 days and 2 years was 0% and 17%.
4HCY, a precursor to phosphoramide mustard, is the principle cytotoxic metabolite of cyclophosphamide. 4HCY area-under-the-curve (AUC) more strongly correlated with PTCy weight-based dosing as calculated by ideal body weight (ρ=0.75, p&lt;0.0001) compared with actual body weight (ρ=0.54, p=0.0011). 4HCY AUC showed significant correlation with the benefits of reduced-dose PTCy, including positive correlation with time to neutrophil engraftment (ρ=0.63, p=0.0013) and duration of mucositis (ρ=0.61, p=0.002) and inverse correlation with CD4+ T-cell recovery on day +14 (ρ=-0.46, p=0.03).
Conclusion
ID-PTCy maintains efficacy at GVHD prevention. PTCy dose reduction has clinical benefits when compared with HD-PTCy with direct correlation of 4HCY exposure with these clinical benefits. Future comparative studies are needed to assess the relative benefits of ID-PTCy vs. HD-PTCy.