Progressive substitution of posttransplant cyclophosphamide with bendamustine: A phase I study in haploidentical bone marrow transplantation

Abstract We have initiated a single center phase I study in patients with hematologic malignancies progressively substituting day +4 posttransplant cyclophosphamide (PT‐CY) with bendamustine (PT‐BEN) following myeloablative conditioning (MAC) and T‐cell replete haploidentical bone marrow transplantation (haplo‐BMT). We report herein our interim analysis of our first three cohorts PT‐CY (mg/kg)/PT‐BEN (mg/m2): 40/20, 20/60, and 0/90. All patients have tolerated PT‐CY/BEN well with no dose limiting toxicities. Compared to contemporaneous controls undergoing haplo‐BMT with the same MAC regimens but only PT‐CY, we have observed earlier trilineage engraftment (P = .002 neutrophils, P = .014 platelets) and a lower incidence of cytomegalovirus reactivation (P = .016) in the PT‐CY/BEN cohorts. After substituting day +4 PT‐CY with PT‐BEN, the registered trial (www.clinicaltrials.gov; NCT02996773) is proceeding to replace day +3 PT‐CY with PT‐BEN with a view to identifying further evidence on the potential advantages of PT‐BEN.


INTRODUCTION
BEN, were asked to participate as PT-CY controls for the clinical endpoints. Moreover, in order to proceed from one phase I cohort to the next, there is a 28-day period of observation. Therefore, patients requiring haplo-BMT during those intervals were also enrolled as PT-CY controls upon consent. All haplo-BMTs were performed at Banner University Medical Center in Tucson, Arizona between January 2017 and October 2019. Ten patients were transplanted on the pediatric service (ages 9.2-24.7 years) and seven on the adult service (ages 26.1-44.6 years).
All acute lymphoblastic leukemia (ALL) patients had negative bone marrow minimal residual disease assessment by flow cytometry prior to initiation of conditioning. Two of the patients with acute myeloid leukemia and two with undifferentiated and bispecific leukemia were in morphologic remission. Two patients with chronic myelogenous leukemia in chronic phase had failed tyrosine kinase inhibitor therapy, while one was in remission following induction chemotherapy for lymphoid blast crisis. One patient in cohort #1 had refractory non-Hodgkin lymphoma (NHL-anaplastic large cell lymphoma) and was in partial remission at the time of transplant.

Transplant procedure
All patients received a MAC regimen. According to diagnosis and disease characteristics, we decided either for fractionated total body irradiation (TBI) followed by fludarabine (FLU) or for busulfan (BU), fludarabine (FLU), and melphalan (MEL) combination (Table 2) [4].

GvHD prophylaxis
All patients received T-replete bone marrow followed by PT-CY 50 mg/kg on day +3. On day +4, cohort #1 patients received 40 mg/kg PT-CY immediately followed by PT-BEN 20 mg/m 2 , cohort #2 patients were treated with PT-CY 20 mg/kg followed by PT-BEN 60 mg/m 2 , and cohort #3 patients received only PT-BEN 90 mg/m 2 ( Table 1). The eight patients in the control group received the standard PT-CY 50 mg/kg on days +3 and +4. Additionally, all patients were given mycophenolate mofetil on day +5 through day +28 and tacrolimus starting on day +5. In the absence of GvHD, tacrolimus was decreased starting day +70 to +90 and discontinued by day +120 to +180. GvHD was graded according to the consensus criteria for grading acute and chronic GvHD [7,8].

Supportive care
Antifungal prophylaxis was administered in all patients. Patients

Donor selection
Donors were first degree relatives who were HLA-haploidentical based on high-resolution typing at HLA-A, -B, -Cw, -DRB1, and -DQB1 ( Table 2). None of the patients had antidonor HLA antibodies.
There were seven major ABO incompatibilities that required donor RBC reduction using Hespan R (6% hetastarch in 0.9% sodium chloride injection) for RBC sedimentation and one minor incompatibility requiring plasma reduction [9]. ABO incompatibilities were comparable between groups.

Engraftment and donor chimerism monitoring
Granulocyte-colony stimulating factor (G-CSF) was started on day +5 at 5 µg/kg/day until an absolute neutrophil count (ANC) of 2.5 × 10 9 /L was achieved for three consecutive days. Day of myeloid engraftment was defined as the first of three consecutive days with an ANC of ≥ 0.5 × 10 9 /L. Day of platelet engraftment was considered the first of three consecutive days with platelet counts of ≥ 20 × 10 9 /L with no platelet transfusions administered in the previous 7 days. Donor chimerism was evaluated on days +28, +100, +180, and +365 by short tandem repeats on peripheral blood or bone marrow.

Patient, disease, and transplant characteristics
Patient characteristics are summarized in

Engraftment and chimerism
PT-CY/BEN patients received a median of 4.05 × 10 6 /kg CD34 + cells compared to 2.7 × 10 6 /kg in the PT-CY group (P = .07) (  Figure S1). The median time to an ANC of 1.0 × 10 9 /L was achieved progressively earlier in each PT-CY/BEN cohort even though the median number of CD34 + cells/kg infused was comparable between cohorts (4.6, 3.7, and 4.05 × 10 6 /kg, P = n.s.) ( Figure 1A). Similarly, the latter PT-CY/BEN cohorts demonstrated earlier platelet engraftment ( Figure 1B). Consequently, PT-CY/BEN patients required fewer platelet and red blood cell transfusions (Fig-ure 1C,D). All PT-CY/BEN patients showed complete donor chimerism in their day +28 bone marrows and in peripheral blood on days +100 and +180 as did all patients that have reached their 1-year follow-up.

Graft versus host disease
The cumulative incidence of grade II-IV aGvHD was 33.3% following PT-CY/BEN (66.7%, 33.3%, and 0% by cohort) compared to 50% in controls ( Figure 1E). No grade III-IV aGvHD was seen in PT-CY/BEN compared to 25% in controls ( Figure 1F and Table S1)  Figure 1G).  (Table S2). In contrast, two PT-CY control patients were admitted to ICU with one requiring mechanical ventilation. Additionally, two control patients developed end-stage renal disease necessitating CRRT followed by chronic renal dialysis, one from BK viral nephritis and cidofovir adverse effect and the other from BK and TA-TMA.

Infections
There were five Gram-positive and no Gram-negative bacteremias in four patients receiving PT-CY/BEN seen between day +5 and day +100 compared to three Gram-positive and one Gram-negative in four PT-CY patients, all of which responded to appropriate antibiotic therapy. All the Gram-positive bacteremias occurred before day +35 (median day +12). There were no documented fungal infections in either group (Table S3). CMV reactivation was significantly less common in trial patients receiving PT-CY/BEN with only one out of eight at risk (seropositive recipient and/or seropositive donor) reactivating CMV (Table 3), compared to 71.4% of at-risk PT-CY patients ( Figure 1H). The majority of CMV reactivations peaked between day +40 and +50 ( Figure S2A) with viral loads of between 10 3 and 10 5 IU/mL ( Figure S2B). All patients responded to antiviral therapy ( Figure   S2C) patients that developed end-stage renal failure requiring dialysis also had significant BK viremia of 6000 and 7500 viral copies. None of the patients had clinically significant reactivation of EBV, HHV-6, or adenovirus warranting therapeutic intervention.

DISCUSSION
Our preclinical murine study in haplo-BMT demonstrated that PT-BEN compared to PT-CY promoted earlier and higher neutrophil counts, similar protection against early GvHD, superior control of late GvHD, and enhanced GvL effects [3]. Building on these preclinical findings, we initiated a phase I clinical trial to progressively substitute PT-CY with PT-BEN following haplo-BMT. PT-CY has been considered safe against hematopoietic stem cells as these cells express high levels of aldehyde dehydrogenase, thus detoxifying CY [10,11]. However, BEN has multiple activities as it contains a mechlorethamine group, butyric acid side chain, and a benzimidazole ring. The alkylating properties provided by the mechlorethamine group are similar to CY, while the butyric acid increases BEN's water solubility and the benzimidazole ring is believed to function as a purine analogue, affording antimetabolic characteris-