Bone marrow haploidentical transplant with post-transplantation cyclophosphamide: does graft cell content have an impact on main clinical outcomes?

ARTICLE IN PRESS Cytotherapy 000 (2020) 1 8

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Bone marrow haploidentical transplant with post-transplantation cyclophosphamide: does graft cell content have an impact on main clinical outcomes? Luciana Teofili1,2,#,*, Patrizia Chiusolo1,2,#, Caterina Giovanna Valentini1, Elisabetta Metafuni1, Silvia Bellesi1, Nicoletta Orlando1, Maria Bianchi1, Sabrina Giammarco1, Simona Sica1,2, Andrea Bacigalupo1,2 1 2

Fondazione Policlinico A. Gemelli IRCCS, Roma, Italy
Cattolica del Sacro Cuore, Roma, Italy Istituto di Ematologia, Universita

A R T I C L E

I N F O

Article History: Received 31 October 2019 Accepted 11 January 2020 Available online xxx Key Words: bone marrow cyclophosphamide engraftment graft-versus-host disease haploidentical transplant

A B S T R A C T

We analyzed data relative to cell content in 88 consecutive patients receiving HLA haploidentical bone marrow (BM) transplants with post-transplantation cyclophosphamide (PT-CY). Median age was 54.5 (range, 17 72); diagnoses were acute leukemia (n = 46), lymphoproliferative disorders (n = 24), myelofibrosis (n = 11) and myelodysplastic syndromes (n = 5). Total nucleated cell (TNC) and CD34+, CD3+, CD4+ and CD8+ cell doses were stratified as higher than first, second and third quartile and the dose effect on various clinical outcomes was assessed. Median time to engraftment was 17 days for neutrophils and 24 days for platelets. To receive a dose of TNC 3.2 x 106/kg or CD34+ cells 2.7 x 106/kg significantly shortened the time to neutrophil and platelet engraftment and reduced the blood product requirements in the 30-day period after transplantation. Overall, TNC and CD34+ cell doses had no effect on acute graft-versus-host disease (GVHD) incidence, whereas patients receiving higher CD3+ and CD8+ cell doses seemed to have less chronic GVHD. No effect on non-relapse mortality, progression-free survival and overall survival was observed at different cell dose thresholds. These data suggest that in HLA haploidentical BM transplant with PT-CY, appropriate cell doses are relevant to the engraftment. The association between low CD3+/CD8+ cells and chronic GVHD deserves further investigation. © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.

Introduction The option to receive hematopoietic stem cells (HSCs) from a haplo-identical donor has significantly expanded the chances to access transplantation for patients with hematologic cancers [1]. This practice was initially accompanied by high rates of graft failure and severe graft-versus-host disease (GVHD) [2 4]. Nevertheless, the introduction of post-transplantation cyclophosphamide (PT-CY) for the prevention of GVHD has profoundly changed the outcome of patients receiving a haplo-HSC transplant [5,6]. The practice of haplo-HSC transplantation (haplo-HSCT) with PT-CY has rapidly sprouted, in association with different types of conditioning regimens or additional GVHD prophylaxis refinements [7 16]. Both peripheral blood (PB) and bone marrow (BM) grafts are currently used as a source for HSCs. The studies comparing the

* Correspondence: Luciana Teofili, MD, Istituto di Ematologia, Fondazione Policlinico A. Gemelli IRCCS, I-00168 Roma, Italy. E-mail address: luciana.teofi[email protected] (L. Teofili). # These author contributed equally to the study.

transplant outcomes according to the graft types indicate no advantage in terms of overall survival and engraftment [17 21]. An increased relapse risk for myeloid malignancies has been reported in BM recipients, whereas a haplo-HSC transplant from PB seems to be associated with a higher incidence of acute and chronic GVHD (aGVHD and cGVHD, respectively) [18 20]. It is, therefore, conceivable that also in the haplo-HSCT setting with PT-CY, the abundant lymphocyte content in the graft might influence GVHD incidence and severity, as occurs in transplants without PT-CY [21]. Considering the great expansion of PB donation, the majority of reports on haplo-HSCT with PT-CY include patients receiving either BM or PB grafts. Nevertheless, in the specific setting of BM haplo-HSCT with PT-CY, the influence of cell graft composition on main clinical outcomes has been rarely investigated. In this study, we retrospectively revised engraftment, GVHD incidence and survival estimates of a cohort of haplo-HSCT patients homogeneously treated at our institution with a PT-CY modified regimen [8]. The main objective was to understand if BM graft characteristics might have a relevant clinical impact in this context.

https://doi.org/10.1016/j.jcyt.2020.01.007 1465-3249/© 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.

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Methods Study population Eighty-eight consecutive patients receiving haplo-BM HSCT at the Hematology Transplant Unit of Fondazione Policlinico A. Gemelli IRCCS of Rome (Italy) between January 2016 and December 2018 were retrospectively investigated. The study followed the tenets of the Declaration of Helsinki received the approval from the Institutional Review Board of Hematology Institute of Fondazione Policlinico A. Gemelli IRCCS (02/2019). Graft characteristics BM was collected from both posterior iliac crests under general anesthesia, with a maximal target volume of 25 mL/kg of the donor body weight. All donors had one or two perioperative autologous blood transfusions during the collection procedure. Total nucleated cell (TNC) counts of BM grafts were analyzed using the ADVIA 2120 (Siemens Healthcare GmbH, Erlangen, Germany). The amount of CD34+ cells was determined using the BD Stem Cell Enumeration Kit (BD Biosciences, Mountain View, CA, USA). BM CD3+ lymphocyte subset was determined using directly conjugated monoclonal antibody CD3-FITC (BD Biosciences). Flow cytometry acquisition was performed using a BD FACS CANTO II instrument. Data were analyzed using the BD CANTO software for CD34+ cell enumeration and using the BD DIVA software for lymphocyte subset quantification. All cell doses were expressed as cell number per kilogram of the recipient’s body weight. ABO major, minor and bidirectional donor-recipient mismatch were also considered. Study endpoints and definitions The following study endpoints were investigated: cumulative incidence of neutrophil and platelet (platelet) engraftment (i.e. the day of achievement of an absolute neutrophil count [ANC]  0.5 x 109/L and a platelet count  20 x 109/L unsupported by transfusion, respectively), with death in absence of engraftment as competing risk; cumulative incidence of aGVHD (grades II IV and grade III IV) and cGVHD (all grades and moderate severe), with death without GVHD as competing risk; transfusion requirements (i.e., the number of red blood cells [RBCs] and platelet units, either as apheresis or pool platelet products transfused over a 30-day or for 6-month period after bone marrow transplantation [BMT]); immune recovery (expressed as the concentration of total lymphocytes and of CD3+, CD4+, CD8+ and natural killer lymphocyte subsets at day +30, +60 and +90); overall survival (OS; i.e., the probability of being alive at any time point, calculated from transplantation until death for any cause, with surviving patients censored at the last follow-up); progression free-survival (PFS; calculated from transplantation until death for any cause or relapse); and non-relapse mortality (NRM; i.e., death without prior relapse). Disease risk index (DRI) was defined according to Armand et al.[22]. Hematopoietic cell transplantation comorbidity index (HCT-CI) was defined according to Sorror et al.[23]. Diagnosis and grading of aGVHD and cGVHD were made according standard criteria [24 27]. Statistical analysis Continuous variables were expressed as median (with range or interquartile ranges [IQRs]) and categorical variables as n (%). Univariate analysis for continuous and categorical variables was performed using the Mann-Whitney U test and the Fisher exact test, as appropriate. OS and PFS were obtained with Kaplan-Meier method. CI of neutrophil and platelet engraftment, acute and chronic GVHD and NRM were calculated using the competing risk analysis. Correlation analysis was performed using nonparametric Spearman correlation test. Comparison between time-dependent curves was performed according to

log-rank test (OS and PFS) or Fine and Grey method (cumulative incidence of engraftment, GVHD and NRM) and expressed as hazard ratio (HR), with relative 95% confidence intervals (95% CI). The combined effect of variables on different outcomes was evaluated using Cox regression analysis in a model including all variables with a P value <0.05 at univariate analysis, adjusted for variables of interest. Results were expressed as odds ratio (OR) with relative 95% CI. A P value <0.05 was considered statistically significant. Analyses were performed using the IBM SPSS Statistics 25.0 and NCSS 10 v 10.0.19. Results Eighty-eight consecutive patients (46 males and 42 females) were included in the study. The characteristics of study population are shown in Table 1. Sixty-four patients were affected by myeloid malignancies (48 acute myeloid leukemia [AML], 11 primary or post-myeloproliferative neoplasm myelofibrosis [MPN] and five myelodysplastic syndrome [MDS]) and 24 by lymphoid malignancies (15 acute lymphoblastic leukemia [ALL], seven Hodgkin’s or non-Hodgkin’s lymphomas and two chronic lymphocytic leukemia [CLL]). All BM donors were haplo-identical relatives. Fifty patients received a myeloablative (MA) regimen, consisting either of total body irradiation (TBI) and fludarabine (Flu-TBI, 10 patients) (TBI 3 4 Gy on days -8, -7 and -6 and fludarabine 30 mg/m2 on days -5, -4, -3 and -2) or TBF (thiotepa 5 mg/kg on day -6 and -5, busulfan [Bu] 3.2 mg/kg on days -4, -3 and -2, fludarabine 50 mg/m2 on days -4, -3 and -2). Twentyeight patients received a non-myeloablative (NMA) regimen consisting of fludarabine 50 mg/m2 on day -4, -3 and -2 followed by 2 Gy TBI. Ten patients received reduced conditioning (RIC) TBF, with a reduced dose of Bu (3.2 mg/kg for 2 days in nine patients, and for Table 1 Characteristics of patients and donors. Patients Age, years, median (range) Males (%)/females (%) Weight, kg median (range) Myeloid neoplasms, patients (%) Acute myeloid leukemia Chronic myeloproliferative neoplasms Myelodysplastic syndromes Lymphoid neoplasms, patients (%) Acute lymphoblastic leukemia Hodgkin/non-Hodgkin lymphoma Chronic lymphocytic leukemia Disease risk index score, patients (%) Low/intermediate High/very high Complete remission at transplantation, patients (%) Positive CMV serology, n (%) HCT-CI score, patients (%) 2 >2 Conditioning regimen, n (%) MA NMA Donors Age, y, median (range) Males (%)/females (%) Donor to recipient relationship, n (%) Offspring Sibling Parent Positive CMV serology, n (%) ABO major mismatch, n (%) ABO minor mismatch, n (%) ABO bidirectional mismatch, n (%)

N = 88 54.5 (17 72) 46 (52.3)/42 (47.7) 70 (45 113) 64 (72.7) 48 (54.5) 11 (12.5) 5 (5.7) 24 (27.3) 15 (17.0) 6 (6.8)/1 (1.1) 2 (2.3) 50 (56.8) 38 (43.2) 48 (54.5) 80 (90.9) 49 (55.7) 39 (44.3) 50 (56.8) 48 (43.2) 34 (14 62) 54 (61.4)/34 (38.6) 48 (54.5) 32 (36.4) 8 (9.1) 56 (63.6) 21 (23.8) 13 (14.7) 3 (3.4)

HCT-CI, hematopoietic cell transplantation-comorbidity index; TNC, total nucleated cells; MNC, mononuclear cells; MA, myeloablative; NMA, non-myeloablative; CMV, cytomegalovirus.

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Table 2 Unmanipulated BM graft cell characteristics.

Age, years Body weight, kg Harvest volume, mL TNC x 109/L CD34+ cells x 106/L CD3+ cells x 109/Lb CD4+ cells x 109/Lb CD8+ cells x 109/Lb

All (n = 88)

Male (n = 54)

Female (n = 34)

34 (29 43) 71.5 (62.0 80.0) 1429 (1176 1525) 19.1 (16.4 22.0) 1.66 (1.39 2.16) 1.47 (1.27 1.94 ) 0.79 (0.64 1.02) 0.63 (0.47 0.86)

34 (2 43) 77.0 (69.5 85.0) 1509 (1364 1631) 18.8 (13.5 21.5) 1.74 (1.41 2.18) 1.47 (1.28 1.93) 0.80 (0.67 1.04) 0.66 (0.46 0.86)

35 (29 43) 62.0 (56.5 68.2) 1278 (1097 1440) 19.7 (15.4 22.8) 1.52 (1.32 2.05) 1.58 (1.17 2.04) 0.80 (057 0.97) 0.59 (0.50 0.92)

Pa 0.401 <0.001 <0.001 0.451 0.208 0.810 0.549 0.907

Median values and IQR range are given. a P values refer to the difference between male and female donors. b Lymphocyte subset data were available only in 64 grafts not subjected to erythrosedimentation.

1 day in one patient). In statistical analysis, conditioning regimens were evaluated as MA versus NMA/RIC. GVHD prophylaxis was carried out according to a modification of the original Baltimora study [5]. In particular, PT-CY was scheduled on days +3 and +5 (instead of +3 and +4) at 50 mg/kg, cyclosporine A (CSA) was given at 2 mg/kg intravenously from day 0 to day +20, with target blood levels of 200 400 ng/mL, and then orally until day +180 and mycophenolate (MMF) was administered at 15 mg/kg every 12 h from day +1 to day +28 [8,15]. Graft characteristics Table 2 shows detailed characteristics of grafts as evaluated by the main features of donors. A median graft volume of 1429 mL (IQR 1176 1525) was collected, corresponding to a median amount of 20.2 mL/kg (IQR 17.4 21.9) of the donor body weight. As expected, a lower amount of BM was collected in female donors, whereas there was no difference in graft cell counts between male and female donors (Table 2). There was a tight correlation between TNC and CD34+ cell content (Spearman correlation coefficient r = 0.528, P < 0.001). The median age was 34 (IQR 29 43): donor age did not correlate with graft cellularity (r = 0.072 for TNC; r = -0.062 for CD34+ cells) and similar cell amounts were harvested in donors aged over or below 34 years (P = 0.977 for TNC and P = 0.378 for CD34+ cells). The thresholds corresponding to 25th, 50th and 75th centiles were selected to analyze the impact of cell doses on transplant outcomes: they were 3.2 x 108/kg, 3.9 x 108/kg and 4.8 x 108/kg for TNC, and 2.7 x 106/kg, 3.7 x 106/kg and 5.2 x 106/kg for CD34+ cells, respectively. Neutrophil and platelet engraftment Among 88 patients, neutrophil engraftment was achieved in 74 cases and platelet engraftment in 69. The median time to engraftment was 17 days (95% CI, 16 18) for neutrophils and 24 days (95% CI, 21 27) for platelet. The estimated cumulative incidence of engraftment was 85.1% (95% CI, 77.8 93.1) for neutrophils and 80.9% (95% CI, 72.6 90.1) for platelet (Figure 1A). Among non-engrafted patients, eight died before day +30 due to transplant-related complications and one at day +32 with persistent disease. Five patients had primary graft failure: four of them underwent successful retransplantation after 38, 53, 34 and 87 days, respectively, whereas one patient died on day +44 without retransplantation. There was a slight trend for primary failure among patients receiving a CD34+ cell dose <2.7 x 106/kg (60% versus 40%; P = 0.086). Both male and female patients with a female donor received fewer CD34+ cells (3.2 x 106/kg, IQR 2.3 4.8 versus 4.0 x 106/kg, IQR 2.9 5.4 for female patients, P = 0.046; 3.0 x 106/ kg, IQR 2.0 4.4 versus 4.0 x 106/kg, IQR 2.7 5.3, for male patients, P = 0.031). On the whole, day 30 engraftment was more frequent among patients receiving a dose of CD34+ cells 2.7 x 106/kg, for

both neutrophils (65.7% versus 16.3%; P = 0.014) and platelets (PLT) (52.5% versus 11.3%; P = 0.060). Table 3 summarizes median times to neutrophil and platelet engraftment according to the different cell doses, and shows no advantages for TNC 3.2 x 106/kg and for CD34+ cells 2.7 x 106/kg. In particular, cumulative incidences of day-30 neutrophil and platelet engraftment were 80.6% (95% CI, 71.7 90.6) versus 61.9% (95% CI, 44.2 86.6) and 62.7% (95% CI, 52.1 75.4) versus 42.8% (95% CI, 26.1 70.2) in patients with and without CD34+ cells 2.7 x 106/kg, respectively (P = 0.030 and P = 0.020 at log-rank test; Figure 1B). Beyond graft characteristics, we observed faster engraftment in patients with ALL (P = 0.012 for neutrophil and P = 0.002 for platelet engraftment), in patients who underwent transplantation while in complete remission (CR) (P = 0.022 for neutrophil and P = 0.045 for platelet engraftment) and after MA conditioning regimen (P = 0.018 for neutrophil and P = 0.004 for platelet engraftment). Moreover, patients with HCT-CI >2 had lower incidence of platelet engraftment (P = 0.029). Including all significant variables in a multivariate regression model and adjusting the model for patient age and cell doses, TNC 3.2 x 108/ kg and CD34+cells 2.7 x 106/kg were reliable predictors of neutrophil engraftment (odds ratio [OR], 2.240, 95% CI, 1.259 3.988, P = 0.006 for TNC and OR, 2.316, 95% CI, 1.207 4.444, P = 0.011 for CD34+ cells; Table 4). Notably, the dose of CD34+ cells, but not that of TNC, was able to overcome the negative impact due to the absence of CR at transplantation (Table 4). TNC and CD34+ cells predicted neutrophil and platelet engraftment also if evaluated as continuous variables (OR, 1.287, 95% CI, 1.057 1,567, P = 0.012, for TNC; OR, 1.178, 95% CI, 1.047 1.326, P = 0.006, for CD34+ cells). We obtained very similar findings regarding platelet engraftment (OR, 2.031, 95% CI, 1.089 3.787, P = 0.025 for TNC and OR, 2.454, 95% CI, 1.266-4.756, P = 0.007 for CD34+ cells; Table 4). Neither the content of CD34+ cells nor that of TNC or T-lymphocyte subsets seemed to be associated to the achievement of full chimerism at day +30 (Table 5). Infections and immune recovery Blood stream infection was documented in 11 (12.5%) patients, and invasive fungal infection in two (2.3%). Among them, seven died due to infection. We found a similar infection rate among patients grouped according to median TNC and CD34+ cell doses. Similarly, we failed to find a protective effect of higher TNC and CD34+ cell counts from infection-related mortality (data not shown). Cytomegalovirus (CMV) viremia developed in 53 patients (48 with positive CMV serology at transplantation); CMV activation rate was similar among groups receiving grafts with different cell quantities. In a subgroup of 33 patients, the effect of graft cell content on the immune recovery was investigated at days +30, +60 and +90. Patients were grouped according to the median doses of TNC and CD34+ cells. Immune reconstitution was similar in patients receiving grafts with high or low TNC and CD34+ cell content.

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Figure 1. Graft cell content and engraftment. Cumulative incidence of neutrophil and platelet engraftment in the entire population (A) and in patients receiving CD34+ cells <2.7 x 106/kg (dotted curves) and CD34+ cells 2.7 x 106/kg (continuous curves) (B). Mean values are shown. Dashed area indicate 95% CI.

GVHD Eighteen patients developed grade II IV aGVHD, which was of grade III IV in six cases. The day +100 cumulative incidence of aGVHD was 34.0% (95% CI, 24.8 46.7) for grade II IV forms and 6.3% (95% CI, 2.7-14.8) for grade III IV forms. We failed to observe any significant effect of TNC and CD34+ cell content on the incidence of aGVHD, either at univariate or multivariate analysis. Moreover, we observed no evidence that higher doses of CD3+, CD4+ and C8+ cells promoted aGVHD. We found a high incidence of grade III IV aGVHD in patients affected by primitive or post-MPN myelofibrosis, as high as 29.5% (95% CI, 11.4 76.7; P = 0.007). The 1-year cumulative incidence of cGVHD was 26.1% (95% CI, 18.0 37.7) for all forms and 9.7% (95% CI, 4.7 19.8) for moderate severe cGVHD. According to the graft cell subsets, we found a trend for a reduced incidence of cGVHD in patients receiving a CD34+ cell dose 2.7 x 106/kg (P = 0.086). Moreover, the incidence of all grade and severe moderate cGVHD was inferior in patients receiving

CD3+ cells 25.8 x 106/kg (19.4%, 95% CI, 11.6 32.2, P = 0.010, and 4.7%, 95% CI, 1.6 14.2, P = 0.008, respectively; Figure 2A). A similar association was observed for CD8+cells 13.6 x 106/kg and all grade cGVHD (14.2%, 95% CI, 6.7 29.8, P = 0.010) and CD8 +cells 10.9 x 106/kg and severe moderate cGVHD (4.7%, 95% CI, 1.6 14.2, P = 0.012; Figure 2B). No association was found between CD4+ cell subpopulation and cGVHD incidence. An increased incidence of all forms of cGVHD was observed in patients with previous II IV aGVHD (60.7%, 95% CI, 44.0 83.9, P < 0.001). At multivariate analysis, neither T-cell subsets nor previous aGVHD were confirmed to predict cGVHD. Transfusion requirements From transplantation to day +30, each patient received an average amount of four RBC units (IQR 2 7) and seven platelet units (IQR 4 14). Table 6 illustrates the effects of different variables on transfusion requirements and clearly shows that increasing thresholds of

Table 3 Times to neutrophil and platelet engraftment according to cell doses. TNC x 108/kg

< 3.2  9

Days to ANC 0.5 x 10 /L Days to PLT 20 x 109/L CD34+ cells x106/kg Days to ANC 0.5 x 109/L Days to PLT 20 x 109/L

20 (17 31) 29 (21 43)

17 (15 21 (17 < 2.7  21 (16 33) 17 (15 31 (20 42) 22 (17

< 3.9 

P 20) 32) 20) 31)

0.008 0.015 P 0.014 0.018

18 (16 26) 26 (20 35)

17 (15 20 (17 < 3.7  18 (6 26) 17 (15 25 (18 37) 21 (17

< 4.8 

P 20) 31) 20) 32)

0.087 0.066 P 0.088 0.142

18 (16 24) 25 (18 34)

17 (15 20 (17 < 5.2  17 (16 24) 17 (16 25 (17 34) 20 (17

Patients were grouped according to thresholds corresponding to the 25th, 50th and 75th percentiles. Engraftment data represent median values with IQR.

P 19) 26) 20) 27)

0.082 0.065 P 0.375 0.181

ARTICLE IN PRESS L. Teofili et al. / Cytotherapy 00 (2020) 1 8 Table 4 Multivariate Cox regression analysis models of neutrophil and platelet engraftment according to delivered doses of TNC and CD34+ cells. Risk ratio Neutrophil engraftment Patient age* ALL Complete remission at transplantation MA conditioning regimen TNC  3.2 x 108/kg Patient age* ALL Complete remission at transplantation MA conditioning regimen CD34+ cells  2.7 x 106/kg Platelet engraftment Patient age* ALL Complete remission at transplantation HCT- CI >2 MA conditioning regimen TNC  3.2 x 108/kg Patient age* ALL Complete remission at transplantation HCT- CI >2 MA conditioning regimen CD34+ cells  2.7 x 106/kg

95% CI

P

0.991 0.927 2.022 1.421 2.240 0.989 1.017 1.557 1.522 2.316

0.975 0.508 1.172 0.814 1.259 0.973 0.549 0.875 0.860 1.207

1.007 1.690 3.487 2.483 3.988 1.007 1.883 2.772 2.694 4.444

0.288 0.804 0.011 0.216 0.006 0.229 0.958 0.132 0.149 0.011

0.991 0.821 1.267 0.854 1.626 2.031 0.982 0.958 0.980 0.845 1.672 2.454

0.972 0.420 0.716 0.443 0.847 1.089 0.964 0.487 0.549 0.442 0.868 1.266

1.006 1.605 2.242 1.645 3.120 3.787 1.008 1.883 1.747 1.615 3.220 4.756

0.223 0.564 0.414 0.638 0.147 0.025 0.061 0.901 0.946 0.611 0.149 0.007

* Considered as continuous variable.

BM cell doses resulted in a significant reduction of the amount of transfused products. The effect on RBC units was evident only for increasing TNC doses, whereas escalating threshold of either TNC or CD34+ cells resulted in a proportional reduction of RBC and PLT requirements. In addition, recipients of major ABO mismatch BM grafts displayed a lower RBC need, probably in consequence of the high amount of RBCs contained in the graft. Regarding further variables, older age, absence of CR at transplantation, HCT-CI >2 and diagnosis of MPN were all associated with higher RBC and PLT requirements, whereas patients with lymphoid neoplasms needed fewer RBCs (patients with ALL) and PLT units (patients with ALL and lymphoma). The favorable effect of increasing graft cell doses on transfusion requirements was confirmed in a multivariate analysis model including transfusion needs categorized as high or low (< or 4 RBC units, < or 7 PLT units) and incorporating either TNC or CD34+ cells as continuous variables. Increasing doses of TNC and CD34+ cells reduced transfusion needs of both RBC units (OR, 0.527, 95% CI 0.323 0.860, P = 0.010 for TNC; OR, 0.722, 95% CI, 0.534 0.757, P = 0.033 for CD34+ cells) and PLT units (OR, 0.483, 95% CI, 0.294 0.797, P = 0.014 for TNC, OR, 0.590, 95% CI 0.428 0.826, P = 0.002 for CD34+ cells). Additional variables predicting low PLT requirement were MA conditioning regimen (OR, 0.255, 95% CI,

Table 5 Day-30 complete chimerism rate according to delivered cell doses. Number of patients

All cases TNC x 108/kg 3.2 3.9 4.8 CD34+ cells x 106/kg 2.7 3.7 5.2 CD3+ cells x 106/kg 25.8 34.9 39.1

Day-30 complete chimerism N (%)

88

9 (10.2)

59 40 20

7 (10.6) 4 (9.1) 2 (9.1)

60 39 19

6 (9.1) 5 (11.4) 3 (13.6)

60 39 19

7 (10.4) 5 (11.4) 3 (13.6)

5

0.088 0.734, P = 0.011) and ALL diagnosis (OR, 0.160, 95% CI, 0.029 0.906, P = 0.038). Finally, patients with HCT-CI >2 required more RBC (OR, 2.980, 95% CI, 1.020 8.711, P = 0.046) and PLT units (OR, 3.390, 95% CI, 1.255 9.153, P = 0.016). OS, NRM and PFS At a median follow-up of 13.7 months (IQR, 5.0 24.9), 11 of 88 patients in our series relapsed (12.1%, 95% CI, 6.4 21.3) and 22 died (25.0%, 95% CI, 16.3 35.3). Causes of death were infections (n = 12), central nervous system hemorrhages (n = 2), relapse (n = 5), GVHD (n = 2) and posterior reversible encephalopathy syndrome (n = 1). The estimated 3-year OS and PFS were 72.3% (95% CI, 62.0 82.6) and 57.2% (95% CI, 42.6 71.9), respectively, with a cumulative incidence of NRM of 22.5% (95% CI, 14.2 35.9). The main factors affecting survival were related to patient and disease status at transplantation. Patients with HCT-CI scores >2 tended to behave worse than others in terms of OS (HR2.25, 95% CI, 0.96 5.30, P = 0.051) and NRM (HR, 2.74, 95% CI, 1.04 7.23, P = 0.040). Similarly, disease status at transplantation significantly affected OS (HR, 2.37, 95% CI, 1.02 5.53, P = 0.043) and PFS (HR, 2.57, 95% CI, 1.21 5.45 for patients not in CR at BMT, P = 0.0125). In our series of patients, TNC, CD34+ cells and T-lymphocyte subsets did not influence the survival outcomes. Indeed, receiving increasing amounts of lymphocytes (either CD3+, CD4+ or CD8+) did not seem to be able to reduce relapse incidence and improve PFS in our patients. Discussion This study shows the outcome data of a cohort of 88 consecutive patients undergoing BM haplo-HSCT followed by a modified PT-CY regimen plus cyclosporine and MMF for GVHD prophylaxis. Our objective was to understand if BM graft composition may have influenced the main clinical outcomes of these patients. According to our results, the quality of graft content, in terms of delivered doses of TNC and CD34+ cell, is relevant to prompt the engraftment. In fact, in our patients, a CD34+ cell dose 2.7 x 106/kg predicted the day-30 neutrophil and platelet engraftment rate, and there was a slight trend for primary failure among patients receiving fewer CD34 + cells. In addition, at multivariate analysis, increasing doses of both TNC and CD34+ cells fostered neutrophil and platelet engraftment, while reducing transfusion requirements. Although several studies explored the role of graft composition in various transplant settings, this issue has been sparsely addressed in the context of BM haploHSCT with PT-CY. A recent study on PB haplo-HSCT with PT-CY reported faster neutrophil but not platelet engraftment in patients receiving a CD34+ cell dose >5.4 x 106/kg [28]. In addition, our data are similar to those obtained in BM transplant settings different from haplo-HSCT [29 32]. Patel et al. reported that higher CD34+ cell doses expedited platelet engraftment in all patients, and both neutrophil and platelet engraftment in the Bu/CY subgroup [29]. In a similar transplant setting including 121 patients receiving BM from HLA identical or matched sibling and matched unrelated donors, Remberger et al. demonstrated that higher TNC and CD34+ cell doses were associated with faster neutrophil engraftment [32]. Bittencourt et al. found that a CD34+ cell dose of >3 x 106/kg significantly influenced neutrophil and platelet recovery and reduced transplantrelated mortality [33]. Our results also indicate that graft compositions may influence the incidence of cGVHD, suggesting that high CD3+ and CD8+ cell doses are associated with a lower incidence of all grade and severe moderate cGVHD. Mussetti et al., in a similar BM transplant setting, found that CD34+ cells, but not CD3+ cells, are associated with an increased incidence of all grade cGVHD in recipients [20]. In line with data recently reported by Mussetti et al. [20] and Granata et al. [28] in

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Figure 2. All grade and severe moderate cGVHD incidence according to CD3+ cells and CD8+ cells. (A) Cumulative incidence of all grade and severe moderate cGVHD in patients receiving CD3+ cells <25.8 x 106/kg (dotted curves) and CD3+ cells 25.8 x 106/kg (continuous curves). (B) Cumulative incidence of all grade and severe moderate cGVHD in patients receiving higher (continuous curves) or lower (dotted curves) CD8+ cell amounts.

patients receiving PB-HSCT and PT-CY, we did not find a detrimental effect of high CD34+ cell dose in promoting cGVHD. Additionally, our observations suggest a possible protective role of CD3+ and CD8+ cells, comparing those recently reported in a larger series of patients by Patel et al. in a different BM transplant background [29]. The authors analyzed the influence of TNC, CD34+, CD3+, CD4+ and CD8+ cell doses in 359 patients accrued from 2002 to 2014, receiving BM HSCT from matched related or unrelated donors. MA conditioning regimens mainly consisted of BU and CY, or TBI and etoposide, with CSA/MMF or CSA/methotrexate (MTX) as GVHD prophylaxis [29]. In this transplant setting, a protective effect from cGVHD for CD3+ cell dose >29.2 x 106/kg was found in the MTX subset analysis (OR, 0.34, 95% CI, 0.16 0.74), whereas CD8+ cell doses >11.2 x 106/kg fostered the lymphocyte recovery in the MTX subset [29]. Indeed, it is conceivable that also in the setting of BM haplo-HSCT with PT-CY, reciprocal changes of CD4+ and CD8+ cell subsets, more than the CD3+ cell dose itself, may influence the recovery of the immune response against allogenic antigens. Alternatively, it could be hypothesized that PT-CY might spare distinct BM graft cell populations (such as plasmocytoid dendritic cells) able to induce a sustained response of alloreactive Tregulatory cells, which mitigate GVHD [29,34]. One unexpected finding of this study is the high incidence of III/IV aGVHD in patients affected by MPN. All of these patients received a RIC TBF regimen, with reduced Bu dose (6.4 or 3.2 instead of 9.6 mg/ kg). We hypothesized that the introduction of MMF and CSA before PT-CY in the setting of RIC regimens might have favored the occurrence of more severe aGVHD. Accordingly, we recently modified the immunosuppressive schedule in patients receiving RIC regimens,

moving PT-CY to day +3 and +4, and postponing the administration of CSA and MMF to day +5 [5]. None of the investigated outcomes was affected by AB0 incompatibility, without difference among matched, major- and major/minormismatched grafts, except for higher RBC transfusion requirement in ABO major-mismatched group. In a previous analysis carried out by the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation in BM haplo-T patients with AML, major ABO incompatibility emerged as a risk factor for poorer OS [34]. Conversely, our data compare with those obtained by Yang et al. in a cohort of patients with AML submitted to haplo-BMT, confirming that AB0 incompatibility does not influence engraftment, transfusion support and survival [35]. Similar findings have been recently obtained in the unrelated transplant setting, with either BM or peripheral blood graft sources [36,37,38]. On the whole, the expected 3-year OS, PFS and NRM in our cohort were 72.3%, 57.2% and 22.5%, respectively. These estimates are in line with those previously reported [11,13,15,16,18 20]. There was no association between graft cell content and main survival outcomes, but disease status and comorbidities at transplantation were the main predictors for PFS and OS. Similarly, patients with active disease at transplantation tended to have higher NRM, a finding that has been previously reported [20]. In conclusion, PT-CY has improved the safety of haplo-BMT, allowing the identification of a suitable donor for most patients. This study evidences that BM graft composition may be relevant in regard to both engraftment and cGVHD development in this transplant setting.

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7

Table 6 Effect of patient, donor and BM graft characteristics on transfusion requirements during the first 30 days from transplantation. RBC units Median (IQR range) All patients Patient age, years 40 50 60 Male gender AML ALL MPN Lymphoma MDS CLL MA conditioning regimen DRI score high/very high Complete remission HCT-CI score >2 Donor age, years 29 34 43 Female donor Female to male Major/minor ABO mismatch Major ABO mismatch TNC x 108/kg 3.2 3.9 4.8 CD34+ cells x 106/kg 2.7 3.7 5.2

P

4.0 (2.0 7.7)

Platelet units Median (IQR range)

P

8.0 (4.0 14.7)

4.5 (2.0 5.0 (3.0 6.0 (3.0 4.0 (1.0 4.0 (2.0 2.0 (0.0 12.0 (5.0 3.0 (0.0 13.0 (2.0 6.0 (6.0 4.0 (0.0 5.0 (2.0 3.0 (0.0 5.0 (3.0

9.0) 9.0) 10.5) 7.0) 6.7) 5.0) 15.0) 6.0) 15.0) 6.0) 5.0) 9.2) 5.0) 10.0)

0.053 0.060 0.004 0.332 0.519 0.019 0.001 0.195 0.183 0.383 0.002 0.062 0.000 0.010

9.0 (5.2 9.0 (6.0 9.0 (6.7 8 (4.7 9.0 (5.0 5.0 (4.0 16.0 (8.0 3.0 (1.0 11.0 (3.5 8.0 (7.0) 6.0 (3.7 9.0 (6.0 6.0 (3.0 9.0 (6.0

17.0) 17.0) 18.2) 16.2) 16.2) 7.0) 21.0) 9.0) 18.5) 11.0) 15.5) 12.5) 18.0)

0.011 0.043 0.007 0.821 0.442 0.026 0.005 0.035 0.752 0.911 0.009 0.169 0.005 0.001

4.0 (2.0 4.0 (1.0 4.0 (0.0 5.0 (3.0 5.0 (1.0 5.0 (2.0 6.0 (4.0

8.0) 9.0) 7.0) 9.2) 9.0) 9.5) 10.0)

0.949 0.507 0.289 0.105 0.665 0.221 0.009

7.0 (4.0 7.0 (3.0 4.0 (3.0 8.5 (4.7 8.0 (4.0 6.0 (4.0 7.0 (4.0

14.0) 17.5) 17.0) 17.0) 15.0) 14.0) 16.2)

0.314 0.513 0.043 0.244 0.847 0.373 0.948

4.0 (1.0 6.0) 4.0 (1.7 7.2) 3.5 (2.0 6.2)

0.031 0.375 0.488

7.0 (4.0 14.0) 6.0 (3.0 13.2) 6.0 (3.7 9.0)

0.023 0.049 0.130

4.0 (2.0 7.0) 4.0 (2.0 5.7) 4.0 (2.0 6.5)

0.195 0.219 0.738

7.0 (4.0 14.0) 6.0 (3.0 12.2) 6.0 (3.0 9.0)

0.017 0.016 0.034

P values refer to the univariate analysis.

Declaration of Competing Interest The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. Author contributions LT handled conception and design of the study, acquisition of data, analysis and interpretation of data, article writing and final approval of the submitted version. PC, CGV and EM handled interpretation of data and article writing. SB, NO, MB and SG did acquisition of data and analysis. SS and AB did the critical revision for important intellectual content. Funding No funding was received. References [1] Kanakry CG, Fuchs EJ, Luznik L. Modern approaches to HLA-haploidentical blood or marrow transplantation. Nat Rev Clin Oncol 2016;13:10–24. [2] Powles RL, Morgenstern GR, Kay HE, McElwain TJ, Clink HM, Dady PJ, et al. Mismatched family donors for bone-marrow transplantation as treatment for acute leukaemia. Lancet 1983;1:612–5. [3] Beatty PG, Clift RA, Mickelson EM, Nisperos BB, Flournoy N, Martin PJ, et al. Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med 1985;313:765–71. [4] Szydlo R, Goldman JM, Klein JP, Gale RP, Ash RC, Bach FH, et al. Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings. J Clin Oncol 1997;15:1767–77. [5] Luznik L, O'Donnell PV, Symons HJ, Chen AR, Leffell MS, Zahurak M, et al. HLAhaploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant 2008;14:641–50.

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