Hematology, National Research Cancer Center - Istituto Tumori Giovanni Paolo II, Bari, Italy;

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1 DCTH CASE REPORT An alternative strategy for cord blood stem cells transplant to reduce time of neutrophils engraftment: case report of co-infusion of haploidentical and cord blood stem cells Rosa Angarano 1, Irene Donnini 2, Alberto Bosi 2 1 Hematology, National Research Cancer Center - Istituto Tumori Giovanni Paolo II, Bari, Italy; 2 Hematology Department, AOU Careggi, Università degli Studi di Firenze, Italy SUMMARY Umbilical cord blood (UCB) stem cells are frequently employed for allogeneic stem cell transplants, but delayed myeloid and lymphoid immune reconstitution leads to an increased risk of transplant related mortality. We present a case-report of a combined haploidentical and umbilical cord blood transplant in a high risk patient affected by refractory acute lymphoblastic leukemia. The possibility of overcoming some of the main limitations of cord blood transplants leads to an improvement in the short-term outcomes of UCB transplantation by quickening hematopoietic recovery. INTRODUCTION Allogeneic-stem cells transplantation (Allo-SCT) is considered a potentially curative treatment for a variety of malignant and non-malignant hematological disorders. Approaches for those lacking matched family donors or well-matched unrelated donors (MUD) include HLA-mismatched unrelated umbilical cord blood (UCB), hap- Key words: Cord blood transplantation, haploidentical hematopoietic stem cell transplantation. Correspondence: Irene Donnini Hematology Department, AOU Careggi Università degli Studi di Firenze, Italy irdonni@tin.it loidentical family donors or partially matched adult UD. Actually in our institution a patient who lacks a related donor goes to alternatives procedures (Figure 1) in a simultaneous research, spending about 30 days. First option is MUD research 8/8 matched, then single cord blood unit option with almost 3x10 6 total nucleated cell dose (TNC)/ kg, a double cord blood unit with a minimum of 2x10 6 TNC/kg each one and then haploidentical transplant, an alternative strategy that needs at least 50% of HLA matched. The use of different options and different conditioning regimens depend on each patient, considering type of disease, age, number of previous treatment, hematopoietic cell transplant co-morbidity index (1) and all anamnestic information that

2 126 R. Angarano, et al. FIGURE 1 Algoritm of alternative options of allogeneic transplant in our Institution. can influence transplant. In the setting of alternative transplants, cord blood transplants (CBT) was introduced into the clinical practice and has evolved into an effective option for the treatment of a number of hematological malignancies and non-malignant disorders (2), above all in children but also in adults. CBT has garnered considerable interest because of its easy availability, low incidence of acute (agvhd) and chronic graft-versushost disease (cgvhd), low incidence of disease recurrence and relatively robust immune reconstitution. Late engraftment, with the subsequent increased risk of serious early neutropenia-related infections, the prolonged length of hospital stay and also high non relapse mortality (NRM), are considered some of the main limitations of CBT (2). One major disadvantage of this procedure is the low dose of TNC/ kg available; TNC transplanted per kg of body weight of recipients correlates with outcomes and NRM (2). Currently, the cut-off value of cord blood TNC at freezing is considered 3x10 7 /kg of recipient weight. The limited numbers of progenitor cells in the UCB units result in delayed and unpredictable recovery count, particularly in adults. The engraftment probability after the CBT depends on time: it peaks at +21 day from transplant, the median halves at +31 and drops to 5% at day +45 (Eurocord data) with a risk of graft failure as well. Currently, many approaches have been explored to facilitate CBT myeloid engraftments. Examples include injecting cord blood cells directly into the bone marrow (3), in vivo o ex vivo amplification of cord blood cells (4), use of reduced intensity conditioning (RIC) regimen (5), use of double unit UCB transplantation (6), co-infusion of mesenchymal stem cells (7) and co-infusion with a haploidentical T-cell depleted graft (8-10). In this context, single UCB transplantation with the co-infusion of mobilized and selected CD34+ cells from a haploidentical HLA-mismatched donor, known as dual SCT, may overcome the late engraftment, the low rate of engraftment using a single CB unit (6)

3 An alternative strategy for cord blood stem cells transplant to reduce time 127 TABLE 1 Haplo-cord summary of main studies. Author No. patients Age Diagnosis Conditioning (no. patients) Bautista et al. (2009) (9) Liu et al. (2011) (10) Chen et al. (2013) (13) Kwon et al. (2014) (12) (14-60) AML 30% ALL 40% Others 30% (10-69) AML/MDS39 ALL 10 NHL 7 CLL/MPD 3 50 ALL 20 AML24 CML 3 MDS 1 LNH (22-62) AML 13 ALL 6 MDS 3 LD 2 Other 2 CyTBIrATG 41 BuCyrATG 11 Flu Mel ratg (RIC) TBI-Cy 13 Bu-Cy 37 ANC; PLT: AML; MDS, ; ALL; NHL; RIC; CLL; MPD; CML; LNH; LD. Bu-Flu-CyATG 25 TT-Flu-ATG 2 TBI regimen 2 Median time to ANC Median time to PLT 10 (9-36) 32 (13-98) 11 (10-14) 19 (15-33) 13 (11-20) 15 (11-180) 14 (10-29) 31 (9-84) and related early morbidity and mortality associated with single CB unit (2, 11). Haploidentical CD34+ cells can be used as a bridge and can help the long term engraftment of the UCB unit reducing the period of post-transplant neutropenia and early related complications associated with single CBT. First results about using this alternative approach of transplant were reported by Fernandez, who showed trials on transplanted NOD/SCID mice and results on cultured cord blood cells (8). Since then, many articles have been published from 2009 up to now showing clinical trials of patients transplanted with dual SCT affected by malignant and non-malignant diseases, using different conditioning regimens (RIC or myeloablative). Regardless of the patient and donor selection or procedural differences, the most important benefit of this transplant procedure is that all groups observe rapid neutrophil recovery with a median time of 10 to 14 days (Table1) (9, 10, 12, 13). The largest group of experiences comes from the Madrid group and from the University of Chicago (9, 10) As far as cord blood is concerned, the primary goal of most trials is to enhance hematopoietic recovery (12). These two groups observe rapid neutrophil recovery that is shorter than the expected value in a single CB unit with a median time of 10 to 11 days (Table 1). To use this alternative transplant we followed the Chicago study (10), where they show a wider survey of patients (Table 1) and also because they use RIC regimen with fludarabine, melphalan and low anti-thymocyte globulin (ATG) that seems to be better tolerated in the high risk patients treated reducing TRM (5). There was no correlation between cell dose of UCB graft or haplograft and time to neutrophil or platelet recovery. In this study the majority of patients had an early haploidentical engraftment replaced by durable engraftment of UCB cells by 100 days, initial engraftment by the third-party donor was ul-

4 128 R. Angarano, et al. timately superseded by permanent UCB engraftment. Common practice for UCB unit selection focuses on cell dose at the expense of matching, but the Chicago study group support the hypothesis that UCB cell dose had no measurable impact on outcome. Analyzing chimerism on unfractionated peripheral blood shows that during the first 100 days after transplantation the haploidentical stem cell chimerism overcame the UCB cells, thereafter a chimeric switch was shown with an evolution from haplo- to cord. CASE REPORT We present a case of a young man, 25-year old, affected by acute lymphoblastic leukemia B common, with normal karyotype, primary refractory that developed an invasive fungal infection arisen during induction therapy and treated by two different antimicotics: voriconazol and caspofungin. The patient lacking HLA identical sibling and unrelated donor, underwent a dual SCT formed by a single CB unit and haploidentical stem cells from a maternal cousin. The CB unit chosen contained a suitable number of TNC for infusion (TNC 4.2x10 7 /kg), but the very high risk patient (previous infection and active disease) and the need to reduce the median time to neutrophil engraftment, led us to consider dual SCT as a new transplantation option. Dual SCT provides a valuable option for high-risk patients who lack a matching adult donor or who require urgent transplantation. In this high risk patient an early engraftment is helpful to overcome the acute septic phase in order to reduce the probability of a life threatening infection. Patient received a RIC regimen with fludarabine 30 mg/m 2 /d IV for 5 consecutive days (days-7, -6, -5, -4, -3) and melphalan 70 mg/m 2 /d for 2 doses on day -3 and -2, granulocite-cell-stimulating-factor (G-CSF) 30 MU/die from day +1. GvHD prophylaxis used consisted of rabbit anti-thymocyte globulin (thymoglobulin, r-atg) at 1.5 mg/kg every other day for 4 doses (days -7, -5, -3, and -1), Tacrolimus 0.03 mg/kg from day -2 and mycophenolate mofetil 1000 mg for 3 doses/d from day 0. The haploidentical cells were infused on day 0 (24 th October 2013) followed by CB unit at the end of haplo-infusion about one hour and an half later in the same day. Haploidentical CD34+ stem cells were selected by CliniMACS method and the value infused was 4.1x10 6 /kg; CB was a single unit HLA matched 4/6, with TNC and CD34+ value infused respectively 4.2x10 7 /kg and 0.9x10 5 /kg. At day +6 from transplant the patient was in a complete aplasia phase; on the same day a septic fever appeared with positive blood culture for Klebsiella pneumoniae carbapenem-resistant (KPC) species, treated by five different antibiotics (gentamicine, tigecicline, meropenem, vancomicine and colistin). During the following days the patient continued to be septic and febrile and needed non-invasive-ventilation (C-PAP) and support therapy with neutrophil granulocytes transfusion (Figure 2). The neutrophil value increased from day +16 until the achievement of a true engraftment at day +18 with a white blood count of 1.79x10 3 /mm 3 (Figure 1) and neutrophil count of 1.73x10 3 / mm 3. The evolution of chimerism, according to literature dates, showed an early engraftment on unfractionated

5 An alternative strategy for cord blood stem cells transplant to reduce time 129 FIGURE 2 Evolution of white blood cells (blue line) and neutrophil value (green line) during dual transplantation from day 0 to +41 in our case report. The red line and points show the granulocyte transfusion, in day +6 sepsis by Klebsiella pneumoniae carbapenem-resistant (KPC) began. The pie charts on the top show the evolution of chimerism in different period of time (day +14, +22,+28,+33,+41). The dark blue line shows the fever evolution during time. peripheral blood cells formed by median percentage of haploidentical cells (90% by day +14 and 79% by day +22) (Figure 2). For eight days (from day +14 to day +22) the patient showed an absolute neutrophil count >0.5x10 3 / mm 3. Subsequently, while the patient was still febrile, the white blood count unexpectedly declined to a secondary aplasia phase and the chimerism at day +28 began to be replaced by durable engraftment of UCB cells. The median percentage of cells of UCB origin increased from 21% by day +22, to 74% by day +28, 98% by day +33 and achieved 100% on day +41. The median time to platelet engraftment at +78 days was very low compared to dates seen in literature, and the reason could be the septic status of the patient that reduced the probability of engraftment and slowed it down. The patient was discharged at day +88 with negative blood culture but with a persistent positive rectal and underarm swab for KPC, without any viral reactivation or signs of acute GvHD. The last bone marrow biopsy, carried out on 25 th of February (day +124 from transplant), showed a complete remission of disease with a negative minimal residual disease and the chimerism was full cord (100% CB). oday, at one year from transplant, our patient is still in a complete remission with a chimerism full cord (100% CB) performed on bone marrow at day The patient had no viral reactivation and is currently in good clinical conditions. Immunosuppression was discontinued five months after transplant. The residual clinical problems in a long way of resolution are a critical illness polyneu-

6 130 R. Angarano, et al. romyophaty and heart failure with an ejection fraction of 49%, probably therapy-related. DISCUSSION AND CONCLUSIONS This combined dual-sct reducing significantly the time to neutrophil engraftment (day +18) respect to a single UCB unit transplantation, helped the patient to overcome the blood infection; the clinical symptoms of sepsis decreased and patient became more responsive to therapy. During hospitalisation, after a first period of aplasia, the white blood cells count increased to a value of 1.79 x 10 3 /mm 3 and the absolute neutrophil count remain >0.5 x 10 3 /mm 3 for eight days (Figure 2). Progressively, the white blood count declined in a second phase of aplasia from day 22 to 35, thereafter the neutrophil count increased permanently again. In this period many support therapies played a role to help the patient overcome the septic phase including a neutrophil granulocyte (Figure 2), platelet and red blood cell transfusion, C-PAP, antibiotic therapy and use of G-CSF. The neutrophil granulocyte transfusions that were carried out in the complete aplasia phase (Figure 2) may well have been important to reduce the duration of the first engraftment phase. We studied the chimeric change in the stem cells to better understand the engraftment roles, and we noticed an almost haploidentical chimerism at day +14 (90% haplo). The switch of chimerism from haplo to cord took place in a shorter median time than that described in literature, maybe due to the large cord blood cell dose (TNC=4.218x10 7 /kg) infused compared to the median dose considered in the Spanish and American groups (9, 10). Indeed, the switch began at day +28 and achieved 100% of cord at day +41. Initial engraftment by the haploidentical donor was ultimately superseded by a permanent UCB engraftment. The characteristics of the umbilical cord grafts include considerable hematopoietic potential and an immune system dominated by effectors of peripheral tolerance. Indeed immunologic mechanisms probably play a part in the UCB dominance (14). Competition between grafts of various sources is commonly observed after double UCB transplantation and, as a rule, leads to the elimination of one of the grafts over time (15). In a study of non myeloablative conditioning and double UCB transplantation, 30% of patients required more than 21 days to have neutrophil recovery (15-17). Our approach therefore may be particularly promising for patients with refractory malignancies who are at high risk of TRM with single or double UCB and at extreme risk for disease recurrence with haplo-sct (18). In summary, haplo-cord transplantation revealed reliable and fast engraftment of neutrophils and this strongly suggests that this procedure could improve the shortterm outcomes of UCB transplantation by hastening hematopoietic recovery (11-14). Our results show that dual SCT provides a valuable option for high-risk patients who lack a matching adult donor or who require urgent transplantation. Many questions and issues remain unresolved, some of which have been briefly discussed here. Thus, this transplantation approach merits broader exploration and validation in different hematological diseases in centers that treat a large number of patients.

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