Bone Marrow Transplantation (2014) 49, 212–218 & 2014 Macmillan Publishers Limited All rights reserved 0268-3369/14 www.nature.com/bmt

ORIGINAL ARTICLE

Early peripheral blood and T-cell chimerism dynamics after umbilical cord blood transplantation supported with haploidentical cells M Kwon, C Martı´nez-Laperche, P Balsalobre, D Serrano, J Anguita, J Gayoso, JL Dı´ez-Martı´n and I Bun˜o Single-unit umbilical cord blood (CB) SCT is limited by low total nucleated cell (TNC) dose. Co-infusion of CD34 þ cells from a third party HLA-mismatched donor, known as dual or haplo-cord transplant, reduces the period of post-transplant neutropenia and related complications. The aim of this study was to analyze the value of early post-transplant peripheral blood (PB) and T cell chimerism after 28 dual transplants regarding CB engraftment. Cumulative incidence of myeloid engraftment at 30 days was 93% with a median time to engraftment of 14 days (10–29). Patients who developed CB graft failure (n ¼ 5) showed very low percentages of CB cells on days þ 14, þ 21 and þ 28 with decreasing dynamics. On the other hand, percentages of CB cells in patients who achieved CB engraftment increased over time. Interestingly, such patients showed two distinct chimerism dynamics in PB, but all of them showed a predominance of CB T cells early after SCT with increasing dynamics over time. Early post-transplant chimerism dynamics in PB and T cells predicts CB graft failure enabling rapid therapeutic measures to be applied. On the other hand, early increasing percentages of CB T cells correlates with ultimate CB engraftment. Bone Marrow Transplantation (2014) 49, 212–218; doi:10.1038/bmt.2013.177; published online 11 November 2013 Keywords: cord blood transplantation; haploidentical donor; chimerism; graft failure

INTRODUCTION Unrelated umbilical cord blood (CB) has been increasingly used as an alternative stem cell source for adult patients eligible for allogeneic SCT, but lacking HLA-matched adult donors.1–3 However, one of the main limitations of CB transplantation is the late engraftment related to low total nucleated cell (TNC) dose, with the subsequent increased risk of serious early neutropenia-related infections and high non-relapse mortality.2,4 Single CB transplantation supported by mobilized and selected CD34 þ cells from a HLA-mismatched third party donor (TPD), known as dual or haplo-cord SCT, has shown to reduce the period of post-transplant neutropenia and related early morbidity and mortality associated with single CB transplantation.5–8 This strategy offers a rapid neutrophil recovery at the expense firstly of TPD cells with subsequent replacement and long-term engraftment of CB cells. Graft failure and graft rejection are major complications after allogeneic SCT, with a frequency as high as 23% using CB transplants.9–11 Outcome of patients who develop graft failure without a second transplantation is dismal with survival rates lower than 10%;12 however, survival rates improve significantly to 30–60% after salvage transplantation using reduced intensity conditioning regimens.13–15 From the extensive experience with CB transplantation, various risk factors have been associated with CB graft failure.16–18 Once transplantation has been performed, early detection of graft failure is important to start rapid therapeutic measures such as immunomodulation and to launch a second transplant when appropriate as soon as possible. With this objective, chimerism studies performed in the early post-transplant period have been shown to facilitate prediction of engraftment outcome in different allogeneic SCT settings including CB SCT.19–22 This is especially

true if chimerism is analyzed in different cell subsets.23 More recently, chimerism studies performed on CD3 þ cells after double CB transplantation with reduced intensity conditioning have been shown to predict unit dominance.24 The aim of this study was to analyze the early post-transplant (first 30 days) chimerism dynamics in peripheral blood (PB) and T cells after dual SCT in order to predict CB engraftment after dual SCT.

MATERIALS AND METHODS Patient and donor characteristics Between March 2004 and May 2012, 29 consecutive patients with high-risk hematological disease received a total of 31 dual transplantations in our institution. One case was excluded from this analysis due to insufficient chimerism data and two patients were excluded due to early toxic death (on days þ 13 years þ 30). Patient, donor and transplantation characteristics of the 28 transplants studied are summarized in Table 1. The study was approved by the institutional review board and all donors and recipients gave written informed consent. CB unit selection criteria included a match of at least four out of six HLA loci considering low resolution for HLA-A and HLA-B and high resolution for HLA-DR. CB units with the highest content of cryopreserved TNC and CD34 þ cells were preferable, with a minimum of 2  107 TNC/kg and 1  105 CD34 þ cells/kg (Table 1). TPD were selected among patients’ first-degree relatives or, alternatively, from unrelated individuals (Table 1). Mobilization and selection of CD34 þ cells have been previously described.7

Conditioning Conditioning regimen in 22 transplants was myeloablative as previously described.7 In two cases, 10 Gy of fractionated total-body irradiation was used instead of busulfan. Two patients received a reduced

Gregorio Maran˜o´n Institute for Health Research, Department of Hematology, Hospital General Universitario Gregorio Maran˜o´n, Madrid, Spain. Correspondence: Dr M Kwon, Gregorio Maran˜o´n Institute for Health Research, Department of Hematology, Hospital General Universitario Gregorio Maran˜o´n, C/Dr Esquerdo 46, Madrid, 28007, Spain. E-mail: [email protected] Received 10 July 2013; revised 19 September 2013; accepted 23 September 2013; published online 11 November 2013

Chimerism after haplo-cord transplantation M Kwon et al

213 Table 1. Characteristics of patients and transplants of the whole cohort and of three groups of patients according to the dynamics of CB engraftment Whole cohort (n ¼ 28)a

CB Graft Failure (n ¼ 5)

Engraftment Standard (n ¼ 14)

Early (n ¼ 9)

42 (32–44)

40 (22–62)

38 (30–51)

Age (years), median (range)

38 (22–62)

Gender, n (%) Female Male

14 (54) 12 (46)

3 (60) 2 (40)

8 (57) 6 (43)

4 (44) 5 (56)

Disease, n (%) AML ALL MDS LD Other

13 6 3 2 2

(50) (23) (11) (8) (8)

3 (60) 0 1 (20) 0 1 (20)

7 (50) 3 (21) 2 (14) 0 2 (14)

4 (44) 3 (33) 0 2 (22) 0

Conditioning, n (%) BU—FDR—CTX—ATG TT—FDR—ATG TBI containing regimen

24 (86) 2 (7) 2 (7)

5 (100) 0 0

11 (78) 2 (14) 1 (7)

8 (89) 0 1 (11)

GVHD prophylaxis, n (%) CsA þ steroids CsA þ /  MMF Recipient weight (Kg), median (range)

27 (96) 1 (4) 64 (42–87.6)

5 (100) 55 (47–77.8)

12 (86) 2 (14) 64 (42–71.8)

9 (100) 0 75 (47–87.6)

CB cells, median (range)b TNC x 107/kg CD34 þ x 105/kg

3 (2–4.7) 1.9 (0.73–3.2)

2.8 (2.2–3.1) 1.6 (0.7–2.4)

3 (2–4.7) 2 (0.9–2.9)

3 (2–4.5) 2.1 (1.5–3.2)

CB HLA match, n (%) 5/6 4/6

8 (29) 20 (71)

2 (40) 3 (60)

5 (36) 9 (64)

1 (11) 8 (89)

TPD, n (%) Related Unrelated

25 (90) 3 (10)

5 (100) 0

12 (86) 2 (14)

8 (89) 1 (11)

TPD cells, median (range)c CD34 þ x 106/kg CD3 þ x 104/kg

2.9 (1.5–3) 0.3 (0.05–1.5)

2.8 (2.4–3) 0.3 (0.05–0.9)

2.9 (2.2–3) 0.3 (0.06–1.5)

2.7 (1.5–3) 0.2 (0.09–1.2)

TPD HLA match, n (%) Haploidentical o Haploidentical

21 (75) 7 (25)

4 (80) 1 (20)

10 (71) 4 (29)

7 (78) 2 (22)

Abbreviations: AML ¼ acute myeloid leukemia; ALL ¼ acute lymphoid leukemia; ATG ¼ antithymocyte globuline; BU ¼ busulfan; CsA ¼ cyclosporine; CTX ¼ cyclophosphamide; FDR ¼ fludarabine; LD ¼ lymphoproliferative disease; MDS ¼ myelodysplastic syndrome; MMF ¼ mycophenolate mofetil; TT ¼ thiotepa; TBI ¼ total body irradiation. a28 transplants performed in 26 patients. bAfter procesing, before cryopreservation. cInfused cells.

intensity conditioning regimen due to age and comorbidities consisting of a 25% dose reduction of busulfan and a 50% dose reduction of cyclophosphamide. Another 2 patients who showed primary CB graft failure after busulfan-based conditioning were rescued with a second dual transplant using tiothepa, fludarabine and ATG as conditioning regimen. Graft versus host disease prophylaxis was previously described (Table 1).7

Chimerism analysis PB samples were obtained weekly from day þ 7 until the achievement of complete CB chimerism (absence of recipient or TPD cells with a sensitivity of 1% in BM and PB samples and 5% in T cells) and once a month thereafter. T cells were isolated by immunomagnetic means (AutoMACS; Miltenyi Biotec, Germany), as previously described.25 Chimerism analysis was performed by short tandem repeat PCR using the AmpFlSTR SGM Plus Kit (Life Technologies, Foster City, CA, USA), as previously described.26 & 2014 Macmillan Publishers Limited

Statistical analysis Quantitative variables were expressed as median and range. Qualitative variables were expressed as frequency and percentage. Analysis of normality was performed with the Kolmogorov–Smirnov test. Categorical data and proportions were analyzed using chi-squared test or Fisher’s exact test as required. Comparisons of continuous variables were evaluated using the non-parametric Mann–Whitney or Kruskal–Wallis test. The optimal cut-off point for percentage of CB cells from the chimerism analysis to predict CB graft failure was investigated using receiveroperating characteristics curves. Areas under the curves for the different receiver-operating characteristics (ROC) curves plotted and their 95% confidence interval (CI) were calculated as a measure of diagnostic accuracy of the cut-off points tested. All statistical tests were performed two sided and a P-value o0.05 was considered statistically significant. Statistical analysis was performed using the PASW Statistic 18 program. Estimates of OS and disease free survival were calculated using the Kaplan–Meier method. Estimates of Bone Marrow Transplantation (2014) 212 – 218

Chimerism after haplo-cord transplantation M Kwon et al

214 engraftment, non-relapse mortality and relapse after transplant were calculated using cumulative incidence (CI) rates. Follow-up of patients was updated on October 2012.

RESULTS Median follow-up of the whole cohort (29 patients) was 13.5 months (range 1.4–92). OS and disease free survival were 56 and 44% at 36 months, respectively. There were 11 deaths, 5 of them due to relapse. The cumulative incidence of non-relapse mortality was 25% at 3 years. Patient, donor and transplantation

CI of engraftment

1.0

Neu

characteristics of the 28 transplants included in this analysis are summarized in Table 1. Engraftment The CI of myeloid engraftment at 30 days was 93% (Figure 1a) achieved in a median of 14 days (range 10–29). The CI of platelet recovery at 60 days was 78% (Figure 1b) achieved in a median of 31 days (range 9–84). The CI of PB complete CB chimerism was 75% at 60 days and 87% at 90 days (Figure 1c), achieved in a median of 29 days

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

Plt

0.0 0

10

20

30

40

50

0

Days after transplant

PB

WBC/µL

60

80

100

600

1300

1900

100

0.8

92% 80

0.6

0.4

69% 60 52% 40

0.2 20 0.0

0 0

1.0

20

40 60 80 Days after transplant

+14 +21 +28 Days after transplant

100

TL

Lym/µL

0

100

100

0.8

80 0.6

% CB cells

CI of complete CB chimerism

40

Days after transplant

% CB cells

CI of complete CB chimerism

1.0

20

0.4

100%

200 98.5%

76%

60 40

0.2 20 0.0

0 0

20

40

60

Days after transplant

80

100

+14

+21

+28

Days after transplant

Figure 1. Cumulative incidence of Neutrophil (a) and Platelet (b) engraftment. Cumulative incidence of complete CB chimerism and box plots of the percentages of CB cells on days þ 14, þ 21 and þ 28 in peripheral blood (PB) (c, d) and T-Lymphocytes (TL) (e, f ). Median white blood cell count at days þ 14, þ 21 and þ 28 were 600/mL (range 100–3,400), 1,300/mL (200–4,100) and 1,900 (range 0–5,800), respectively. Median absolute lymphocyte count at days þ 14, þ 21 and þ 28 were 0/mL (0–500), 100/mL (0–800) and 200/mL (0–900), respectively. Grey boxes in the box plots represent the interquartile range, the horizontal bar within the grey boxes represent the median value, whiskers show maximum and minimum values, and open circles represent outlier values (more than 3/2 times of upper or lower quartiles). Neu: neutrophils; Plt: platelets; WBC/mL: Absolute white blood cells/mL, median; Lym/mL: Absolute Lymphocytes/mL, median. Bone Marrow Transplantation (2014) 212 – 218

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Chimerism after haplo-cord transplantation M Kwon et al

215 (range 14–71). The CI of T cell complete CB chimerism was 76% at 60 and 90 days (Figure 1e), achieved in a median of 20 days (range 12–49). All patients who achieved full CB chimerism showed full sustained CB engraftment in the last follow-up. Graft failure was documented in 2/28 (7%) cases. Additionally, in another 3/28 patients, engraftment occurred with TPD cells only. Overall, CB graft failure (absence of CB-specific alleles by short tandem repeat PCR at day 30) was documented in 5 cases (17%). One of the patients who showed engraftment of TPD cells only, died due to toxicity and infection after a second CB transplantation (despite CB myeloid engraftment). A second patient died due to relapse after multiple infectious complications derived from poor immune reconstitution. The third patient underwent a second haploidentical transplant but relapsed and died early after transplantation. From the two patients that showed failure of both CB and TPD grafts, one underwent a second dual transplantation (on day þ 43) with favorable outcome and the other was successfully rescued with a haploidentical graft. In our series, CB units failing to engraft matched our cellular content criteria for CB unit selection in all cases except one (0.7  105 CD34 þ cells/kg). Although the cause for CB graft failure was unknown in 1 case, poor post-thawing growth of colony forming units was most likely related to CB graft failure in 3 cases, as well as HLA-antibodies directed against CB antigens in one case. Chimerism dynamics On day þ 7 Median white blood cell counts of the whole cohort was 100/mL (0–100). Sufficient cells/DNA for chimerism analysis could be obtained from PB in 12 cases and from T cells in 1 case (Supplementary Table 1). Most determinations showed a predominant proportion of recipient cells with a median of 80% (9%–100%). CI of complete CB chimerism and percentages of CB cells on days þ 14, þ 21 and þ 28 in PB and T-Lymphocytes is shown in Figure 1 and Supplementary Table 1. Interestingly, 11 out of the 12 patients with chimerism results on day þ 7 showed an increase (from 9% to 40%) in the median percentage of TPD PB cells on day þ 14. On day þ 21 and þ 28, most samples showed dominance of CB cells both in PB and T cells. BM chimerism at day þ 28 of 24 transplants showed a median of 73% CB cells (range 0–100%). Chimerism dynamics was different between patients who experienced CB graft failure (n ¼ 5) and those who showed CB engraftment (n ¼ 23), (Figure 2). Patients who developed CB graft failure, showed very low percentages of CB cells in all three determinations with decreasing dynamics, both in PB and T cells (Figure 2). On the other hand, percentages of CB cells in those

100

PB

patients who achieved CB engraftment increased over time in different determinations. Cut-off values of CB cell percentages for the prediction of CB graft failure obtained from receiver-operating characteristics curves generated for PB and T cells at different time points within the first month after transplantation are shown in Table 2. Therefore, patients who showed o15% PB CB cells or o30% T cells CB cells on days þ 14 and þ 21 had a higher probability of developing CB failure. Moreover, among the 23 patients who showed CB engraftment, two groups with distinct chimerism dynamics were observed (Figure 3) with different times to hematological engraftment. Standard engraftment group (n ¼ 14). This group of patients showed high percentages of PB CB cells from day þ 14 (median 62%) with lower proportions of TPD cells (median 31%), maintaining high values at day þ 21 (Figure 3). Only two patients showed o10% of CB cells on day þ 14; however, both exhibited a significant increase in the following determination. Chimerism dynamics in T cells exhibited a similar pattern. BM chimerism on day þ 30 of these cases showed a median of 100% of CB cells (range 75–100%). Median time to neutrophil engraftment in this group was 19 days (range 11–29). Early engraftment group (n ¼ 9). These patients showed lower percentages of CB (median 30%) with high percentages of TPD cells (median 70%) on day þ 14 (Figure 3). On day þ 21, the

Table 2. ROC curves plot sensitivity (y-axis) versus false positive (x-axis) for different cut-off values day þ 14

PB TL

day þ 21

AUC (95% CI)

Cut-off (% CB)

AUC (95% CI)

Cut-off (% CB)

0.862 (0.699–1.000) 0.923 (0.778–1.000)

15 26

0.983 (0.939–1.000) 1.000 (1.000–1.000)

12 30

Abbreviation: CI ¼ confidence interval. Cut-off values of the percentage of CB in both peripheral blood (PB) and T lymphocytes (TL), 14 and 21 days after transplantation, for the prediction of CB graft failure obtained from the ROC curves generated. AUC indicates the relationship between sensitivity and false positive (1-specificity) of a single test. Greater AUC indicates higher sensitivity and specificity.

TL

% CB cells

80 60 40 20 0

+14 +21 +28 CB failure

+14 +21 +28 CB engraftment

+14 +21 +28

+14 +21 +28

CB failure

CB engraftment

Figure 2. Box plot of the percentages of CB cells at days þ 14, þ 21 and þ 28 in peripheral blood (PB, left) and T-lymphocytes (TL, right) in patients who experienced CB graft failure and patients who showed CB engraftment. Patients who developed CB graft failure showed very low percentages of CB cells in all three determinations with decreasing dynamics, both in PB and T cells. Percentages of CB cells in those patients who achieved CB engraftment increased over time in different determinations. & 2014 Macmillan Publishers Limited

Bone Marrow Transplantation (2014) 212 – 218

Chimerism after haplo-cord transplantation M Kwon et al

216 % TPD PB

% CB PB

% CB TL

Standard engraftment

100 80 60 40 20 0 +14

+21

+28

+14

+21

+28

+14

+21

+28

+14

+21

+28

+14

+21

+28

+14

+21

+28

Early engraftment

100 80 60 40 20 0

Figure 3. Percentages of cord blood (CB) cells at days þ 14, þ 21 and þ 28 in peripheral blood (PB) and T-lymphocytes (TL) in the standard engraftment group (upper panels) and the early engraftment group (lower panels) of patients. Patients in the Standard engraftment group showed low proportions of TPD cells and higher percentages of PB CB cells. Chimerism dynamics in T cells exhibited a similar pattern. Patients in the early engraftment group showed lower percentages of CB and high percentages of TPD cells at day þ 14. On day þ 21, the proportion of CB cells was stable or even decreased compared to the previous sample. However, T cell chimerism dynamics showed a significant increase or remained near 100% CB cells in both determinations.

proportion of CB cells was stable or even decreased (median 30%) compared to the previous sample. However, T cell chimerism showed a significant increase or remained near 100% CB cells in both determinations. BM chimerism on day þ 30 in this group showed a median of 54% of CB cells (range 27–92%). Median time for neutrophil engraftment in this group was 13 days.10–19 There were no significant differences between patients from standard and early engraftment groups regarding age, gender, diagnosis, disease status at transplant, previous therapy, CB-recipient ABO compatibility, CB-recipient HLA mismatch, TPD-recipient HLA mismatch, CB TNC and CD34 þ cells, TPD TNC and CD3 þ cells, CB colony forming units and CB bank origin. In terms of outcome, frequency of infectious complications during the first 60 days after transplantation was similar between both groups, including bacterial (92 vs 100%), viral (85 vs 88%) and probable/proven fungal infections (34 vs 22%). Early mortality was associated to infectious complications in one patient in the standard engraftment group (invasive fungal infection in a second dual transplant after primary graft failure) and in one patient in the early engraftment group (disseminated adenovirus in a heavilytreated patient with Hodgkin lymphoma). Incidences of acute graft versus host disease grade II-IV (14%) and moderate-severe chronic graft versus host disease (6%) were very low and without statistically significant differences between both groups. There were no differences in the incidence of relapse between both groups (38 vs 41% at 2 years). On the other hand, transplantrelated death occurred in one patient in the standard engraftment group and in 3 patients in the early engraftment group. DISCUSSION Chimerism studies performed in the early post-transplant period have been shown to facilitate prediction of engraftment in different allogenic SCT settings including CB SCT.19–22 After conventional allogeneic SCT, early detection of graft failure enables early salvage interventions such as manipulation of immunosuppressive therapy and/or donor lymphocyte infusion.27,28 However, in the CB SCT setting, since additional cells are not available, once graft failure is detected, a second transplant has to be planned in most cases. This is the first report on the utility of the analysis of early chimerism dynamics in whole PB and T cells for the prediction of CB engraftment after dual SCT. Our series showed a rapid neutrophil and platelet recovery, comparable to those obtained after HLA-matched PB SCT.7 CB graft failure rate (17%) was similar to Bone Marrow Transplantation (2014) 212 – 218

that previously reported.28,29 Various mechanisms have been associated with CB graft failure, including unit cell content, clonogenic capacity of CB progenitors, degree of HLA mismatch, presence of host anti-HLA antibodies, disease status at trasplant and so on.16–18 In our series, poor post-thawing growth of colony forming units was most likely related to CB graft failure in 3 cases, as well as HLA-antibodies directed against CB antigens in one case. Therefore, we believe that both post-thawing CB colony forming units assays and detection of HLA-antibodies should be used as criteria for unit selection, as previously suggested.17,18 Chimerism results showed TPD myeloid engraftment in two of these cases, which maintained them free of severe aplasia, allowing to arrange and perform a second CB transplantation in one of them. However, both cases showed a dismal outcome with multiple infectious complications most likely due to poor immune reconstitution. Therefore, rescue measures may be appropriate in this setting as soon as CB failure is detected. Dual transplants show a particular chimerism status due to the eventual coexistence of 3 cell parties: recipient, TPD and CB. The rapid neutrophil engraftment achieved is composed firstly of variable proportions of TPD superseded by permanent CB engraftment. The majority of the results at day þ 7 mostly before neutrophil engraftment, showed significant proportions of recipient cells. Neither the presence nor the absence of CB cells or recipient cells on day þ 7 from studied PB samples show correlation with CB engraftment. However, an increase in the percentage of TPD PB cells was observed from day þ 7 to day þ 14, consistently with its contribution to engraftment. On the other hand, results from subsequent determinations ( þ 14, þ 21) were clearly more informative regarding engraftment, especially in terms of chimerism dynamics more than considering absolute values. Patients who showed o15% of CB cells in PB or o30% in T cells on days þ 14 and þ 21 showed a higher probability of developing CB failure (Table 2; Supplementary Table 1). Additionally, chimerism showed opposite dynamics between patients who achieved CB engraftment and those who developed CB graft failure. Hence, patients who show either low percentages of CB in PB/T cells or a decreasing dynamics in the percentage of CB cells are likely (80% of cases) to develop CB graft failure. In this context, changes in immunosuppressive therapy (tapering) and/or a second salvage graft must be considered. Interestingly, two distinct chimerism dynamics were observed among the 23 patients who did show CB engraftment. Patients in & 2014 Macmillan Publishers Limited

Chimerism after haplo-cord transplantation M Kwon et al

217 the early engraftment group achieved faster neutrophil engraftment compared to those from the standard engraftment (13 vs 19 days), with higher percentages of TPD cells in PB. In this group, an early T cells full CB chimerism enables CB engraftment and ultimate replacement of TPD cells. The relative delay in neutrophil engraftment of the standard engraftment group could be due to an early CB unit dominance, which leads to a lower contribution of the TPD cells. However, time to engraftment of this group was still short compared to other CB transplant modalities, including double CB SCT. Therefore, even low proportions of TPD cells at early stages after transplantation contribute to improve leukocyte engraftment. Prior studies have shown a critical role for T cells in facilitating engraftment after BM and PB SCT, with higher graft failure rates after T cell-depleted allografts.29 In the double CB transplantation setting using reduced intensity conditioning regimen, the number of infused T cells seems to be also associated with enhanced engraftment and unit dominance.30 Moreover, early T cells chimerism predicts the long-term engrafting unit in the double CB setting.24 Similarly, our findings show that a high early posttransplantation CB T cells chimerism, and more importantly, an increasing T cells chimerism dynamics between days þ 14 and þ 21, correlates with long-term CB engraftment. Therefore, CB T cells also seem to play a key role in the ultimate CB engraftment in the dual transplant setting. Further studies analyzing the number of infused T cells from a single CB unit in this context could render practical information regarding additional cell criteria for unit selection. In summary, these findings have important practical clinical implications in the early management of patients undergoing dual SCT. Early chimerism dynamics predicts the probability of CB graft failure enabling rapid therapeutic measures to be applied. On the other hand, early increasing percentages of CB T cells, correlates with ultimate CB engraftment, leading to a conservative strategy irrespectively of the speed of hematological engraftment.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We wish to thank Jose´ Marı´a Bello´n from the Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n for statistical analysis. This work was partially supported with grants (PI05-2505, PI08-1463, PI11-007089, RD12/0036/0061) from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain and the Spanish Association Against Cancer.

AUTHOR CONTRIBUTIONS Conception and design: MK and IB. Provision of study materials or patients: MK, CML and IB. Collection and assembly of data: MK, CML, PB and IB. Data analysis and interpretation: All authors. Manuscript writing: MK and IB. Final approval of manuscript: All authors.

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