Allogeneic transplantation of multiple umbilical cord blood units in adults: role of pretransplant-mixed lymphocyte reaction to predict host-vs-graft rejection

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Reduced-intensity conditioning (RIC) with conventional adult donor allogeneic graft sources is increasingly utilized for patients deemed unable to tolerate fully myeloablative conditioning due to advanced age and/or comorbidity; with the advantages of lower early treatment-related mortality and faster hematopoietic recovery.1 Use of unrelated allogeneic umbilical cord blood (UCB) grafts in the reduced-intensity setting, however, has been limited due to concerns of possible higher rates of primary graft failure.2, 3 Limited cell dose in a single UCB graft provides sound rationale for consideration of multiple UCB unit transplantation in adult patients. Early experience with two unit UCB infusions has included specified requirements for human leukocyte antigens (HLA) matching between UCB units as well as to the patient.4, 5, 6 In UCB multiunit studies to date, there has been no unit characteristic (cell dose, degree of HLA matching and so on) that predicts donor engraftment of one UCB unit over another.

This exploratory phase I prospective clinical trial was designed to determine if a minimum nucleated cell dose (5 × 107 per kg) provided by infusion of multiple (3–5) unrelated donor UCB units would improve rates and kinetics of allogeneic donor engraftment for adult recipients treated with RIC. This strategy was based on a review of UCB engraftment after myeloablative conditioning7 indicating 100% probability of allogeneic donor engraftment in pediatric recipients receiving single UCB graft cell doses exceeding 5 × 107 cells per kg.

The clinical objectives of the study were to determine the safety of this approach in adults with advanced hematologic malignancies, and to determine the rates and kinetics of UCB donor-derived engraftment. Correlative laboratory studies included measurement of host-vs–graft (HvG) T- and natural killer (NK)-cell reactivity in mixed lymphocyte cultures and their relation to clinical observations of donor engraftment.

A total of seven patients were consecutively enrolled and treated during March 2002 to March 2003 (Table 1). The median age of enrolled study patients was 56 years (range 20–69), and median weight was 77 kg (range 71–99). Four study patients had received intensive chemotherapy within 3 months prior to multi UCB unit transplantation.

Table 1 Patient demographics

The UCB graft characteristics are summarized in Tables 2a and b. Graft units (3–5) were at least an HLA 4/6 match to the patient and no matching was required between the cord blood units. UCB grafts and patient HLA typing was performed using standard molecular analyses including antigen level for HLA-A and -B loci, and allele level for HLA-DR loci. To determine overall degree of HLA matching, the total number of HLA matched loci of all grafts was divided by all HLA loci. The median overall proportion of HLA matching for the entire patient cohort was 72% (range 67–83, Table 2a). HLA matching between individual UCB units was not required. Patients received a combined median nucleated cell dose of 5.4 × 107 cells per kg (range 4.2–8.9 × 107 cells per kg), which provided a median CD34+ cell dose of 2.3 × 105 cells per kg (range 1.7–6.2 × 105 cells per kg). A median CD3+ T-cell dose of 11.5 × 106 cells per kg (range 8.8–23.7 × 106 cells per kg) was infused. Additionally, a median NK and NK/T-cell dose of 1.2 × 106 (range 0.4–9.8 × 106 cells per kg) and 5.3 × 106 cells per kg (range 4.5–12.3 × 106 cells per kg) was infused (Table 2b). Individual unit total nucleated cell doses pre-freeze included a median of 1.2 × 107 cells per kg (range 0.6–3.6 × 107 cells per kg). It is noteworthy that 2 of the 24 individual UCB units contained >2.5 × 107 nucleated cells per kg and 11 of 24 individual units contained >1.5 × 107 total nucleated cells per kg (Supplementary Table 1). No trend in overall HLA matching (67–83% in engrafting and non-engrafting patients) combined nucleated cell dose (average 5.5 × 107 cells per kg non-engrafting; and 4.9 × 107 cells per kg in engrafting patients), CD34+ graft cell dose (average 3.69 × 105 cells per kg non-engrafting; 2.13 × 105 cells per kg engrafting patients) or colony-forming units of granulocyte/macrophage (CFU-GM) infused (average 1.25 × 104 kg−1 non-engrafting; 1.97 × 104 kg−1 engrafting patients) was observed to be clearly associated with donor engraftment. The small number of study patients in this phase I cohort however, prohibited statistical comparative analyses.

Table 2 Graft characteristics

The median day to achieve an absolute neutrophil count (ANC)>500 μl−1 (either patient or donor derived) was 11.5 days (range 11–41) in the seven study patients. There were no early deaths prior to day 42, study patient 4 did not achieve ANC>500 prior to day 42, but did achieve measurable donor chimerism. Chimerism analyses of peripheral blood mononuclear cells (PBMCs) revealed predominance of donor chimerism (>90% donor) attained in only three study patients by day +100 (Figure 1). Mixed chimerism indicative of more than one UCB donor was noted only during early donor engraftment prior to day +100, with the emergence of one sustained single engrafting unit in all patients attaining donor engraftment. No secondary graft failures were observed (Figure 1). No trend in number of units infused or infusion order was observed to be associated with donor engraftment. Two patients did not achieve platelet recovery, both of whom died prior to day 100. Two patients achieved platelet recovery by day 8 and 34, respectively, and three patients never dropped below 20 000 cells per μl. The four study patients with failed donor engraftment all demonstrated autologous hematopoietic recovery. Overall, infusion of multiple UCB units was not associated with improved rates or kinetics of donor-derived hematopoietic engraftment in this limited phase I cohort.

Figure 1
figure1

Donor umbilical cord blood (UCB) unit engraftment. Percentages of donor chimerism, engrafting UCB units are represented for donor-engrafting patients. Those patients reaching an absolute neutrophil count (ANC)500 by day 42 without achieving full (>90%) donor chimerism were considered to have failed donor engraftment. Human leukocyte antigens (HLA) typing was performed on posttransplant bone marrow or peripheral blood by PCR using a panel of polymorphic short-tandem repeat DNA marker loci.

Six of seven study patients had sufficient cells available to perform carboxyfluorescein diacetate succinimidyl ester (CFSE) analyses (Figure 2). CFSE analyses of patient-derived CD3+ T cells in mixed lymphocyte reactions (MLRs) on the four study patients with graft failure demonstrated significantly increased host T-cell proliferation (ranging 4–34% CD3+ T cells cycling) in the presence of UCB graft cells over that of baseline (patient PBMC alone) host proliferation, indicating HvG antigen response (Figures 2a–d). CFSE analyses of two study patients demonstrating UCB donor engraftment, for whom sufficient cells were available, demonstrated negligible increase in host T-cell proliferation over that of baseline (patient PBMC alone) host T-cell proliferation (Figures 2e and f). Positive mitogen controls (data not shown) confirmed the ability of all patient cells to generate a proliferative response in vitro. Mean values of patient T-cell proliferation in the presence of UCB grafts (HvG) for those patients with failed donor engraftment was high: 19.4% (s.e.m. 6.0%) compared to −0.6% (s.e.m. 2.6%) HvG T-cell proliferation in patients demonstrating attained UCB donor engraftment. Patient T-cell proliferation threshold was defined as CD3+ T cells demonstrating more than one cell division by CSFE labeling at 72 h MLR (Figure 2).

Figure 2
figure2

Cell proliferation of recipient T cells. (af) Carboxyfluorescein diacetate succinimidyl ester (CFSE) proliferation analyses of patient T cells in the presence and absence of umbilical cord blood (UCB) graft units. Patient (host) CD3+ cells were gated out of total mononuclear cell (MNC) populations and CFSE-negative cells were gated out based on stimulator cell auto fluorescence (data not shown). Decrease in fluorescence intensity (peak shift to the left) indicates one T-cell division. Numbers of cells going through one or two cell divisions were measured. Symbol (Δ) indicates the change in overall proliferation between baseline patient cell cycling and patient cycling in the presence of 3–5 UCB graft units. Histograms are prepared each such that baseline proliferation activity is above that of the HvG response. Each panel represents an individual patient. Only two divisions can be shown due to high auto fluorescence of stimulator cells (data not shown). (g) Average of donor UCB engraftment (n=2 had cells available for studies) and graft failure (n=4 had cells available for studies) increase in T-cell proliferation over baseline (Δ) ±s.e.m. Patient cohort size precludes statistical analysis.

CD56+ cell cycle analyses showed variable NK proliferation in the HvG direction without clear trends to indicate subsequent UCB graft rejection. UCB graft T- and NK-cell proliferation studies were also performed in the graft-vs-graft and graft-vs-host (GvH) directions; these analyses also showed variable increases in proliferation regardless of attainment of UCB donor engraftment (data not shown). Cytokine analysis of MLR supernatants demonstrated trends in upregulation of TH1 cytokines elicited by UCB grafts in the presence of patient cells over that of baseline UCB graft–graft immune reactivity. However, there was significant interpatient variability between measured TH1 cytokine production in 72 h MLR supernatant and attainment of UCB allogeneic engraftment (Table 3).

Table 3 Cytokine production

Acute GvHD was absent to mild in all of the study patients. Three of seven patients developed grade II aGvHD (patients 2, 3 and 7), no patient developed severe aGvHD, grade III/IV. Of four patients surviving past day 100, no patient developed chronic GvHD.

Survival reflected high-risk study patients with advanced hematologic malignancies enrolled in this phase I study of UCB engraftment. Three patients died prior to day 100, of whom two died due to opportunistic infection and one with persistent leukemia. The predominant cause of death in the seven study patients was disease relapse, with average time to relapse 414 days (range 29–1016 days). Patient overall survival was 13.1 months post transplant (range 1.6–45 months). Patient-limited cohort size did not allow for comparative statistical analyses of patient outcome and survival.

We observed very unfavorable rates of UCB donor allogeneic engraftment in this phase I cohort with donor engraftment occurring in only three of seven patients indicating that transplant of 3–5 unmatched UCB units is not feasible in this setting. Additionally, these results support the hypothesis that allogeneic engraftment may not be solely determined by combined UCB graft hematopoietic stem and progenitor cell content. Our results indicate a lower rate of engraftment as compared to that observed in patients undergoing UCB transplantation after fully myeloablative conditioning (ranging 65–92%) or two UCB unit nonablative conditioning incorporating HLA matching between UCB grafts (ranging 80–95%).6 In this limited phase I study cohort, we observed robust host T-cell proliferation by CSFE staining in 72 h MLR in study patients demonstrating failure of UCB allogeneic donor engraftment. Minimal host T-cell proliferation indicative of HvG immune reactivity was observed in patients attaining UCB allogeneic engraftment. However, patient cohort size did not allow for more than exploratory analyses.

Therefore, with further study of larger patient cohorts, consideration may be given to test host T-cell proliferation in MLR as a potency assay in UCB graft selection and to serve as adjunct to HLA and UCB graft cell dose criteria, in attempt to overcome current 10–15% primary graft failure rates observed after UCB transplantation in the reduced-intensity setting. Additionally, this assay may be of particular relevance to assess patient capability to mount adverse HvG reactions if not treated with intensive chemotherapy within 3 months of attempted UCB allogeneic transplant in the nonablative setting.8 Though the logistics of cord blood freezing does not allow for prospective testing of all cryopreserved units, consideration can be taken at the time of HLA confirmatory typing, to perform short term in vitro lymphocyte reactivity studies in attempt to better predict UCB transplant outcomes. This noteworthy MLR host T-cell measurement approach, if verified in larger patient study cohorts, also has important implications for patients with nonmalignant hematopoietic disorders requiring UCB allogeneic transplant who do not receive intensive immunosuppressive chemotherapy pretransplant as that required in patients with hematologic malignancies.

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Acknowledgements

This work was supported by the Abraham J & Phyllis Katz Foundation and Donald J & Ruth Weber Goodman Philanthropic Fund.

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Correspondence to M J Laughlin.

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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Fanning, L., Hegerfeldt, Y., Tary-Lehmann, M. et al. Allogeneic transplantation of multiple umbilical cord blood units in adults: role of pretransplant-mixed lymphocyte reaction to predict host-vs-graft rejection. Leukemia 22, 1786–1790 (2008) doi:10.1038/leu.2008.55

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