Double-unit Cord Blood Transplantation Recipients Research Articles

Robust Vaccine Responses in Adult and Pediatric Cord Blood Transplantation Recipients Treated for Hematologic Malignancies.

Because cord blood (CB) lacks memory T and B cells and recent decreases in herd immunity to vaccine-preventable diseases in many developed countries have been documented, vaccine responses in CB transplantation (CBT) survivors are of great interest. We analyzed vaccine responses in double-unit CBT recipients transplanted for hematologic malignancies. In 103 vaccine-eligible patients, graft-versus-host disease (GVHD) most commonly precluded vaccination. Sixty-five patients (63%; engrafting units median HLA-allele match 5/8; range, 2 to 7/8) received protein conjugated vaccines, and 63 patients (median age, 34 years; range, .9 to 64) were evaluated for responses. Median vaccination time was 17 months (range, 7 to 45) post-CBT. GVHD (n = 42) and prior rituximab (n = 13) delayed vaccination. Responses to Prevnar 7 and/or 13 vaccines (serotypes 14, 19F, 23F) were seen in children and adults (60% versus 49%, P = .555). Responses to tetanus, diphtheria, pertussis, Haemophilus influenzae, and polio were observed in children (86% to 100%) and adults (53% to 89%) even if patients had prior GVHD or rituximab. CD4(+)CD45RA(+) and CD19(+) cell recovery significantly influenced tetanus and polio responses. In a smaller cohort responses were seen to measles (65%), mumps (50%), and rubella (100%) vaccines. No vaccine side effects were identified, and all vaccinated patients survived (median follow-up, 57 months). Although GVHD and rituximab can delay vaccination, CBT recipients (including adults and those with prior GVHD) have similar vaccine response rates to adult donor allograft recipients supporting vaccination in CBT recipients.

Evaluation of Initial Telomere Length and Changes after Transplantation in Adult Double-Unit Cord Blood Transplant Recipients

Cord blood (CB) leukocytes have inherent telomere length (TL) variation, and CB hematopoietic stem cells (HSC) can maintain high telomerase levels preventing telomere attrition in vitro. We evaluated TL changes in 13 adult double-unit CB transplant (CBT) recipients. In the 26 units, we observed a marked variation in CB TL at thaw (median, 9.99 kilobases [kb]; range, 6.85 to 13.5). All 13 patients engrafted. Of 11 engrafting with 1 unit, there was no correlation between unit dominance and TL (mean dominant unit TL, 8.84 kb ± 1.76; mean nonengrafting unit TL, 10.3 kb ± 1.81; P = .77). Serial measurements of TL up to 1 year after CBT demonstrated an overall mean 3.04 kb ± .16 TL decrease with only 1 patient exhibiting telomere maintenance. In summary, initial TL does not predict CB unit dominance. Moreover, our analysis suggests neonatal hematopoiesis makes a transition to an HSC characterized by changes in average TL and potentially low telomerase asymmetric cell division in adult CBT recipients. Further investigation of alterations in telomere length and its clinical implications after transplantation of this observation are indicated.

General and Virus-Specific Immune Cell Reconstitution after Double Cord Blood Transplantation

Cord blood transplantation (CBT) is curative for many patients with hematologic malignancies but is associated with delayed immune recovery and an increased risk of viral infections compared with HLA-matched bone marrow or peripheral blood progenitor cell transplantation. In this study we evaluated the significance of lymphocyte recovery in 125 consecutive patients with hematologic malignancies who underwent double-unit CBT (DUCBT) with an antithymocyte globulin–containing regimen at our institution. A subset of 65 patients was prospectively evaluated for recovery of T, natural killer (NK), and B cells, and in 46 patients we also examined viral-specific T cell recovery against adenovirus, Epstein-Barr virus, cytomegalovirus, BK virus, respiratory syncytial virus, and influenza antigen. Our results indicate that in recipients of DUCBT, the day 30 absolute lymphocyte count is highly predictive of nonrelapse mortality and overall survival. Immune recovery post-DUCBT was characterized by prolonged CD8+ and CD4+ T lymphopenia associated with preferential expansion of B and NK cells. We also observed profound delays in quantitative and functional recovery of viral-specific CD4+ and CD8+ T cell responses for the first year post-CBT. Taken together, our data support efforts aimed at optimizing viral-specific T cell recovery to improve outcomes post-CBT.

Disease-Free Survival in Adult Patients with Acute Leukemia and Advanced CML Supports Use of Double-Unit Cord Blood Grafts As an Immediate Alternative to 8/8 HLA-Matched Unrelated Donors (URD)

Background: Double-unit cord blood transplantation (DCB-T) is a rapidly available alternative to unrelated donor transplantation (URD-T) for patients with high-risk acute leukemia or advanced CML. Retrospective analyses in adult DCB-T suggest that double-unit CB grafts may be associated with improved disease-free survival (DFS). However, the prioritization of URD-T vs DCB-T is controversial.Methods: We evaluated 175 consecutive adult allograft recipients (120 URD-T and 55 DCB-T) aged 16-60 years transplanted 10/2005-11/2012 for acute leukemia in morphologic remission or aplasia (113 AML/ biphenotypic, 50 ALL), or advanced CML (n = 12). URD grafts were 7-8/8 HLA-matched (74 8/8, 46 7/8). CB grafts were 4-6/6 donor-recipient HLA-matched (4 6/6, 51 5/6, 55 4/6). All patients received either high dose or reduced intensity myeloablative conditioning. The majority of URD-T recipients (n = 111, 93%) received T-cell depleted (TCD) grafts with rabbit ATG, whereas GVHD prophylaxis for DCB-T was calcineurin-inhibitor/mycophenolate mofetil.Results: The median ages of URD-T (43 years) and DCB-T (42 years) recipients were similar (p = 0.713). Distributions of gender, recipient CMV positivity, HCT-CI scores, time from diagnosis or relapse to transplant, diagnoses, disease risk, and percentage of patients with minimal residual disease pre-transplant were also similar. Neutrophil engraftment was slower in DCB-T (95%, median 24 days) than URD-T (100%, median 11 days) (p <0.001). While the incidence of grade II-IV acute GVHD at day 100 was lower in TCD URD-T recipients (15%) than in unmodified URD-T (56%) and DCB-T (55%), p = 0.002, the incidence of day 100 grade III-IV acute GVHD was similar in TCD URD-T, unmodified URD-T, and DCB-T recipients (p = 0.794). With a comparable survivor follow-up [URD-T median 51 months (range 15-99) vs DCB-T median 46 months (range 15-92)], transplant-related mortality was similar (3-year estimates: URD-T 25% vs DCB-T 24%, p = 0.838) whereas the relapse risk was decreased after DCB-T (3-year estimates: URD-T 23% vs DCB-T 9%, p = 0.008). Overall, the 3-year DFS after URD-T was 52% and 68% after DCB-T (p = 0.056). When split into 3 groups, the 3-year DFS was 59% in 8/8 URD-T, 40% in 7/8 URD-T, and 68% in DCB-T, p = 0.043 (Figure). Multivariate analysis was performed to determine risk factors for disease relapse or death in the 175 patients (Table). Female gender (HR 1.65, p = 0.029), diagnosis of ALL (HR 2.11, p = 0.002), and mismatched URD-T (HR 1.97, p = 0.027) were each significantly associated with treatment failure.Conclusions: DCB-T can achieve favorable DFS in adults with acute leukemia and CML with low relapse rates. In this series, multivariate analysis demonstrated that mismatched URD-T was independently associated with lower DFS. Our findings support use of DCB-T as an immediate alternative for high-risk acute leukemia and advanced CML in adult patients without a readily available 8/8 allele HLA-matched unrelated volunteer donor. This could have the additional benefit of speeding time to transplant in high-risk patients.TableVariableMultivariateHR (95% CI)P-valueMale FemaleReference 1.65 (1.05-2.59)0.029 Recipient CMV Negative Recipient CMV PositiveReference 1.34 (0.85-2.12)0.201HCT-CI score 0-2 HCT-CI score > 3Reference 1.56 (0.98-2.47)0.059AML/CML ALLReference 2.11 (1.30-3.41)0.002DCB-T 8/8 URD-T 7/8 URD-TReference 1.32 (0.72-2.41) 1.97 (1.08-3.60)- 0.365 0.027 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantitative Assessment of T Cell Repertoire Recovery after Double Unit Cord Blood Transplantation Demonstrates Early T Cell Diversity and Correlation Between CMV Reactivation and CD8 Clonal Domimance

Introduction: Double-unit cord blood transplantation (DCBT) is a viable therapy for adults with high-risk hematologic malignancies who lack an adult donor. However, lack of transfer of memory T cells in the graft is associated with an increased risk of viral infections. To study immune reconstitution, we recently described a novel method that combines 5′ rapid amplification of complementary DNA ends (RACE) PCR and deep sequencing to quantify T cell receptor (TCR) diversity after allogeneic hematopoietic stem cell transplant (van Heijst, Nat Med 2013). In that study, we showed that recipients of DCBT recover TCR diversity comparable to healthy donors by 12 months. We now report results of a prospective analysis of CD4+ and CD8+ T cell repertoire recovery in DCBT recipients and correlation with clinical outcomes.Methods: We prospectively collected samples from 33 DCBT recipients. The median age was 45 years (range 26-71), 18 (55%) were CMV seropositive, and the majority (n = 17, 52%) had non-European ancestry. Diagnoses included 20 (61%) acute leukemias and 13 (39%) lymphomas. Conditioning was myeloablative (n=1, 3%), reduced intensity (n=28, 85%), or non-myeloablative (n=4, 12%), and all patients received GVHD prophylaxis with cyclosporine-A and mycophenolate mofetil and no ATG. Patients received double-unit CB grafts (4-6/6 HLA-A,-B antigen, -DRB1 allele donor-recipient matched); this was supplemented with haploidentical CD34-selected PBSC in 18 patients. The 66 units had a median donor-recipient HLA-allele match of 6/8 (range 3-8). Infused total nucleated cell doses were 2.3 (1.7-3.3) and 1.9 (1.3-2.5) for the larger and smaller units, respectively. Samples were collected from the DCB grafts, recipient day+21 bone marrow, and peripheral blood at days +30, 60, 90, 120, 180 and 365 post-transplant. TCR-β sequences from each sample were amplified and sequenced using the Illumina/MiSeq sequencing platform after isolation of CD4+ and CD8+ T cells. TCR abundances were assessed at the level of clonotype and TCR diversity was calculated using inverse Simpson index.Results: Of the 33 patients, long-term samples were obtained in 25 patients, short-term samples (≤ day 100) in 6 patients who died early after DCBT, and no samples other than the graft for 2 patients. The remainder of the results focuses on 25 patients with complete samples. As previously shown, there is a 1-2-log increased diversity in CD4+ vs. CD8+ T cells (Figure). Furthermore, median CD4+ steady-state diversity is achieved early by day 60. In contrast, there is a higher rate of clonal dominance in CD8+ compared to CD4+ T cells (24/25 vs. 11/25, p=0.0001 by Fisher test). Several patterns of clonal dominance were observed, including two main patterns in CD8+ T cells. In 7/24 patients, clonal dominance is established by day 60 and persists throughout, whereas in 12/24 patients, clonal dominance fluctuates throughout follow-up. In CD4 +T cells, where less dominance is observed, a similar distribution is seen, though prolonged clonal dominance is rare. Interestingly, some of the dominant clones can be detected in the graft and are present in the day 21 marrow sample. Persistent clonal dominance in CD8+ T cells was seen 6/9 patients with CMV reactivation, whereas ongoing fluctuation was seen in 9/12 patients without CMV reactivation. In 2 patients with fluctuating clones who reactivated CMV, 1 had low level and the other a late viremia. In contrast, no link to a specific pattern was observed in patients with HHV6 viremia or acute GVHD. Finally, when we assessed similarity in clonal distribution between time points, there was more similarity in CD8+ than CD4+ T cells.Conclusions: This novel deep TCR repertoire sequencing provides a quantitative picture of T cell recovery after DCBT and supports the following: 1) separate analysis of CD4+ and CD8+ T cell populations is critical given different patterns of recovery in T cell subsets; 2) there is significant turnover in CD4+ clones but with overall limited dominance, whereas there is less turnover in CD8+ clones; 3) although the grafts contain predominantly naïve T cells, the clonal evolution of CD8+ T cells strongly suggests generation of virus-specific T cells that control viral infection; and 4) CMV appears to be an important driver of CD8+ T cell clonal expansion after CBT. Ongoing analyses are correlating immune recovery with cord blood unit dominance as well as the biology of GVHD and relapse. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of 129 Myeloablative Double-Unit Cord Blood Transplantation Recipients Demonstrates an Independent Association Between Non-Dominant Unit TNC Dose and Engraftment Suggesting a Facilitation Effect

▪Introduction: Double-unit cord blood (CB) transplantation (dCBT) has been investigated to augment engraftment in patients without an adequately dosed single CB unit. However, only one unit engrafts in the majority of patients and the role, if any, of the non-dominant unit is not established.Methods: To investigate whether the non-engrafting unit facilitates engraftment of the dominant unit, we studied 129 patients (median age 34 years, 76% > 16 years) who underwent myeloablative dCBT for hematological malignancies at our center in 10/2005-8/2013. Chimerism testing at 21 and 28 days after dCBT was used to identify dominant and non-dominant CB units which could be assigned even in cases of clinical graft failure based on bone marrow chimerism.Results: The cumulative incidence of neutrophil engraftment at day 45 was 95% (95%CI: 90-98) and occurred at a median of 24 days (range 12-43). The dominant CB unit was the only unit detected in the peripheral blood by day 28 in 111 patients (86%) whereas both the dominant and non-dominant units were detected in the remaining 18 patients [median non-dominant unit contribution to chimerism 20% (range 6-48)]. Recipients of units that had close unit-unit HLA- allele match (7-9/10 vs 1-6/10) were more likely to have both CB units detected at day 28 (27% vs 9%, respectively, p = 0.018). However, there was no engraftment advantage associated with persistence of the non-dominant unit 21-28 days post-dCBT with a median neutrophil recovery of 27 days (range 14-34) in those co-engrafting with both units vs 24 days (range 12-43) in those engrafting with a single unit. In univariate analyses, dominant unit infused total nucleated cell (TNC) dose, infused viable CD34+ cell dose, infused viable CD3+ cell dose, infused viable CD3-56+16+ cell dose, and percentage of viable CD34+ cells post-thaw (CD34+ cell viability) were all significantly associated with neutrophil engraftment. However, the TNC (Figure) and CD3+ cell doses of the non-dominant unit were also associated with neutrophil engraftment on univariate analysis [hazard ratio (HR) 1.35, p < 0.001 and HR 1.16, p < 0.001, respectively] whereas non-dominant unit CD34+ cell and CD3-56+16+ cell doses were not (HR 1.18, p = 0.107 and HR 1.06, p = 0.159, respectively). Neither donor-recipient HLA-match of dominant or non-dominant units, nor unit-unit HLA-match, were associated with time to neutrophil engraftment. Notably, on multivariate analysis, the dominant unit infused viable CD34+ cell dose, dominant unit CD34+ cell viability, and the non-dominant unit infused TNC dose were each independently associated with the speed and success of neutrophil engraftment (Table). To further assess potential effects of the non-dominant unit TNC dose, we evaluated the subset of patients in whom haematopoiesis by day 28 was purely derived from the dominant unit. Among these 111 patients, non-dominant unit TNC dose remained independently associated with engraftment in multivariate analysis (HR 1.21, p = 0.026). Finally, we hypothesized that the non-dominant unit TNC dose effect may be seen primarily in patients whose dominant units had a relatively low infused viable CD34+ dose. Indeed, analysis revealed the non-dominant unit TNC effect was only evident in the two-thirds of patients (n = 86) whose dominant unit infused viable CD34+ cell dose was low (< 1.20 x 105/kg, HR 1.37, p = 0.006). There was no association in patients whose dominant unit had a higher infused viable CD34+ cell dose (HR 1.03, p = 0.799).Conclusions: While infused viable CD34+ cell dose of the dominant unit is critical, the non-dominant unit TNC dose was also independently associated with neutrophil engraftment after dCBT. Therefore, despite their lack of engraftment, non-dominant units may facilitate engraftment of the dominant units in myeloablative dCBT recipients. These data suggest dCBT is associated with interactions that foster engraftment of the dominant unit. This finding supports continued investigation of dCBT, especially in adults who are more likely to receive units with low CD34+ cell doses. Furthermore, the mechanisms that underlie this observation warrant investigation.TableTable (n = 129 dCBT recipients)Multivariate AnalysisHR95% CIp valueDominant unit viable CD34+ cell x 105/kg (continuous)1.721.41-2.10< 0.001Dominant unit CD34+ cell viability (%)1.031.00-1.050.026Non-dominant unit TNC x 107/kg (continuous)1.191.01-1.400.035 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Frequent Human Herpesvirus-6 Viremia But Low Incidence of Encephalitis in Double-Unit Cord Blood Recipients Transplanted Without Antithymocyte Globulin

Cord blood transplantation (CBT) is a known risk factor for human herpesvirus-6 (HHV-6) infection. We analyzed the nature of HHV-6 infections in 125 double-unit CBT recipients (median age, 42 years) transplanted for hematologic malignancies with calcineurin inhibitor/mycophenolate mofetil prophylaxis and no antithymocyte globulin. One hundred seventeen patients (94%) reactivated HHV-6 by quantitative plasma PCR (median peak, 7600 copies/mL; range, 100 to 160,000) at a median of 20 days (range, 10 to 59) after transplantation. HHV-6 encephalitis occurred in 2 patients (1.6%), of whom 1 died and 1 recovered with therapy. No association was found between high-level HHV-6 viremia (≥10,000 or ≥25,000 copies/mL) and age, diagnosis, conditioning intensity, or dominant unit characteristics or between high-level viremia and transplant outcomes (engraftment, cytomegalovirus reactivation, day 100 grades II to IV acute graft-versus-host disease, day 100 transplant-related mortality, or 1-year disease-free survival). HHV-6 therapy delayed the onset of cytomegalovirus reactivation. Interestingly, HHV-6 resolution was observed in untreated patients, and resolution of viremia correlated with absolute lymphocyte count recovery. We observed a low incidence of encephalitis and no association with CBT outcomes. Our data suggest therapy in uncomplicated viremia may not be warranted. However, further investigation of the risk-to-benefit of HHV-6 viremia treatment and standardization of PCR testing is required.

Intensified Mycophenolate Mofetil (MMF) Dosing Every 8 Hours Is Safe From The Standpoint Of Engraftment and May Ameliorate Severe Acute Graft-Versus-Host Disease (GVHD) After Double-Unit Cord Blood Transplantation (CBT)

Background Acute graft-versus-host disease (aGVHD) is common after double-unit cord blood transplantation (CBT) with an incidence of grade II-IV aGVHD as high as 55% by day 180 in patients transplanted without ATG. aGVHD is associated with increased morbidity and transplant-related mortality (TRM). Mycophenolate mofetil (MMF) combined with a calcineurin-inhibitor is commonly used to prevent GVHD after CBT. However, unlike the 1 gram (gm) every 8 hours dosing that is now standard in adult donor allografts, MMF dosing in CBT has traditionally been every 12 hours. Our center has increased MMF dose from 1 gm every 12 (q12) to 1 gm every 8 (q8) hours in an effort to reduce severe aGVHD after double-unit CBT. However, the efficacy of this intervention is not established and a theoretical concern is that intensified MMF dosing could result in an increased risk of delayed engraftment or graft failure. Methods We evaluated 171 double-unit CBT recipients (median age 39 years, range 0.9-71) transplanted with either myeloablative (MA, n = 133) or non-myeloablative (NMA, n = 38) conditioning for high-risk hematologic malignancies between 10/2005 and 4/2013. CB units were 4-6/6 HLA-A, -B antigen, -DRB1 allele matched to the recipient (16 6/6, 171 5/6, 155 4/6). All patients received GVHD prophylaxis with intravenous calcineurin-inhibitor (predominantly CSA) and MMF from day -3 without ATG. Prior to 9/2009, 80 patients (47%) received MMF 1 gm IV q12 (and those &lt;12 years received 15 mg/kg/dose). From 9/2009, 91 patients (53%) received MMF at 1 gm IV q8 for patients both &gt;12 years and ≥50 kg (or 15 mg/kg/dose if &gt;12 years but &lt;50 kg, or 20 mg/kg/dose if &lt;12 years). The Gray's test compared the incidence of GVHD across the q12 and q8 dosing while the log-rank test was used to compare progression-free survival (PFS). Results Patient characteristics and infused viable CD34+ cell doses were similar in the q12 and q8 MMF groups. A comparison of transplant outcomes by MMF dosing is shown (Table). There were no differences in the time to neutrophil or platelet engraftment and the median time to count recovery was also similar. The incidences of grade II-IV aGVHD at day 100 were similar in the groups (46% vs 52%). However, there was a suggestion of decreased grade III-IV aGVHD at day 100 in the q8 MMF group (12%) versus the q12 group (21%), although this comparison did not reach significance. With a median follow-up of 41 months (range 3-94), the PFS in each dosing group was not significantly different. Conclusions We demonstrated that intensified q8 MMF dosing is safe from the standpoint of engraftment and toxicity. There was a suggestion of decreased severe grade III-IV aGVHD in recipients of 1 gm IV every 8 hours. While a larger analysis is needed to further investigate these findings, the results are encouraging. They suggest that, as with adult donor allografts, intensified q8 hour MMF dosing should be the dose investigated for the prevention of aGVHD in CBT recipients transplanted with CSA/ MMF prophylaxis without ATG. Finally, aGVHD prophylaxis could be further optimized in CSA/ MMF-based CBT by mycophenolic acid trough level drug monitoring, early post-transplant biomarker measurements such as ST2, and delayed taper post-transplant. Disclosures: No relevant conflicts of interest to declare.

Graft-versus-Host Disease after Double-Unit Cord Blood Transplantation Has Unique Features and an Association with Engrafting Unit-to-Recipient HLA Match

Manifestations of and risk factors for graft-versus-host disease (GVHD) after double-unit cord blood transplantation (DCBT) are not firmly established. We evaluated 115 DCBT recipients (median age, 37 years) who underwent transplantation for hematologic malignancies with myeloablative or nonmyeloablative conditioning and calcineurin inhibitor/mycophenolate mofetil immunosuppression. Incidence of day 180 grades II to IV and III to IV acute GVHD (aGVHD) were 53% (95% confidence interval, 44 to 62) and 23% (95% confidence interval, 15 to 31), respectively, with a median onset of 40 days (range, 14 to 169). Eighty percent of patients with grades II to IV aGVHD had gut involvement, and 79% and 85% had day 28 treatment responses to systemic corticosteroids or budesonide, respectively. Of 89 engrafted patients cancer-free at day 100, 54% subsequently had active GVHD, with 79% of those affected having persistent or recurrent aGVHD or overlap syndrome. Late GVHD in the form of classic chronic GVHD was uncommon. Notably, grades III to IV aGVHD incidence was lower if the engrafting unit human leukocyte antigen (HLA)-A, -B, -DRB1 allele match was >4/6 to the recipient (hazard ratio, 0.385; P = .031), whereas engrafting unit infused nucleated cell dose and unit-to-unit HLA match were not significant. GVHD after DCBT was common in our study, predominantly affected the gut, and had a high therapy response, and late GVHD frequently had acute features. Our findings support the consideration of HLA- A,-B,-DRB1 allele donor–recipient (but not unit–unit) HLA match in unit selection, a practice change in the field. Moreover, new prophylaxis strategies that target the gastrointestinal tract are needed.

Graft-Versus-Host Disease After Double-Unit Cord Blood Transplantation Has Unique Features and an Association with Engrafting Unit Recipient HLA-Match

AbstractAbstract ▪4197▪This icon denotes a clinically relevant abstract Background:Graft-versus-host disease (GVHD) has emerged as a major cause of morbidity and mortality after double-unit cord blood transplantation (DCBT), but data concerning its manifestations, treatment response, and risk factors are limited. Moreover, the incidence and clinical characteristics of GVHD after day 100 in DCBT recipients have not been well described. Methods:We evaluated the incidence and nature of GVHD in 115 DCBT recipients (median 37 years, range 0.9–69) transplanted for hematologic malignancies with either myeloablative (n = 88) or non-myeloablative conditioning (n = 27). CB units were 4–6/6 human leukocyte antigen (HLA)-A,-B antigen, -DRB1 allele match to the recipient, and all patients received calcineurin-inhibitor/mycophenolate mofetil immunosuppression without anti-thymocyte globulin. Results:With a median follow-up of 33 months (range 8–73), the cumulative incidences of grade II-IV and III-IV acute GVHD (aGVHD) at day 180 were 53% (95% CI: 44–62) and 23% (95% CI: 15–31), respectively. Among patients with grade II-IV aGVHD, the median onset was 40 days (range 14–169), but earlier for those with grade III-IV (median 35 days). The gastrointestinal (GI) tract was the most commonly affected organ (80%, 14 upper gut, 9 lower gut, 26 both), followed by skin (39%), and liver (18%). Among patients with grade II-IV aGVHD, 29 (48%) were treated with systemic corticosteroids, 27 (44%) with budesonide alone, and 4 (7%) with topical corticosteroids. Budesonide was used as the sole treatment exclusively in adults for grade II disease predominantly affecting the gut. Treatment response by day 28 was 79% and 85% to systemic corticosteroids or budesonide, respectively. Of 89 engrafted patients disease-free at day 100, 54% subsequently had active GVHD with 79% of those affected having persistent/recurrent aGVHD or overlap syndrome. Classical chronic GVHD was quite uncommon affecting only 10 patients (21%) in the study. To take into account potential confounding variables associated with day 180 grade III-IV aGVHD incidence, a multivariate Cox regression analysis was performed (Table). Grade III-IV aGVHD incidence was lower if the engrafting unit-recipient human HLA-A,-B,-DRB1 allele match was > 4/6 (HR 0.385, p = 0.031) (Figure), whereas engrafting unit nucleated cell dose and unit-unit HLA-match were not significant. Conclusions:GVHD after DCBT was common in our study, predominantly affected the gut, and had a high rate of successful response to treatment. Late GVHD frequently had acute features. Our findings support the consideration of HLA-A,-B,-DRB1 allele donor-recipient (but not unit-unit) HLA-matching in unit selection. This would represent a practice change for most transplant centers. Moreover, new prophylaxis strategies that target the gut are needed.TableMultivariate AnalysisPatient and Graft CharacteristicsHazard Ratio*P valueAge (years)0-15 (n = 23)Reference>16 (n = 92)0.920 (0.358–2.36)0.862CMV serostatusSero-negative (n = 51)ReferenceSero-positive (n = 64)0.570 (0.255–1.275)0.171Engrafting unit TNC ×107/kg< 3.0 (n = 94)Reference> 3.0 (n = 21)2.040 (0.802–5.217)0.134Engrafting unit-recipient 6 allele HLA–match1–3/6 (n = 36)Reference4–6/6 (n = 79)0.385 (0.162–0.915)0.031Unit-unit 6 allele HLA-match0–4/6 (n = 88)Reference5–6/6 (n = 27)2.204 (0.866–5.609)0.097*Includes 95% confidence interval. [Display omitted] Disclosures:Off Label Use: The use of Budesonide for acute gastrointestinal graft-versus-host disease. Giralt:Celgene: Honoraria, Research Funding.

HHV-6 Viremia Is Frequent but the Incidence of Encephalitis Is Low in Double-Unit Cord Blood Transplant Recipients Transplanted without ATG

AbstractAbstract 459 Background:While cord blood transplantation is a known risk factor for Human Herpesvirus-6 (HHV-6) reactivation, recent studies have yielded inconsistent results in regard to the level of viremia which is associated with a high risk of HHV-6 encephalitis. Moreover, the association with graft failure or transplant-related mortality is controversial. Methods:We conducted a retrospective analysis of 125 patients who underwent double-unit cord blood transplantation (DCBT) for the treatment of hematologic malignancies from 2/2006-3/2012 who were monitored for HHV-6 reactivation to examine the incidence and severity of HHV-6 viremia, the incidence of encephalitis, and the association with DCBT outcome. HHV-6 viremia was measured by quantitive PCR of HHV-6 DNA from plasma (lower limit of detection 100 DNA copies/ml). Results:Of the 125 patients (median age 42, range 1–69), 93 (74%) received myeloablative conditioning and 32 (26%) received a non-myeloablative regimen followed by 4–6/6 HLA-A, B antigen, DRB1 allele matched DCBT for the treatment of AML (N = 43, 34%), ALL (N = 24, 19%), MDS/CML/other leukemia (N = 12, 10%), or lymphoma/CLL (N = 46, 37%). No patient received anti-thymocyte globulin (ATG). Of 125 monitored patients, 117 (94%) reactivated HHV-6 to a median peak of 7,600 (range 100–160,000) copies/ml at a median onset of 20 days (range 10–59) post-DCBT. The median time to peak viremia was 23 days (range 12–62) with a median viremia duration of 10 days (range 1–60 days). Fifty-one patients (41% of total, 44% of viremic patients) developed HHV-6 > 10,000 copies/ml (median peak viremia 31,200 copies/ml at 20 days, range 12–57). Only 6 patients (5% of total, 5% of viremic patients) developed HHV-6 > 100,000 copies/ml (median peak 130,000 copies/ml at 19 days, range 14–29). HHV-6 encephalitis occurred in 2 patients (1.6%, peak viremias 13,000 and 118,000, respectively). One patient died from encephalitis and the other had a complete recovery following therapy. Four additional viremic patients had HHV-6 isolated from bronchoalveolar lavage but did not meet criteria for HHV-6 pneumonia. Using a high level viremia definition of > 10,000 copies/ml from days 14–60 (a level reportedly associated with end-organ disease and decreased survival, Dulery, BBMT 2011), viremia was not associated with diagnosis or conditioning regimen. Engrafting unit-recipient HLA-match and TNC, CD34+ and CD3+ cell dose were not associated with high level viremia. Treating viremia as a time-dependent covariate in Cox regression analysis, no association was found between viremia and neutrophil or platelet engraftment. Specifically, there were two graft failures in patients with no or low level viremia and two in patients with high level viremia. There was also no association between viremia and CMV reactivation, day 100 grade II-IV aGVHD, day 100 TRM, relapse or overall survival. A second analysis was performed to examine effects of high level viremia defined as a level > 25,000 copies/ml (N = 31 patients, the highest peak viremia quartile during days 14–60) and no associations with DCBT outcomes were detected. Conclusions:In our DCBT population, nearly all patients have reactivated HHV-6. However, the incidence of end-organ disease is relatively low. We postulate the difference between our findings and other studies reporting higher rates of HHV-6 encephalitis in DCBT recipients could be due to our exclusion of ATG from the conditioning regimen. At this time our understanding of the significance of HHV-6 after CBT is incomplete. We are currently evaluating anti-viral treatment responses, and ultimately a prospective trial is needed to better define the causality between HHV-6 viremia and transplantation outcomes, and to investigate the risk-benefits of pre-emptive therapy. Disclosures:No relevant conflicts of interest to declare.

Telomere Length Plays No Role in Clinical Unit Dominance After Double-Unit Cord Blood Transplantation

Abstract 4661 Background:Umbilical cord blood (CB) is an alternative source of allogeneic hematopoietic stem cells for transplantation and has the advantage of a reduced stringency of the required human leukocyte antigen-match. It is limited, however, by low total nucleated cell (TNC) dose and a low progenitor number in single CB units, thus restricting the use of CB transplantation (CBT) in larger children and adults. One strategy to augment engraftment is to combine 2 units from 2 different donors in a double-unit graft. We have previously shown that in most cases, one unit emerges as the sole source of hematopoiesis long-term, but as yet the mechanism of unit dominance remains unknown (Blood, 2010, 116(19):3999–4006). CB units are known to have an inherent biological variation in telomere length, the repeat sequence capping the ends of chromosomes. Telomere length variation and progressive shortening of telomere could later hematopoietic potential. Methods:We evaluated if telomere length has a role in unit dominance and how telomere length progresses over time post-transplant. We purified mononuclear cells from small aliquots of each unit of double-unit grafts and post-transplant peripheral blood (days +28, 100, 180 and 1 year) in 12 adult double-unit CBT recipients transplanted for hematologic malignancies at MSKCC. Average telomere length was measured using the TeloTTAGGG Telomere Length Assay (Roche) which utilizes Southern analysis of terminal restriction fragments (TRF) that are obtained by the digestion of isolated genomic DNA. Results:All 12 patients engrafted with one unit showing predominance. While there was a range of telomere length (5.57–12.09 kb) in the 24 units evaluated on the day of transplant, when comparing the telomere length of engrafting to non-engrafting units, there was no association between telomere length at day 0 and subsequent unit dominance. In 5 patients the engrafting unit had longer telomeres and in 7 patients the non-engrafting unit had longer telomeres. In serial assays, 7 of 12 patients demonstrated telomere length stabilization post-transplant in the dominant unit with a mean percentage loss of telomere length of 7.15% ± 2.33%. A second group (n = 5) demonstrated a decrease in telomere length with a mean percentage loss of 29.48% ± 5.12%. While this difference is significant (p = 0.0015) the clinical significance of this finding is uncertain. We are currently following these patients in order to correlate telomere length (stabilization versus decrease) with clinical outcomes. Conclusions:This data suggests that unit dominance is not influenced by telomere length. It is likely, based on emerging data from our laboratory as well as others, that unit dominance is immune mediated. However, the influence of telomere length on the quality of engraftment is of interest and analysis of this is ongoing with larger numbers of patients required to also consider the infused cell dose. It is notable that 7/12 patients had telomere length stabilization which correlates with the high levels of telomerase activity previously reported in the in vitro expansion of CB (Blood, 2004, 103(12):4440-8). Disclosures:No relevant conflicts of interest to declare.

Graft-Specific HLA-Antibodies Do Not Influence Unit Dominance and Do Not Prevent High Rates of Sustained Donor Engraftment in Recipients of Double-Unit Cord Blood Transplantation

AbstractAbstract 960 Background:Pre-transplant human leukocyte antigen (HLA)-antibodies have an established role in failed engraftment after single-unit cord blood (CB) transplantation (CBT). Their role in double-unit CBT (DCBT) engraftment, however, is more controversial. Some transplant centers advocate pre-transplant screening of the recipient for HLA-antibodies, and avoidance of units with the corresponding antigens if antibodies are detected. Methods:We evaluated the influence of pre-transplant HLA-antibodies on sustained donor neutrophil engraftment and unit dominance in 82 recipients (median 48 years, range 2–69) of 4–6/6 HLA-A, B antigen, DRB1 allele matched double-unit CB grafts, and transplanted for hematologic malignancies at MSKCC from July 2008 to July 2012. HLA-antibodies were measured by the HLA Laboratory of the American Red Cross Blood Service using Single Antigen Luminex beads and analyzed by HLA Fusion software. The cut-off for positive results was a normalized mean fluorescence intensity value > 1000. Results:Overall, 28/82 (34%) patients were positive for HLA-antibodies [16 (19.5%) had antibodies without graft specificity, and 12 (14.5%) had antibodies with graft specificity]. These patients were more likely to be female with acute leukemia and cytomegalovirus seropositive. All patients with graft-specific antibodies received myeloablative conditioning; 5 had antibodies against class I HLA, 6 against class II, and one patient had antibodies to both Class I/II. Moreover, of these 12 patients, 6 had antibodies against one unit, and 6 had antibodies against both units. Neutrophil engraftment according to antibody presence is summarized in the Table. 64 of 66 (97%) evaluable myeloablative DCBT recipients (Table 1A) engrafted at a median of 24 days (range 12–40); one patient without antibodies and one with antibodies against both units had primary graft failure, both in the setting of early onset multi-organ failure. Both patients were 100% donor with one unit but did not recover counts. Of the 6 patients with antibodies to one unit, 3 engrafted with that unit and 3 with the opposite unit. Of the 6 patients with antibodies against both units, one had clinical graft failure as described above, and the 5 others had sustained donor engraftment (4 with one unit and one with both). In engrafting myeloablative recipients, the median time to neutrophil recovery was 8 days slower in patients with graft-specific antibodies, but the engrafting unit in these patients also had the lowest infused dose: median CD34+ cell dose/kg if no antibodies 0.83 × 105/kg, non-specific antibodies 1.09 × 105/kg, and antibodies specific to graft 0.65 × 105/kg. In non-myeloablative recipients, 15/16 (94%) engrafted (Table 1B). The single patient with graft rejection and autologous recovery had HLA-antibodies that were not graft specific. Conclusions:11 of 12 double-unit CBT recipients with graft-specific antibodies engrafted successfully. While myeloablative recipients with graft-specific antibodies engrafted more slowly, this may be explained by a lower infused CD34+ cell dose of their engrafting units. Multivariate analysis of larger patient numbers will be required to further evaluate the effect of graft-specific antibodies on engraftment speed. At this time, however, the presence of graft-specific antibodies should not preclude DCBT, and whether their presence should influence graft selection is not clear. Furthermore, there is no suggestion that the presence of HLA-antibodies influences unit dominance after DCBT.TableSustained Donor Neutrophil Engraftment According to HLA-antibodies1A) Myeloablative Conditioning (n=67)Median (Range) Time to Neutrophil Recovery (> 0.5)N (%) Sustained EngraftmentNo antibodies (n=41)23 days (range 12–38)39/40 (1 non-evaluable)Antibodies: not graft-specific (n=14)23 days (range 12–37)14/14Antibodies: graft-specific (n=12)31 days (range 13–40)11/12Against engrafting unit (n=3)28 days (range 24–31)3/3Against non-engrafting unit (n=3)31 days (range 25–32)3/3Antibodies against both units (n=6)33 days (range 13–40)5/61B) Non-Myeloablative Conditioning (n=15)Median (Range) Time to Neutrophil Recovery (> 0.5)N (%) Sustained EngraftmentNo antibodies (n=13)10 days (range 8–20)13/13Antibodies: not graft specific (n=2)19 days in 1 patient (other had graft failure/autologous recovery)1/2Antibodies: graft specific (n=0)-- Disclosures:Giralt:Celgene: Honoraria, Research Funding.

Importance of day 21 BM chimerism in sustained neutrophil engraftment following double-unit cord blood transplantation

Delayed or failed engraftment remains a concern after cord blood transplantation (CBT) even when using double-unit grafts. Therefore, we analyzed the association between BM assessment performed approximately 21 days after transplantation, and the speed and success of sustained donor-derived neutrophil engraftment in 56 myeloablative double-unit CBT (DCBT) recipients. Overall, the cumulative incidence of sustained neutrophil engraftment was 95% (95% confidence intervals (CI): 89-100). Of the percentage of myeloid precursors, the BM cellularity and the total donor chimerism the total donor chimerism percentage had the most critical association with the speed and success of engraftment. DCBT recipients who were 100% donor achieved a 98% engraftment rate at a median of 22 days. This compared with 100% engraftment in patients who were 90-99% donor, but at a delayed median of 29 days and only 68% engraftment in patients <90% donor at a median of 37 days (P=0.001). Multivariate analysis was performed in the subgroup of patients who had not engrafted at the time the BM analysis was performed, the subgroup of most clinical concern. This confirmed donor chimerism was predictive of subsequent neutrophil recovery (P=0.004). These findings demonstrate the importance of the day 21 BM chimerism determinations after DCBT.

Graft-Versus-Host Disease (GVHD) After Double-Unit Cord Blood Transplantation (DCBT) Is Associated with Unique Clinical Features Including a Higher Incidence of Grade III-IV Acute GVHD in Children

Abstract 3044While the GVHD incidence after unrelated donor CBT is lower than expected for the degree of human leukocyte antigen (HLA)-mismatch, GVHD can be a serious complication and at our center has been the second most common cause of transplant-related mortality after DCBT. However, relatively little is known about DCBT GVHD manifestations, treatment response, and risk factors. Therefore, we evaluated 108 DCBT recipients (median 37 years, range 0.9–69) transplanted for hematologic malignancies. The majority had acute leukemia and high-risk disease. Patients received either myeloablative (n = 81) or non-myeloablative (n = 27) conditioning and 4–6/6 HLA-matched grafts. GVHD prophylaxis consisted of a calcineurin-inhibitor with mycophenolate mofetil, and no patient received anti-thymocyte globulin (ATG). With a median follow-up of 28 months (range 9–64), the cumulative incidences of day 180 grade II-IV and III-IV acute GVHD (aGVHD) were 52% (95%CI :42–62) and 24% (95%CI :15–32), respectively. The median onset was 40 days (range 14–161); the gut was most commonly affected (43/54, 80%) followed by skin (35/54, 65%). Twenty-five patients with mainly grade II gut aGVHD were treated with budesonide alone, 26 patients with predominantly grade III-IV aGVHD received systemic corticosteroids, and complete or partial treatment response was achieved in over 80% by day 56 of therapy. However, 41 patients had active GVHD after day 100 with the majority (25/41, 61%) having aGVHD (persistent, recurrent or late onset), particularly of the gut. Overlap syndrome and classical chronic GVHD were uncommon. Only 1 patient had oral ulceration, and no patient had moderate or severe ocular or sclerotic skin involvement, joint, or pulmonary GVHD manifestations. Univariate analysis of the association between patient/ graft characteristics and grade III-IV aGVHD showed the only significant factor associated with a higher severe aGVHD incidence was age 0–15 years (Figure). Diagnosis, patient ancestry, cytomegalovirus seropositivity, conditioning intensity, and infused cell doses/kg (total graft and engrafting-unit nucleated cell, CD34+ and CD3+) were not significant. A higher engrafting unit-recipient HLA-match of 8–9/10 was associated with a lower incidence of severe aGVHD, and a better unit-unit HLA-match of 6–10/10 was associated with a higher incidence of severe aGVHD, although these differences were not significant (p = 0.128 and 0.266, respectively). To further investigate these findings multivariate Cox regression analysis was performed (Table). Younger age was independently associated with a higher incidence of severe aGVHD (p = 0.042) whereas better engrafting unit-recipient match at 8–9/10 HLA-alleles was protective (p = 0.053). There was a trend toward better unit-unit HLA-match being associated with a higher incidence of grade III-IV aGVHD, but, surprisingly, total infused TNC/kg had no relationship. The 2-year PFS of 72% (95%CI :51–94) in children was higher than the 56% (95%CI :45–66) in adults despite their greater incidence of severe aGVHD. Nine patients (all adult) have died of GVHD including 5 patients initially treated with systemic corticosteroids and 4 with budesonide. We conclude that aGVHD after DCBT is common in the absence of ATG, predominantly affects the gut, and has a high rate of treatment response. Furthermore, GVHD after day 100 frequently has acute features. While the GVHD incidence does not preclude a high rate of survival, improved prophylaxis and treatment are needed. Notably, in contrast to single-unit CBT and adult hematopoietic stem cell transplantation, children receiving DCBT are at a higher risk for severe disease. A possible approach to reduce aGVHD in pediatric DCBT recipients with adequate CB units doses would be to prioritize high resolution HLA-match. Moreover, our data does not currently support an upper limit of infused TNC/kg in DCBT recipients. Further investigation of the biology underlying these unique observations (including the role of specific cellular subsets) should be a major priority. [Display omitted] [Display omitted] Disclosures:Off Label Use: Mycophenolate Mofetil as GvHD prophylaxis.

Double-Unit Cord Blood Transplantation (DCBT) for Acute Leukemia: High Disease-Free Survival in Adults and Children with Comparable Survival in European and Minority Patients

Abstract 3080As compared to single-unit CBT, DCBT may improve engraftment and protect against relapse. Therefore, we have adopted DCBT for both children and adults with acute leukemia, myelodysplasia (MDS), and myeloproliferative diseases (MPD). However, determinants of disease-free survival (DFS) have yet to be fully established. Furthermore, whether DFS after DCBT is comparable in patients of European and non-European ancestry is of special interest. Therefore, we analyzed the DFS of 75 DCBT recipients with acute leukemia in morphologic remission or aplasia (n = 69), and MDS/MPD with ≤ 5% blasts (n = 6) transplanted from 10/2005-4/2011. Nearly all patients had high-risk disease. Children 0–15 years (n = 23) had the following characteristics: median age 9 years (range 0.9–15); median weight 37 kg (range 7–72); 30% European; and 26% CMV sero-positive. Diagnoses were 43% AML (or biphenotypic), 52% ALL, and 4% MDS/MPD, and all received high-dose conditioning. The children received grafts with a median infused TNC × 107/kg of 3.3 (larger unit) and 2.6 (smaller unit), and 2% of units were 6/6 HLA-A, -B antigen, -DRB1 allele matched, 63% 5/6, and 35% 4/6. Adults ≥ 16 years (n = 52) had the following characteristics: median age 41 years (range 16–69); median weight 69 kg (range 47–105); 48% European; 69% CMV sero-positive; and diagnoses were 63% AML (or biphenotypic), 27% ALL, and 10% MDS/MPD. Fifty percent received high-dose and 50% reduced intensity conditioning. Their units had a median infused TNC/kg of 2.7 and 1.9, and 3% were 6/6 HLA-matched, 47% 5/6, and 50% 4/6. All patients (pediatric and adult) received calcineurin-inhibitor/ mycophenolate mofetil immunosuppression, and none received anti-thymocyte globulin. Sustained donor neutrophil engraftment was seen in 91% of children and 94% of adults at medians of 20 and 26 days, respectively. The incidence of grade II-IV acute GVHD by day 180 was 44% in children and 58% in adults. Day 100 transplant-related mortality (TRM) was 9% in children and 19% in adults. The 2-year relapse incidence was 9% in children and 6% in adults. With a median follow-up of survivors of 26 months (range 4–70), 2-year Kaplan-Meier estimate of DFS was 78% in children and 64% in adults (Figure). Differences in survival by age did not reach significance. Univariate analysis of variables potentially influencing 2-year DFS (with log rank estimates of significance) in all patients is shown in the Table. There were no differences in 2-year DFS according to ancestry, remission status, and conditioning intensity. There was also no difference in 2-year DFS according to engrafting unit-recipient HLA-match (4-6/6 or 10 allele), or engrafting unit infused TNC dose/kg. However, patients who were CMV seronegative had a higher 2-year DFS (85% vs 55%, p = 0.018). Multivariate analysis revealed recipient CMV serostatus was a predictor of DFS independent of patient age, and its effect was mediated by an influence on TRM. We have previously shown that DCBT extends transplant access to minority patients. We now demonstrate that DCBT can achieve high and comparable DFS in both European and non-European pediatric and adult patients with acute leukemia and MDS/MPD. While these are very encouraging results further investigation in racial/ethnic sub-groups is needed. Nonetheless, our findings support DCBT as an immediate alternative therapy for high-risk acute leukemia in patients without suitable unrelated volunteer donors, especially given the very low incidence of relapse. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Single-unit dominance after double-unit umbilical cord blood transplantation coincides with a specific CD8+ T-cell response against the nonengrafted unit

AbstractWe investigated the potential role of an immune reaction in mediating the dominant engraftment of 1 cord blood unit in 14 patients who received a double-unit cord blood transplantation (CBT). In 10 patients, dominant engraftment of a single donor unit emerged by day 28 after CBT. In 9 of these 10 patients, a significant subset of CD8+ CD45RO+/−CCR7− T cells, present in peripheral blood mononuclear cells and derived from the engrafting cord blood unit, produced interferon-γ (IFN-γ) in response to the nonengrafting unit. No significant population of IFN-γ–secreting cells was detectable when posttransplantation peripheral blood mononuclear cells were stimulated against cells from the engrafted unit (P < .001) or from a random human leukocyte antigen disparate third party (P = .003). Three patients maintained persistent mixed chimerism after CBT, and no significant IFN-γ–secreting cells were detected after similar stimulations in these patients (P < .005). Our data provide the first direct evidence in human double-unit CBT recipients that immune rejection mediated by effector CD8+ T cells developing after CBT from naive precursors is responsible for the failure of 1 unit to engraft. Future investigations based on these findings may result in strategies to predict a dominant unit and enhance graft-versus-leukemia effect.

Pre-Engraftment Syndrome after Double-Unit Cord Blood Transplantation: A Distinct Syndrome not Associated with Acute Graft-Versus-Host Disease

Pre-engraftment syndrome (PES) occurring after cord blood transplantation (CBT) is poorly characterized. We reviewed 52 consecutive double-unit CBT recipients treated for high-risk hematologic malignancies. PES was defined as unexplained fever >38.3 degrees C (101 degrees F) not associated with infection and unresponsive to antimicrobials, and/or unexplained rash occurring before or at neutrophil recovery. CBT recipients (median age, 38 years; range, 3-66 years) received either myeloablative (MA; n=36) or nonmyeloablative (NMA; n=16) conditioning. Sixteen patients (31%) fulfilled PES criteria: 15 with fever (median at onset, 39 degrees C [102.2 degrees F]), 13 of whom also had rash, and 1 with rash alone. The median onset was 9 days (range, 5-12 days) posttransplantation (a median of 14 days before neutrophil recovery). Sixteen patients (14 with PES and 2 with infection and possible PES) received intravenous methylprednisolone (median dose, 1mg/kg; median duration, 3 days); 15 (94%) experienced resolution of fever within 24 hours. Recurrent PES (n=3) resolved with retreatment. There was no association between the development of PES and the likelihood of sustained donor engraftment, speed of neutrophil recovery, grade II-IV acute graft-versus-host disease (aGVHD), day-180 treatment-related mortality (TRM), or survival. PES is common after CBT, precedes neutrophil recovery, is distinct from and does not predict for aGVHD, and responds promptly to short-course corticosteroid therapy.

An Animal Model That Predicts Unit Engraftment in Double Unit Cord Blood Transplant Recipients.

Patients receiving double unit cord blood transplant (DCBT) have demonstrated improved engraftment and survival compared to single unit historical controls despite the fact that only one unit is responsible for sustained donor engraftment. However, the mechanism of this advantage and unit predominance is not understood. Therefore, we investigated DCBT in NOD/SCID/IL2Rγc−/− (NOG) mice using mononuclear cells (MNC) from each unit of clinical double unit allografts with correlation of the murine engraftment with which unit engrafted in the patients. Our hypothesis was that the animal model would correlate with clinical unit engraftment. Nine patients with high-risk hematologic malignancies received myeloablative (n=5) or non-myeloablative (n=4) conditioning according to age and diagnosis, and 4–6/6 HLA-matched double unit allografts. Patients consented to ≤ 5% of each unit being used for research purposes. A minimum of 1 million MNC per mouse were transplanted by tail vein injection into sublethally irradiated NOG mice: each unit alone and then in combination in the same proportion as transplanted into the patients according to the relative TNC/kg of each unit. Experiments were established in duplicate or triplicate according to cell number available. Mice were sacrificed at 4–6 weeks and engraftment was determined by FACS analysis for human CD45+. In both human and murine DCBT recipients the contribution of each unit to engraftment was measured by quantitative PCR for informative short tandem repeat (qSTR) regions that distinguished each donor. Consistent with prior clinical studies all patients demonstrated donor engraftment by STR evaluation of day +21 bone marrow (BM) with one unit predominating (designated the “winner”). The average percent human engraftment in the murine experiments is shown in the Table (W = winning and L = losing unit as seen in the patients, DCBT is W+L). Notably, in 9/9 (100%) experiments, while each unit engrafted when transplanted alone, a single unit predominated when given as a double unit transplant which correlated with clinical unit predominance. Interestingly, there was no improvement in engraftment of the winner with the double unit co-transplant as compared to the winner transplanted alone.No.BM: (W)Spleen: (W)BM: (L)Spleen: (L)BM: DCBT (W+L)Spleen: DCBT (W+L)BM & Spleen: DCBT (W+L) Winner %131%34%27%33%24%30%100% & 100%212%12%10%2%2%16%100% & 100%35%6%1%12%15%2%100% & 100%421%29%17%24%20%26%100% & 100%533%52%20%32%31%45%100% & 100%67%11%7%14%15%25%100% & 100%725%44%46%57%45%72%100% & 100%822%53%48%78%27%22%83% & 91%988%74%68%70%38%82%100% & 100%Therefore, for the first time we have established a reliable in vivo model of DCBT using clinical specimens that predicts clinical engraftment. To date, the data suggests that unit predominance is due to an inherent advantage of the winning unit (exact mechanism to be determined) and that the presence of the losing unit does not augment the engraftment of the winner. This experimental model should permit further investigation of double unit biology.