Optimal Donor for African Americans with Hematologic Malignancy: HLA-Haploidentical Relative or Umbilical Cord Blood Transplant.

Although hematopoietic cell transplantation from an HLA-matched unrelated donor is potentially curative for hematologic malignancies, survival is lower for African Americans compared with Caucasians. Because only approximately 20% of African Americans will have an HLA-matched unrelated donor, many of these patients undergo HLA-haploidentical relative or umbilical cord blood transplantation. In this study, we analyzed outcomes after HLA-haploidentical related donor (n = 249) and umbilical cord blood (n = 118) transplantations in African American patients with hematologic malignancy between 2008 and 2016. The predominant disease was acute myelogenous leukemia for recipients of both types of donor grafts. The incidences of grade II-IV and III-IV acute graft-versus-host disease were higher after umbilical cord blood transplantation compared with HLA-haploidentical relative transplantation (56% and 29%, respectively, versus 33% and 11%, respectively; P < .0001). The 2-year incidence of transplantation-related mortality adjusted for age and conditioning regimen intensity was higher after umbilical cord blood transplantation compared with HLA-haploidentical related donor transplantation (31% versus 18%; P = .008); however, there were no between-group differences in the 2-year adjusted incidence of relapse (30% versus 34%; P = .51), overall survival (54% versus 57%; P = .66), or disease-free survival (43% versus 47%; P = .46). Our findings show that the use of HLA-haploidentical and umbilical cord blood transplants expands the access to transplantation with comparable leukemia-free and overall survival for African Americans with hematologic malignancies.

The clinical impact of donor-specific antibodies on antibody-mediated rejection and long-term prognosis after heart transplantation.

PURPOSE OF REVIEW: Outcomes after cardiac transplantation have improved over past decades, but long-term graft survival remains limited in part because of uncertainty regarding clinical implications of donor-specific antibodies (DSAs). The purpose of this review is to consolidate recent advances in knowledge on the topic of DSA and their potential to impact long-term prognosis after heart transplantation. RECENT FINDINGS: The presence of persistent DSA increases the risk of poor outcome after heart transplantation, including development of antibody-mediated rejection (AMR), graft failure, cardiac allograft vasculopathy, and mortality. Importantly, different DSA vary in clinical significance. DSA capable of activating the complement cascade portend a higher risk of developing AMR. human leukocyte antigen class I and class II antigens are expressed differently within the heart, and so, clinical manifestations of class I and class II DSA vary accordingly. Further, compared with class I, class II DSA carry an increased risk of graft loss and mortality. When comparing preexisting DSA with formation of de-novo DSA, de-novo DSA are associated with worse outcome. SUMMARY: DSAs are generally associated worse long-term prognosis after heart transplantation but vary in their clinical significance. Recognition of specific risk profiles is essential for guiding posttransplant antibody management.

A Phase I Study of Reduced-Intensity Conditioning and Allogeneic Stem Cell Transplantation Followed by Dose Escalation of Targeted Consolidation Immunotherapy with Gemtuzumab Ozogamicin in Children and Adolescents with CD33+ Acute Myeloid Leukemia.

Myeloablative conditioning and allogeneic hematopoietic stem cell transplant (alloHSCT) in children with acute myeloid leukemia (AML) in first complete remission (CR1) may be associated with significant acute toxicity and late effects. Reduced-intensity conditioning (RIC) and alloHSCT in children is safe, feasible, and may be associated with less adverse effects. Gemtuzumab ozogamicin (GO) induces a response in 30% of patients with CD33+ relapsed/refractory AML. The dose of GO is significantly lower when combined with chemotherapy. We examined the feasibility and toxicity of RIC alloHSCT followed by GO targeted immunotherapy in children with CD33+ AML in CR1/CR2. Conditioning consisted of fludarabine 30 mg/m2 × 6 days, busulfan 3.2 to 4 mg/kg × 2 days ± rabbit antithymocyte globulin 2 mg/kg × 4 days followed by alloHSCT from matched related/unrelated donors. GO was administered ≥60 days after alloHSCT in 2 doses (8 weeks apart), following a dose-escalation design (4.5, 6, 7.5, and 9 mg/m2). Fourteen patients with average risk AML received RIC alloHSCT and post-GO consolidation: median age 13.5 years at transplant (range, 1 to 21), male-to-female 8:6, and disease status at alloHSCT 11 CR1 and 3 CR2. Eleven patients received alloHSCT from 5-6/6 HLA-matched family donors: 8 received peripheral blood stem cells, 2 received bone marrow, and 1 received related cord blood transplantation. Three patients received an unrelated allograft (two 4-5/6 and one 9/10) from unrelated cord blood unit and bone marrow, respectively. Neutrophil and platelet engraftment was observed in all assessable patients (100%), achieved at median 15.5 days (range, 7 to 31) and 21 days (range, 10 to 52), respectively. Three patients received GO at dose level 1 (4.5 mg/m2 per dose), 5 at dose level 2 (6 mg/m2 per dose), 3 at dose level 3 (7.5 mg/m2 per dose), and 3 at dose level 4 (9 mg/m2 per dose). Three of 14 patients received only 1 dose of GO after alloHSCT. One patient experienced grade III transaminitis, which resolved; no grade IV transaminitis, no grade III/IV hyperbilirubinemia, or sinusoidal obstructive syndrome were observed. The second dose of GO was given at median of 143 days (range, 120 to 209) after alloHSCT. Probability of grades II to IV acute and chronic graft-versus-host disease were 21% and 33.5%, respectively. Probability of overall survival after RIC alloHSCT and GO consolidation at 1 and 5 years was 78% and 61%, respectively. Probability of 5-year event-free survival after RIC alloHSCT and GO consolidation in patients in CR1 was 78%. No dose-limiting toxicities probably or directly related to GO were observed in this cohort. This preliminary data demonstrate that RIC followed by alloHSCT and consolidation with GO appears to be safe in children and adolescents with CD33+ AML in CR1/CR2. A phase II trial is currently underway investigating this approach with a GO dose of 9 mg/m2 per dose.

Impact of allele-level HLA matching on outcomes after myeloablative single unit umbilical cord blood transplantation for hematologic malignancy.

We studied the effect of allele-level matching at human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 in 1568 single umbilical cord blood (UCB) transplantations for hematologic malignancy. The primary end point was nonrelapse mortality (NRM). Only 7% of units were allele matched at HLA-A, -B, -C, and -DRB1; 15% were mismatched at 1, 26% at 2, 30% at 3, 16% at 4, and 5% at 5 alleles. In a subset, allele-level HLA match was assigned using imputation; concordance between HLA-match assignment and outcome correlation was confirmed between the actual and imputed HLA-match groups. Compared with HLA-matched units, neutrophil recovery was lower with mismatches at 3, 4, or 5, but not 1 or 2 alleles. NRM was higher with units mismatched at 1, 2, 3, 4, or 5 alleles compared with HLA-matched units. The observed effects are independent of cell dose and patient age. These data support allele-level HLA matching in the selection of single UCB units.

Identification of a permissible HLA mismatch in hematopoietic stem cell transplantation.

In subjects mismatched in the HLA alleles C*03:03/C*03:04 no allogeneic cytotoxic T-lymphocyte responses are detected in vitro. Hematopoietic stem cell transplantation (HSCT) with unrelated donors (UDs) showed no association between the HLA-C allele mismatches (CAMMs) and adverse outcomes; antigen mismatches at this and mismatches other HLA loci are deleterious. The absence of effect of the CAMM may have resulted from the predominance of the mismatch C*03:03/C*03:04. Patients with hematologic malignancies receiving UD HSCT matched in 8/8 and 7/8 HLA alleles were examined. Transplants mismatched in HLA-C antigens or mismatched in HLA-A, -B, or -DRB1 presented significant differences (P < .0001) in mortality (hazard ratio [HR] = 1.37, 1.30), disease-free survival (HR = 1.33, 1.27), treatment-related mortality (HR = 1.54, 1.54), and grade 3-4 acute graft-versus-host disease (HR = 1.49, 1.77) compared with the 8/8 group; transplants mismatched in other CAMMs had similar outcomes with HR ranging from 1.34 to 172 for these endpoints. The C*03:03/C*03:04 mismatched and the 8/8 matched groups had identical outcomes (HR ranging from 0.96-1.05). The previous finding that CAMMs do not associate with adverse outcomes is explained by the predominance (69%) of the mismatch C*03:03/03:04 in this group that is better tolerated than other HLA mismatches.

Multiple mismatches at the low expression HLA loci DP, DQ, and DRB3/4/5 associate with adverse outcomes in hematopoietic stem cell transplantation.

A single mismatch in highly expressed HLA-A, -B, -C, and -DRB1 loci (HEL) is associated with worse outcomes in hematopoietic stem cell transplantation, while less is known about the cumulative impact of mismatches in the lesser expressed HLA loci DRB3/4/5, DQ, and DP (LEL). We studied whether accumulation of LEL mismatches is associated with deleterious effects in 3853 unrelated donor transplants stratified according to number of matches in the HEL. In the 8/8 matched HEL group, LEL mismatches were not associated with any adverse outcome. Mismatches at HLA-DRB1 were associated with occurrence of multiple LEL mismatches. In the 7/8 HEL group, patients with 3 or more LEL mismatches scored in the graft-versus-host vector had a significantly higher risk of mortality (1.45 and 1.43) and transplant-related mortality (1.68 and 1.54) than the subgroups with 0 or 1 LEL mismatches. No single LEL locus had a more pronounced effect on clinical outcome. Three or more LEL mismatches are associated with lower survival after 7/8 HEL matched transplantation. Prospective evaluation of matching for HLA-DRB3/4/5, -DQ, and -DP loci is warranted to reduce posttransplant risks in donor-recipient pairs matched for 7/8 HEL.

Amino acid substitution at peptide-binding pockets of HLA class I molecules increases risk of severe acute GVHD and mortality.

HLA disparity has a negative impact on the outcomes of hematopoietic cell transplantation (HCT). We studied the independent impact of amino acid substitution (AAS) at peptide-binding positions 9, 99, 116, and 156, and killer immunoglobulin-like receptor binding position 77 of HLA-A, B, or C, on the risks for grade 3-4 acute graft-versus-host disease (GVHD), chronic GVHD, treatment-related mortality (TRM), relapse, and overall survival. In multivariate analysis, a mismatch at HLA-C position 116 was associated with increased risk for severe acute GVHD (hazard ratio [HR] = 1.45, 95% confidence interval [CI] = 1.15-1.82, P = .0016). Mismatch at HLA-C position 99 was associated with increased transplant-related mortality (HR = 1.37, 95% CI = 1.1-1.69, P = .0038). Mismatch at HLA-B position 9 was associated with increased chronic GVHD (HR = 2.28, 95% CI = 1.36-3.82, P = .0018). No AAS were significantly associated with outcome at HLA-A. Specific AAS pair combinations with a frequency >30 were tested for association with HCT outcomes. Cysteine to tyrosine substitution at position 99 of HLA-C was associated with increased TRM (HR = 1.78, 95% = CI 1.27-2.51, P = .0009). These results demonstrate that donor-recipient mismatch for certain peptide-binding residues of the HLA class I molecule is associated with increased risk for acute and chronic GVHD and death.

Pilot trial of risk-adapted cyclophosphamide intensity based conditioning and HLA matched sibling and unrelated cord blood stem cell transplantation in newly diagnosed pediatric and adolescent recipients with acquired severe aplastic anemia.

BACKGROUND: Cyclophosphamide-based conditioning regimens and allogeneic hematopoietic stem cell transplantation (AlloHSCT) from matched related donors (MRD) has resulted in the highest survival rates in children and adolescents with acquired severe aplastic anemia (SAA). Time to transplant has consistently been associated with decreased overall survival. Reduced toxicity conditioning and AlloHSCT has been used successfully in other pediatric non-malignant diseases. PROCEDURE: We piloted a risk-adapted AlloHSCT approach, using fludarabine and anti-thymocyte globulin based conditioning with high (200 mg/kg) and low (60 mg/kg) dose cyclophosphamide as upfront treatment in newly diagnosed pediatric patients with acquired SAA incorporating alternative donor sources, including cord blood. Average risk for non-engraftment patients with <10 transfusions received low dose cyclophosphamide (60 mg/kg); High Risk, those with ≥10 transfusions received conditioning regimen with higher intensity cyclophosphamide (200 mg/kg). RESULTS: Seventeen patients were enrolled and underwent AlloHSCT including 12 males and 5 females with mean age of 8 years (range 3-16), and median follow-up time of 39 months (range 1-135). Donor sources included MRD BM (6/6 [n = 9], 5/6 [n = 2]) and unrelated CB (5/6 [n = 4], 4/6 [n = 2]). Five year OS was 67.6% (37.9-85.4). Three secondary graft failures (17.6%) occurred in the low dose cyclophosphamide arm. CONCLUSIONS: Upfront treatment with risk-adapted cyclophosphamide conditioning AlloSCT is well tolerated for the management of newly diagnosed pediatric and adolescent patients with acquired SAA. However, the increased risk of graft rejection in the lower dose arm warrants additional research regarding the optimal intensity of cyclophosphamide-based conditioning regimen to reduce toxicity without increasing graft failure.

Transplantation-related mortality, graft failure, and survival after reduced-toxicity conditioning and allogeneic hematopoietic stem cell transplantation in 100 consecutive pediatric recipients.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with myeloablative conditioning is associated with a 10%-40% risk of day +100 transplantation-related mortality (TRM). We evaluated the feasibility and safety of reduced-toxicity conditioning and allo-HSCT in 100 consecutive children and adolescent recipients (mean age, 9.2 ± 6.8 years). The mean duration of follow-up was 1278 ± 1042 days. Fifty patients had malignant disease. The median time to neutrophil recovery was 18 days, and the median time to platelet recovery was 43 days. Median donor chimerism in engrafted patients was 98% on day +100 and 98% on day +365. The cumulative incidence of acute graft-versus-host disease (GVHD) was 20% (95% confidence interval [CI], 12.1%-27.9%), and that of chronic GVHD was 13.5% (95% CI, 6.6%-20.4%). TRM was 3% (95% CI, 0%-6.4%) by day +100 and 13.6% (95% CI, 6.7%-20.5%) for the entire study period. The incidence of primary graft failure (PGF) was 16% overall, 31.4% after umbilical cord blood transplantation (UCBT), and 0% after allo-HSCT with matched unrelated or matched sibling donors (P < .0001). The incidence of PGF in UCBT recipients was 46.7% (14 of 30) in chemotherapy-naive recipients, versus 9.5% (2 of 21) in non-chemotherapy-naive recipients (P = .019). Five-year event-free survival was 59.5% ± 5%, and 5-year overall survival was 72.9% ± 5%. Only PGF and poor-risk disease status were significantly associated with decreased overall survival (P = .03). Reduced-toxicity conditioning allo-HSCT in pediatric recipients is associated with low TRM; however, chemotherapy-naive UCBT recipients have a significantly higher incidence of PGF.

Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design.

Digital PCR (dPCR) is a powerful tool for research and diagnostic applications that require absolute quantification of target molecules or detection of rare events, but the number of nucleic acid targets that can be distinguished within an assay has limited its usefulness. For most dPCR systems, one target is detected per optical channel and the total number of targets is limited by the number of optical channels on the platform. Higher-order multiplexing has the potential to dramatically increase the usefulness of dPCR, especially in scenarios with limited sample. Other potential benefits of multiplexing include lower cost, additional information generated by more probes, and higher throughput. To address this unmet need, we developed a novel melt-based hairpin probe design to provide a robust option for multiplexing digital PCR. A prototype multiplex digital PCR (mdPCR) assay using three melt-based hairpin probes per optical channel in a 16-well microfluidic digital PCR platform accurately distinguished and quantified 12 nucleic acid targets per well. For samples with 10,000 human genome equivalents, the probe-specific ranges for limit of blank were 0.00%-0.13%, and those for analytical limit of detection were 0.00%-0.20%. Inter-laboratory reproducibility was excellent (r 2 = 0.997). Importantly, this novel melt-based hairpin probe design has potential to achieve multiplexing beyond the 12 targets/well of this prototype assay. This easy-to-use mdPCR technology with excellent performance characteristics has the potential to revolutionize the use of digital PCR in research and diagnostic settings.

HLA diversity in ethnic populations can affect detection of donor-specific antibodies by single antigen beads.

Introduction: In solid-organ transplantation, human leukocyte antigen (HLA) donor-specific antibodies (DSA) are strongly associated with graft rejection, graft loss, and patient death. The predominant tests used for detecting HLA DSA before and after solid-organ transplantation are HLA single antigen bead (SAB) assays. However, SAB assays may not detect antibodies directed against HLA epitopes that are not represented in the SAB. The prevalence and potential impact of unrepresented HLA epitopes are expected to vary by ethnicity, but have not been thoroughly investigated. To address this knowledge gap, HLA allele frequencies from seven ethnic populations were compared with HLA proteins present in SAB products from two manufacturers to determine unrepresented HLA proteins. Materials: Allele frequencies were obtained from the Common, Intermediate, and Well Documented HLA catalog v3.0, and frequencies of unrepresented HLA types were calculated. Next-generation sequencing was used to determine HLA types of 60 deceased solid-organ donors, and results were used to determine if their HLA-A, -B, -C, and -DRB1 proteins were not present in SAB reagents from two vendors. Unrepresented HLA proteins were compared with the most similar protein in SAB assays from either vendor and then visualized using modeling software to assess potential HLA epitopes. Results: For the seven ethnic populations, 0.5% to 11.8% of each population had HLA proteins not included in SAB assays from one vendor. Non-European populations had greater numbers of unrepresented alleles. Among the deceased donors, 26.7% (16/60) had at least one unrepresented HLA-A, -B, -C, or -DRB1 protein. Structural modeling demonstrated that a subset of these had potential HLA epitopes that are solvent accessible amino acid mismatches and are likely to be accessible to B cell receptors. Discussion: In conclusion, SAB assays cannot completely rule out the presence of HLA DSA. HLA epitopes not represented in those assays vary by ethnicity and should not be overlooked, especially in non-European populations. Allele-level HLA typing can help determine the potential for HLA antibodies that could evade detection.

Antibiotic-refractory Lyme arthritis is associated with HLA-DR molecules that bind a Borrelia burgdorferi peptide.

An association has previously been shown between antibiotic-refractory Lyme arthritis, the human histocompatibility leukocyte antigen (HLA)-DR4 molecule, and T cell recognition of an epitope of Borrelia burgdorferi outer-surface protein A (OspA163-175). We studied the frequencies of HLA-DRB1-DQA1-DQB1 haplotypes in 121 patients with antibiotic-refractory or antibiotic-responsive Lyme arthritis and correlated these frequencies with in vitro binding of the OspA163-175 peptide to 14 DRB molecules. Among the 121 patients, the frequencies of HLA-DRB1-DQA1-DQB1 haplotypes were similar to those in control subjects. However, when stratified by antibiotic response, the frequencies of DRB1 alleles in the 71 patients with antibiotic-refractory arthritis differed significantly from those in the 50 antibiotic-responsive patients (log likelihood test, P = 0.006; exact test, P = 0.008; effect size, Wn = 0.38). 7 of the 14 DRB molecules (DRB1*0401, 0101, 0404, 0405, DRB5*0101, DRB1*0402, and 0102) showed strong to weak binding of OspA163-175, whereas the other seven showed negligible or no binding of the peptide. Altogether, 79% of the antibiotic-refractory patients had at least one of the seven known OspA peptide-binding DR molecules compared with 46% of the antibiotic-responsive patients (odds ratio = 4.4; P < 0.001). We conclude that binding of a single spirochetal peptide to certain DRB molecules is a marker for antibiotic-refractory Lyme arthritis and might play a role in the pathogenesis of the disease.

Scoring HLA class I mismatches by HistoCheck does not predict clinical outcome in unrelated hematopoietic stem cell transplantation.

Currently, there is no well-accepted rating system for reliably predicting which HLA-mismatched (MM) unrelated donor should be selected for a patient without an HLA allele-matched donor. We evaluated the ability of an MM ranking system, HistoCheck, to predict the risk associated with HLA class I disparity in a population of 744 single allele or antigen HLA-A, -B, or -C MM myeloablative unrelated donor hematopoietic stem cell transplantation recipients with acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, or myelodysplastic syndrome, facilitated through the National Marrow Donor Program between 1988 and 2003. Multivariate models were used to adjust for other significant clinical risk factors. HLA MMs were scored using the HistoCheck Web-based tool, and the patients were divided into 4 quartiles: dissimilarity score (DSS) 1.04-2.84 (allele MM), DSS >2.84-13.75 (allele and antigen MM), DSS >13.75-19.39 (antigen MM), and DSS >19.39-36.62 (antigen MM). Using the lowest scoring quartile as the reference, the DSS groups were evaluated for associations with relapse, treatment-related mortality, acute and chronic graft-versus-host disease, leukemia-free survival, and overall survival in the entire cohort and also in subset analyses by disease and disease stage. No significant associations were found between DSS and any outcomes in the overall cohort using the quartile categories or treating DSS as a continuous variable. Higher DSS scores were associated with decreased engraftment in early-stage disease (P = .0003), but not in other disease stages. In summary, DSS does not correlate with transplantation outcomes, and the HistoCheck scoring system does not provide an effective technique for ranking HLA class I MM. The dataset used in this study is available to evaluate new algorithms proposed for donor selection.

Busulfan, fludarabine, and alemtuzumab as a reduced toxicity regimen for children with malignant and nonmalignant diseases improves engraftment and graft-versus-host disease without delaying immune reconstitution.

For children receiving allogeneic hematopoietic stem cell transplants (HSCTs), the toxicity of the conditioning regimen and graft failure remain challenges. We previously reported that targeted i.v. busulfan, fludarabine, and rabbit anti-thymocyte globulin (rATG) decreased toxicity but had a graft failure rate of 21%. To improve the engraftment rate, we replaced ATG with alemtuzumab, a monoclonal Ab targeting CD52. Thirty-five children with malignant and nonmalignant diseases were enrolled in this phase II prospective study. Twelve children had HLA-matched related donors (MRDs), 16 had 10 of 10, and 7 had 9 of 10 HLA allele-matched unrelated donors (MUDs). Thirty-one of 34 evaluable patients (91%) achieved a durable engraftment. All 3 patients who rejected had a nonmalignant disease and received a MUD transplantation (2 mismatched at 1 antigen). Three patients died of a transplantation-related complication (9% ± 5.2%). Seven patients had disease relapse or progression, 2 of whom died. At a median follow-up of 35 months (range, 15-85 months), the event-free survival (EFS) was 61% ± 9.0% and the overall survival (OS) was 78% ± 7.5%. The median time to neutrophil recovery, B cell, and T cell reconstitution were 16 days, 3 months, and 6 months, respectively. At 1 year, the median donor chimerism in whole blood, CD3, CD14/15, and CD19 subsets were 97%, 87%, 100%, and 99%, respectively. Six patients (17% ± 6.6%) developed acute graft-versus-host disease (aGVHD), only 2 of which were >grade II. Two patients (8% ± 5.4%) progressed to chronic GVHD (cGVHD). These results suggest that replacement of rATG with alemtuzumab may improve engraftment as well as decrease cGVHD rates without resulting in delays in immune reconstitution.

A Phase I study of gemtuzumab ozogamicin (GO) in combination with busulfan and cyclophosphamide (Bu/Cy) and allogeneic stem cell transplantation in children with poor-risk CD33+ AML: a new targeted immunochemotherapy myeloablative conditioning (MAC) regimen.

Children with high-risk acute myelogenous leukemia (AML) (induction failure [IF], refractory relapse [RR], third complete remission [CR3]) have dismal outcomes. Over 80% of AML patients express CD33, a target of gemtuzumab ozogamicin (GO). GO is an active drug in childhood AML but has not been studied in a myeloablative conditioning regimen. We sought to determine the safety of GO in combination with busulfan/cyclophosphamide (Bu/Cy) conditioning before allogeneic hematopoietic stem cell transplantation (alloSCT). GO was administered on day -14 at doses of 3.0, 4.5, 6.0, and 7.5 mg/m(2), busulfan on days -7, -6, -5, -4 (12.8-16.0 mg/kg), and cyclophosphamide on days -3 and -2 (60 mg/kg/day). GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil. We enrolled 12 patients: 8 IF, 3 RR, 1 CR3; median age: 3 years (1-17); median follow-up: 1379 days (939-2305). Nine received umbilical cord blood (UCB), 2 matched unrelated donors (MUDs) and 1 HLA-matched sibling donor: 3 patients each at GO doses of 3.0, 4.5, 6.0, or 7.5 mg/m(2). No dose-limiting toxicities secondary to GO were observed. Day 100 treatment-related mortality (TRM) was 0%. Myeloid and platelet engraftment was observed in 92% and 75% of patients at median day 22 (12-40) and 42 (21-164), respectively. Median day +30 donor chimerism was 99% (85%-100%). The probability of grade II-IV acute graft-versus-host disease (aGVHD) was 42% and chronic GVHD (cGVHD) was 28%. One-year overall survival (OS) and event-free survival (EFS) was 50% (95% confidence interval [CI], 20.8-73.6). GO combined with Bu/Cy regimen followed by alloSCT is well tolerated in children with poor-risk AML. GO at 7.5 mg/m(2) in combination with Bu/Cy is currently being tested in a phase II study.

Effect of HLA-matching recipients to donor noninherited maternal antigens on outcomes after mismatched umbilical cord blood transplantation for hematologic malignancy.

Transplantation-related mortality (TRM) is high after HLA-mismatched umbilical cord blood (UCB) transplantation (UCBT). In utero, exposure to noninherited maternal antigen (NIMA) is recognized by the fetus, which induces T regulator cells to that haplotype. It is plausible that UCBTs in which recipients are matched to donor NIMAs may alleviate some of the excess mortality associated with this treatment. To explore this concept, we used marginal matched-pair Cox regression analysis to compare outcomes in 48 NIMA-matched UCBTs (ie, the NIMA of the donor UCB unit matched to the patient) and in 116 non-NIMA-matched UCBTs. All patients had a hematologic malignancy and received a single UCB unit. Cases and controls were matched on age, disease, disease status, transplantation-conditioning regimen, HLA match, and infused cell dose. TRM was lower after NIMA-matched UCBTs compared with NIMA-mismatched UCBTs (relative risk, 0.48; P = .05; 18% versus 32% at 5 years posttransplantation). Consequently, overall survival was higher after NIMA-matched UCBT. The 5-year probability of overall survival was 55% after NIMA-matched UCBTs versus 38% after NIMA-mismatched UCBTs (P = .04). When faced with the choice of multiple HLA-mismatched UCB units containing adequate cell doses, selecting an NIMA-matched UCB unit may improve survival after mismatched UCBT.

T cell depletion utilizing CD34(+) stem cell selection and CD3(+) addback from unrelated adult donors in paediatric allogeneic stem cell transplantation recipients.

CD34-selected haploidentical and unrelated donor allogeneic stem cell transplantation (AlloSCT) in paediatric recipients is associated with sustained engraftment and low risk of acute graft-versus-host disease (aGVHD), but limited by delayed immune reconstitution and increased risk of viral and fungal infection. The optimal dose of donor T cells to prevent graft failure and minimize risk of early opportunistic infection and post-transplant lymphoproliferative disorder (PTLD), while avoiding severe aGVHD, remains unknown. We prospectively studied CD34-selected 8-10/10 human leucocyte antigen (HLA)-matched unrelated donor (MUD) peripheral blood stem cell transplantation (PBSCT) in a cohort of 19 paediatric AlloSCT recipients with malignant (n = 13) or non-malignant (n = 6) diseases. T cells were added back to achieve total dose 1·0-2·5 × 10(5)  CD3(+) /kg. GVHD pharmacoprophylaxis consisted only of tacrolimus. All patients engrafted neutrophils. Probabilities of grade II-IV aGVHD, limited chronic GVHD (cGVHD), and extensive cGVHD were 15·8%, 23·3%, and 0%, respectively. One patient developed PTLD. One-year infection-related mortality was 5·6%. T cell immune reconstitution was delayed. One-year overall survival was 82·3%. Five patients with malignant disease ultimately died from progressive disease. CD34-selected MUD PBSCT using a defined dose of T cell add-back resulted in high rates of engraftment and low risk of grade II-IV aGVHD, early transplantation-related mortality, and extensive cGVHD.

Cell-free DNA as a solid-organ transplant biomarker: technologies and approaches.

High-quality biomarkers that detect emergent graft damage and/or rejection after solid-organ transplantation offer new opportunities to improve post-transplant monitoring, allow early therapeutic intervention and facilitate personalized patient management. Donor-derived cell-free DNA (DD-cfDNA) is a particularly exciting minimally invasive biomarker because it has the potential to be quantitative, time-sensitive and cost-effective. Increased DD-cfDNA has been associated with graft damage and rejection episodes. Efforts are underway to further improve sensitivity and specificity. This review summarizes the procedures used to process and detect DD-cfDNA, measurement of DD-cfDNA in clinical transplantation, approaches for improving sensitivity and specificity and long-term prospects as a transplant biomarker to supplement traditional organ monitoring and invasive biopsies.

Donor chimerism and immune reconstitution following haploidentical transplantation in sickle cell disease.

Introduction: We previously reported the initial results of a phase II multicenter transplant trial using haploidentical parental donors for children and aolescents with high-risk sickle cell disease achieving excellent survival with exceptionally low rates of graft-versus-host disease and resolution of sickle cell disease symptoms. To investigate human leukocyte antigen (HLA) sensitization, graft characteristics, donor chimerism, and immune reconstitution in these recipients. Methods: CD34 cells were enriched using the CliniMACS® system with a target dose of 10 x 106 CD34+ cells/kg with a peripheral blood mononuclear cell (PBMNC) addback dose of 2x105 CD3/kg in the final product. Pre-transplant HLA antibodies were characterized. Donor chimerism was monitored 1-24 months post-transplant. Comprehensive assessment of immune reconstitution included lymphocyte subsets, plasma cytokines, complement levels, anti-viral T-cell responses, activation markers, and cytokine production. Infections were monitored. Results: HLA antibodies were detected in 7 of 11 (64%) evaluable patients but rarely were against donor antigens. Myeloid engraftment was rapid (100%) at a median of 9 days. At 30 days, donor chimerism was 93-99% and natural killer cell levels were restored. By 60 days, CD19 B cells were normal. CD8 and CD4 T-cells levels were normal by 279 and 365 days, respectively. Activated CD4 and CD8 T-cells were elevated at 100-365 days post-transplant while naïve cells remained below baseline. Tregs were elevated at 100-270 days post-transplant, returning to baseline levels at one year. At one year, C3 and C4 levels were above baseline and CH50 levels were near baseline. At one year, cytokine levels were not significantly different from baseline. Discussion: These results suggest that haploidentical transplantation with CD34-enriched cells and peripheral blood mononuclear cell addback results in rapid engraftment, sustained donor chimerism and broad-based immune reconstitution.