Recipient Alleles Research Articles

Combination of risk alleles of PNPLA3, TM6SF2, and HSD17B13 of donors can predict recurrence of steatotic liver disease after liver transplantation.

This study aimed to identify the genetic risk factors from donors or recipients that contribute to postliver transplantation (LT) steatotic liver disease (SLD), focusing on the genetic risk score (GRS) based on single nucleotide polymorphisms (SNPs) in SLD patients. This retrospective study included 55 Japanese SLD recipients and their respective donors. Genotyping of PNPLA3, TM6SF2, and HSD17B13 was undertaken, and the combined GRS was calculated. The relationship between the GRS and the incidence of posttransplant SLD was also evaluated. The SLD recipients had a high prevalence of post-LT graft steatosis/steatohepatitis (76.4% and 58.2%, respectively). Although the recipients had a high frequency of risk alleles, there was no relationship between the number of risk alleles for each SNP and the incidence of posttransplant SLD. In contrast, an increased number of risk alleles for any SNP in the donor was correlated with high incidence rates of both post-LT steatosis and steatohepatitis. A multivariable analysis showed that a high donor GRS was an independent risk factor for graft steatosis (odds ratio 8.77; 95% CI, 1.94-52.94; p=0.009). Similarly, a high donor GRS was an independent risk factor (odds ratio 6.76; 95% CI, 1.84-30.78; p=0.007) for post-LT graft steatohepatitis. Donor risk alleles of PNPLA3, TM6SF2, and HSD17B13, rather than recipient risk alleles, have been implicated in the development of posttransplant SLD. The combination of these donor risk alleles into a GRS could predict the development of posttransplant SLD.

MICB Genetic Variants and Its Protein Soluble Level Are Associated with the Risk of Chronic GvHD and CMV Infection after Allogeneic HSCT.

The aim of the present study was to determine the associations between the MICB genetic variability and the expression and the risk of development of post-transplant complications after allogeneic hematopoietic stem cell transplantation (HSCT). HSCT recipients and their donors were genotyped for two MICB polymorphisms (rs1065075, rs3828903). Moreover, the expression of a soluble form of MICB was determined in the recipients' serum samples after transplantation using the Luminex assay. Our results revealed a favorable role of the MICB rs1065075 G allele. Recipients with donors carrying this genetic variant were less prone to developing chronic graft-versus-host disease (cGvHD) when compared to recipients without any symptoms of this disease (41.41% vs. 65.38%, p = 0.046). Moreover, the MICB rs1065075 G allele was associated with a lower incidence of cytomegalovirus (CMV) reactivation, both as a donor (p = 0.015) and as a recipient allele (p = 0.039). The MICB rs1065075 G variant was also found to be associated with decreased serum soluble MICB (sMICB) levels, whereas serum sMICB levels were significantly higher in recipients diagnosed with CMV infection (p = 0.0386) and cGvHD (p = 0.0008) compared to recipients without those complications. A protective role of the G allele was also observed for the rs3828903 polymorphism, as it was more frequently detected among donors of recipients without cGvHD (89.90% vs. 69.23%; p = 0.013). MICB genetic variants, as well as serum levels of sMICB, may serve as prognostic factors for the risk of developing cGvHD and CMV infection after allogeneic HSCT.

Blast resistance (Pi54) introgression in temperate rice (Oryza sativa L.) K343 using marker assisted backcrossing

In hill rice ecologies, Magnaporthe oryzae causes rice blasts and a significant biotic constraint. The study was aimed to develop rice blast-resistant lines/varieties through marker-assisted selection (MAS) by using the Pi54 gene, which provides resistance against the most prominent blast fungus isolate (PLP-1). The blast resistance gene Pi54 from the indica rice genotype DHMAS has been inserted into the genetic makeup of the temperate rice variety K343 in the current study. Three SSR markers (TRS26, TRS33, and RM206) are closely linked to the Pi54 locus used in this study. Marker RM206, located 0.7cM from the Pi54 gene, distinguished between donor and recipient alleles and co-segregated with the target gene. Thus, RM206 is used for Pi54 gene foreground selection. Sixty-one plants have the homozygous allele for the Pi54 in BC1F2, accounting for roughly 50% of the homozygous population. Polymorphic genome-wide SSR assessment of the BC1F2 genetic stock (K343*2/DHMAS) revealed recurrent parent genome (RPG) recovery above 75% in 4 plants. Therefore, M. oryzae race PLP-1, the dominant race used in this study, showed high resistance to the resistant response after inoculation.

Immunoinformatic Evaluation of the Cross-presentation of Donor Major Histocompatibility Derived Epitopes by Recipient Major Histocompatibility Class I Molecules in Transplantation Rejection

Aim: The aim of this study was to analyze the cross-presentation of allopeptides derived from donor major histocompatibility (MHC) molecules by recipient class I MHC molecules in the context of transplantation rejection. Materials and Methods: The MHC molecules were selected based on frequency distribution in the Kerala (India) population using the “allelefrequency. net” database. The most frequent alleles were chosen as donor and recipient alleles. Different immunoinformatics servers such as Netcytotoxic T lymphocyte, NetMHCpan, and Immune Epitope Database MHC I were used for predicting potential T-cell epitopes, and MHC class I epitopes, respectively, from the chosen donor MHC molecule. Three peptides with strong binding affinities in all three predictions were selected for further analysis. The binding of these peptides to the second MHC class I molecule, the recipient one, and transporter associated with antigen processing protein was confirmed by molecular docking using the “AutoDockVina” tool. The binding interactions were visualized using the “Discovery Studio Visualizer” platform. Results: The ability of the selected class I MHC molecule, human leukocyte antigens (HLAs)-A* 11:01, considered the donor allele, to act as an exogenous antigen and to get processed into peptides and presented on the second class I MHC molecule, HLA-A*02:01, considered the recipient allele was confirmed. This immunoinformatics approach could help us to identify potential epitopes from an HLA class I molecule and to measure its ability to get cross-presented on a second HLA class I molecule to trigger the process of cell-mediated rejection. Conclusion: This immunoinformatics approach could help us to identify potential epitopes from an HLA class I molecule and to measure its ability to get cross-presented on a second HLA class I molecule to trigger the process of cell-mediated rejection. Our study provides an easy in silico method for predicting the chances of cell mediated rejection in organ transplantations involving an unrelated donor. We hope that this computational analysis will pave the way for simplifying the process involved in organ rejection prediction.

STR typing of skin swabs from individuals after an allogeneic hematopoietic stem cell transplantation

One of the pre-requisites for forensic DNA analysis is the fact that all nucleated cells of a person carry the same genetic information. However, this is not the case for individuals who have received an allogeneic hematopoietic stem cell or bone marrow transplantation, as all new cells formed by the bone marrow no longer show the genetic information of the recipient but that of the donor, while all other cells still carry the original information before transplantation. Thus, STR typing of a blood sample after successful transplantation yields a DNA profile that differs from the recipient’s original profile and corresponds to the donor genotype instead. Evidence from a routine case suggests that transplanted individuals may show donor alleles in skin swabs, as well. In order to examine this issue more closely, various skin swabs from 28 patients who have received an allogeneic hematopoietic stem cell transplantation were examined in this study. Swabs from the right and left palm, the back of the hand, one of the two upper arms, and the neck were collected from each person. Ninety-one of the 140 resulting swabs delivered useful results. All of those samples showed mixtures of recipient and donor DNA with different mixture ratios and the proportions of donor and recipient alleles revealed inter- and intra-individual differences. Those results were discussed with respect to graft versus host disease.

Recipient APOL1 risk alleles associate with death-censored renal allograft survival and rejection episodes.

Apolipoprotein L1 (APOL1) risk alleles in donor kidneys associate with graft loss, but whether recipient risk allele expression affects transplant outcomes is unclear. To test whether recipient APOL1 risk alleles independently correlate with transplant outcomes, we analyzed genome-wide SNP genotyping data on donors and recipients from 2 kidney transplant cohorts: Genomics of Chronic Allograft Rejection (GOCAR) and Clinical Trials in Organ Transplantation 01/17 (CTOT-01/17). We estimated genetic ancestry (quantified as the proportion of African ancestry, or pAFR) by ADMIXTURE and correlated APOL1 genotypes and pAFR with outcomes. In the GOCAR discovery set, we noted that the number of recipient APOL1 G1/G2 alleles (R-nAPOL1) associated with an increased risk of death-censored allograft loss (DCAL), independent of ancestry (HR = 2.14; P = 0.006), as well as within the subgroup of African American and Hispanic (AA/H) recipients (HR = 2.36; P = 0.003). R-nAPOL1 also associated with an increased risk of any T cell-mediated rejection (TCMR) event. These associations were validated in CTOT-01/17. Ex vivo studies of PMBCs revealed, unexpectedly, high expression levels of APOL1 in activated CD4+/CD8+ T cells and NK cells. We detected enriched immune response gene pathways in risk allele carriers compared with noncarriers on the kidney transplant waitlist and among healthy controls. Our findings demonstrate an immunomodulatory role for recipient APOL1 risk alleles associated with TCMR and DCAL. We believe this finding has broader implications for immune-mediated injury to native kidneys.

The Presence of Recipient CMV Immunodominant Human Leukocyte Antigen (HLA) Alleles Affect Outcomes after Haploidentical (HI) Hematopoietic Stem Cell Transplantation (HSCT)

CMV reactivation is associated with increased treatment-related mortality (TRM), decreased overall survival (OS), and in some analyses, protection from relapse after HSCT. We previously showed that CMV reactivation was significantly higher in HI versus matched related HSCT recipients receiving an identical preparatory regimen, implicating HLA mismatch as a contributor to reactivation. It is known that specific CMV immunodominant HLA alleles (CMV ID alleles) are associated with more efficient antigen presentation and protection from CMV. We hypothesized that a match at a CMV ID allele in the shared haplotype of the donor and recipient pairs may overcome the disadvantage proffered by HI HSCT with respect to CMV. As a control, we tested whether the presence of a CMV ID allele in either the donor or recipient, as opposed to a match at the CMV ID allele, was the more important impactor of outcomes. This information would potentially impact donor selection and identify HI recipients with greater risk for CMV-related complications.MethodsThis is a retrospective, analysis of 138 HI HSCT recipients at risk for CMV reactivation as defined by the presence of positive CMV serologic status in the recipient, donor, or both. All recipients were treated on the Jefferson 2 step approach in which after conditioning, a fixed T cell dose (2 x 108/kg) is administered (step 1), followed 2-3 days later by cyclophosphamide (CY) for bidirectional tolerization. Two days after CY, patients receive a CD34-selected donor product (step 2). To be included in the analysis, patients had to successfully engraft and be alive for at least 1 month post HSCT. Outcomes related to CMV, 1) reactivation incidence, 2) peak copy number of initial CMV reactivation occurring by d+100, 3) TRM, 4) relapse related mortality (RRM), and 5) OS, were tested in 3 contexts: the presence vs the absence of a CMV ID allele in the recipient or the donor, or the presence or absence of a donor/recipient (R/D) match at the same CMV ID allele. CMV ID alleles were defined as HLA A01:01, A02:01, A24:02, B07:02, B08:01, B35:01 and C07:02. Statistics used: incidence of CMV reactivation=Chi-Square; comparison of peak CMV copy numbers=T-test; assessment of RRM and NRM=cumulative incidence (CI); OS=Kaplan-Meier method.Results:A CMV ID allele was present in 68.1% recipients and 66.7% donors. 41.7% of D/R pairs were matched at the same CMV ID allele. The incidence of CMV reactivation was not associated with a D/R CMV ID allele match or recipient or donor CMV ID allele positivity. Peak CMV levels were lower in the presence of any recipient or donor CMV ID allele, but only approached significance in D/R pairs matched at the same CMV ID allele (p=0.065) with a mean of 8.7 x 10e3 vs 4.1 x10e4 copies/ml by PCR in matched vs non-matched pairs respectively. At 3 years, in recipients who matched their donor at 1 or more CMV ID alleles vs those who had no match, CI of NRM was 28% vs 26% (p=0.48) and 16% vs 32% (p=0.07) for RRM. In recipients with vs without a CMV ID allele CI of NRM was 23% vs 36% (p=0.059) and 20% vs 36% for RRM (p= 0.02) (Figure). In recipients with donors who possessed a CMV ID allele vs not, CI of NRM was 27% vs 28% (p=0.80) and 21% vs 31% for RRM (p= 0.12). OS in recipients who were matched at the same CMV ID allele with their donors or who possessed a CMV ID allele was 51% vs 37% (p=0.062) and 52% vs 27% (p=0.001) respectively as compared to recipients negative for these alleles.Conclusion:This analysis suggests that matching HI D/R pairs at a CMV ID epitope may reduce CMV peak copy number but has no significant effect on post HSCT reactivation. Recipient positivity for a CMV ID allele was strongly associated with improvement in NRM, RRM, and OS. Whether the benefit is specific to improved CMV control or to a more efficient antigen presentation in general by these alleles is unknown, but should be further investigated. This data requires confirmation with multivariate analysis. [Display omitted] DisclosuresWagner:Novartis: Research Funding.

Cfcloud: A Cloud-Based Workflow for Cell-Free DNA Data Analysis

Introductions:Cell-free DNA (cfDNA) are extracellular nucleic acids circulating in blood and other biological fluids. They are non-randomly fragmented, mainly short (< 200 bp) DNA molecules released as a result of apoptosis, necrosis or active secretion from cells. They are believed to be protected from degradation by nucleases due to nucleosome structure. cfDNA is a promising biomarker for prenatal testing, hemoglobinopathies, inflammatory progress, and detection of tumor-derived DNA in plasma. The amount of donor derived cell-free DNA circulating in the recipient's plasma can be used as a signature of dying cells from the transplanted organ. Donor derived cell-free DNA percent (%dd-cfDNA) can be quantified using next generation sequencing (NGS)-based method that utilizes genotype information of recipient and donor by measuring single nucleotide polymorphism (SNP) differences. This method was used in Genome Transplant Dynamics (GTD) cohort study. This study showed that elevated dd-cfDNA levels correlate with rejection of the transplanted organ. GTD method was shown to be a rigorous and highly reproducible across different genotyping or sequencing platforms. Here, we present thecfCloud, an open source, cloud-based implement of the GTD method in a Snakemake pipeline which automates the quantification of dd-cfDNA amount. Methods:As illustrated in Figure 1, the analysis workflow contains several modules and rules; 1) Align reads rule - aligns sequencing reads against the reference genome (hg19), and marks duplicated reads. 2) Prepare genotype rule - given the input VCF file, for every recipient/donor pair, generates a SNP table by classifying SNPs as i) Group A, SNPs where both recipient (R) and donor (D) are homozygous with the same allele, like R=AA, D=AA, and ii) Group B, SNPs where both recipient and donor are homozygous with different alleles, like R=AA, D=BB. 3) Make CallsTable rule - for every SNP position, if the observed base of an overlapping read matches the recipient allele, it classifies that read as recipient-derived, if the observed base matches the donor allele then it classifies as donor-derived, otherwise as background-error. CallsTable lists chromosome, position, base quality and mapping quality scores of all the sites as well as the assigned category. 4) Call % dd-cfDNA rule - using the CallsTable, filters out the reads based on the input parameters. Then, calculates % dd-cfDNA by using SNPs in Group A and estimates background-error rate by using SNPs in Group B. 5) Prepare FinalReport rule - generates a final report showing total counts, donor counts, dd-cfDNA percentages and estimated background-error rates for all the samples. Results:Using the workflow management system, Snakemake, we have developed a user friendly, efficient, and comprehensive pipeline that is fully automated, reproducible, and scalable. While the pipeline is fully customizable, it can be run using a simple command line. Upon execution, Snakemake infers the combination of rules necessary to achieve a specific output and or a report. With a basic understanding of the underlying framework of Snakemake, cfCloud is highly scalable and can be easily customized. In addition to local implementation, cfCloud also runs as an Amazon Machine Instance (AMI) in the Amazon Elastic Compute Cloud (EC2). The operating system, software tools and all settings, are encapsulated into a single cloud image of the computing system that is ready to deploy and can be easily saved and restored for later use. A snapshot is also an executable machine image that can be shared with other users of the cloud, allowing collaborators to share their configurations and analysis results as a single image. Conclusions:Here we present a fully automated pipeline to systematize the quantification of donor derived cell-free DNA amount. The Amazon Web Machine Image is available from the AWS Market Place. The cfCloud provides users with improved usability, efficiency, and scalability in local and cloud environment. The cfCloud software, installation instructions, tutorials and example data can be freely obtained from https://github.com/NHLBI-BCB/cfCloud under the MIT license. Figure Disclosures No relevant conflicts of interest to declare.

Human leukocyte antigen disparities reduce relapse after hematopoietic stem cell transplantation in children with juvenile myelomonocytic leukemia: A single-center retrospective study from China.

HSCT is the only proven curative therapy for JMML. Matching donor and recipient HLA alleles is considered optimal to reduce the risk of GVHD after HSCT but is not always possible. Only a limited number of studies have compared the influence of HLA disparities on HSCT outcomes for patients with JMML. We conducted a retrospective study among 47 children with JMML who received related or unrelated unmanipulated HSCT (March 2010-October 2018). Among our participants, 27 (57.4%) donor-recipient pairs had 0-1 HLA disparities (Group 1: HLA-matched or ≤1 allele/antigen mismatch donor) and 20 (42.6%) had ≥2 HLA disparities (Group 2: 2-3 mismatched/haploidentical donors). The median follow-up period was 26.0months (range: 1-105months), and the 5-year probabilities of DFS and RI for the whole cohort were 54.6±7.7% and 34.8±15.0%, respectively. Compared to Group 1, Group 2 patients had a significantly lower RI (5.3±10.5% vs 55.5±20.9%, P˂.001), though similar rates of grade II-IV acute GVHD (60.0±22.4% vs 33.3±18.2%, P=.08), grade III-IV acute GVHD (25.0±19.5% vs 7.4±10.1%, P=.08), chronic GVHD (30.0±20.9% vs 34.9±18.8%, P=.85), NRM (20.0±18.0% vs 3.9±7.7%, P=.07), and DFS (74.4±9.9% vs 41.3±10.0%, P=.08). Disease relapse remains the major cause of treatment failure in JMML patients, especially in patients receiving HLA-matched and limited HLA-mismatched HSCT. Our findings suggest that donor-recipient HLA disparities may improve the outcome of HSCT in children with JMML.

Epitope matching in kidney transplantation: recent advances and current limitations.

Evolution of human leukocyte antigen (HLA) molecular typing techniques has progressively enabled more accurate determination of the three-dimensional building blocks that form the antibody accessibility and binding sites of each HLA allele. These immunogenic HLA regions known as epitopes are composed of polymorphic sequences of amino acid residues termed eplets. This review provides a critical appraisal of the current understanding of epitope compatibility in kidney transplantation. There is a tendency to suggest that epitope matching is likely to be superior to broad antigen HLA matching such that the allocation of donor kidneys to patients with a more favorable epitope compatibility profile may lead to better allograft outcomes. A growing body of work has highlighted the association between a greater number of eplet mismatches and adverse allograft outcomes, and approaches using eplet matching have been successfully implemented in organ allocation programs. However, our understanding of epitope compatibility remains in its infancy, requiring further and more in-depth evaluation. Critically, it remains unclear how best to translate findings derived at the population level to the care of individual patients. Questions that need to be answered include a lack of consensus in the definition and interpretation of epitope compatibility, are class I and II compatibility of similar clinical importance, how best to define predetermined mismatch thresholds for utilization in organ allocation, and whether other properties such as differences in electrostatic potential between donor and recipient HLA alleles are also important in determining immunological compatibility. Epitope matching likely represents a valid progression in understanding donor-recipient HLA compatibility. However, more clinical data and a better understanding about differences in methods to determine epitope compatibility are required before the approach can be widely applied in clinical practice.

Identification of the rs9277534 HLA-DP expression marker by next generation sequencing for the selection of unrelated donors for hematopoietic cell transplantation

Mismatching of an unrelated donor against a high-expression HLA-DPB1 recipient allele is associated with a high risk of graft-versus-host disease and mortality. The Seattle Cancer Care Alliance (SCCA) and Fred Hutchinson Cancer Research Center transplant program employs an algorithm to match for HLA-A, B, C, DRB1, DQB1 and DPB1 alleles (12/12) and to avoid, whenever possible, donor mismatching against a recipient high-expression HLA-DPB1 allele. HLA-DPB1 expression is associated with the rs9277534 A/G polymorphism located in the 3′UTR of the HLA-DPB1 gene. Next generation sequencing of HLA-DPB1 using the Illumina TruSight HLA V2 Sequencing Panel and Conexio Assign software analyses provides information on rs9277534 variants without the need for any additional SNP testing. Here we present the molecular location of rs9277534 in NGS data and discuss the challenges to resolve HLA-DPB1 ambiguities.

Personalized Chimerism Test that Uses Selection of Short Tandem Repeat or Quantitative PCR Depending on Patient's Chimerism Status

Chimerism testing is used to monitor engraftment and risk of relapse after allogeneic hematopoietic stem cell transplantation for hematologic malignancies. Although short tandem repeat (STR) method is widely used among clinical laboratories, quantitative PCR (qPCR) provides better sensitivity (0.1%) than STR (1% to 5%) but is less accurate than STR for patients in mixed chimerism. qPCR chimerism allows evaluation of residual recipient cells as a surrogate of measurable residual disease. To achieve higher sensitivity and accuracy, we applied qPCR or STR based on patient chimerism status (recipient alleles <5% or ≥5%, respectively). Of the 230 patients tested by STR in a 1-year period, excluding 10 deceased patients, 30 qPCR markers were genotyped and 167 patients converted to qPCR chimerism (76%), including eight patients undergoing multiple-donor transplantation. STR was continued on 53 patients (24%) for the following reasons: mixed chimerism (n = 23), lack of donor or pretransplantation DNA (n= 22), and insufficient qPCR informative markers [8 of 60 patients with related donors (13.3%)]. qPCR detected residual recipient chimerism in 85.5% of patients with complete chimerism by STR (<5% recipient). Selecting STR or qPCR testing based on each patient's chimerism status facilitates sensitive and accurate chimerism testing in clinical settings. In addition, we discuss clinical relevance of chimerism testing for measurable residual disease detection in various hematologic malignancies.

Efficacy of Donor and ‘Third Party’ (Tabelecleucel) EBV-Specific T Cells for Treatment of Central Nervous System (CNS) EBV-PTLD

Background EBV+PTLD is a life-threatening complication after hematopoietic cell transplantation (HCT) and solid organ transplantation (SOT). Introduction of EBV-specific T-cells (EBV+CTLs) as second line treatment after rituximab has reduced overall EBV+PTLD related mortality. Due to limited efficacy of rituximab for EBV+PTLD disease involving the CNS there remains a clear clinical need. We studied the ability of EBV+CTLs to mediate responses in CNS EBV+PTLD. Methods Patients with CNS EBV+PTLD, with or without systemic disease, after HCT or SOT were eligible to receive either donor-derived or ‘third party' Tabelecleucel between 1996 and 2016 on two IRB approved MSKCC protocols (95-024: NC2663 and 11-130: NCT01498484). Donor EBV+CTLs were generated from EBV seropositive HCT-donors, while Tabelecleucel were selected from a bank of 330 lines from volunteer HCT donors. Selection of Tabelecleucel lines was based on matching for > 2/10 recipient alleles and HLA restriction by at least one HLA allele shared by the patient's tumor. Patients received 3 weekly infusions of ∼1-2 × 106 T cells/kg. Responses were assessed 21-35 days after the start of each cycle of EBV+CTLs based on Lugano criteria with CNS disease being assessed by MRI, CSF (EBV-PCR) or thalium scan. Patients not achieving a complete response (CR) to an initial cycle of EBV+CTLs were eligible to receive a subsequent cycle. Main outcome of interest was response to EBV+CTLs. Other outcomes of interest were 1-year overall survival (OS) and infusion-related toxicities. Results Nineteen patients were treated: 12 after HCT (T-cell depletion 6, cord blood 3, T-replete 3) and 7 after SOT (renal 4, heart 1, liver 1, heart/liver 1). 7 patients received EBV+CTLs from their primary donor, 11 from a third-party donor and 1 from both donor types. Ten were treated for had isolated-CNS disease while 9 had CNS and systemic disease. All of these patients had received prior therapy including rituximab (N = 17), radiation therapy (N = 6), and/or systemic chemotherapy (N = 9). The overall response rate was 63% and the one-year OS was 60% (70% for isolated-CNS, 56% for combined). Two patients with combined disease had simultaneous radiation therapy and achieved response, and thus, the CNS specific response could not be attributed to cell therapy alone. Toxicities associated with infusions were limited; 8 patients experienced ≥ grade 3 events with one patient experiencing a possibly related grade 3 event. Interpretation This study demonstrates clear responses and promising survival among patients with otherwise refractory and lethal CNS EBV+PTLD. Adoptive therapy with EBV directed cellular therapy (either primary donor or third party derived Tabelecleucel is a promising treatment modality for CNS EBV+PTLD.

Adoptive Therapy with EBV-Specific T Cells for Treatment of CNS EBV Post-Transplant Lymphoproliferative Disease Arising after Hematopoietic Stem Cell Transplant or Solid Organ Transplant

Adoptive immunotherapy with EBV-specific T-cells (EBV-CTLs) derived from primary hematopoietic transplant donors is effective in the treatment of EBV disease complicating allogeneic hematopoietic stem cell transplant (HCT). In addition, third party donor-derived EBV-CTLs (tabelecleucel) have demonstrated efficacy in the treatment of EBV post-transplant lymphoproliferative in the setting of both HCT and solid organ transplant (SOT). While the introduction of first line therapy with rituximab has reduced the mortality associated with EBV-PTLD, EBV PTLD involving the central nervous system (CNS) remains a particularly ominous event as the efficacy of rituximab for CNS disease is limited by poor CNS bioavailability.Between 1996 and 2016 we treated nineteen patients for EBV-PTLD involving the CNS arising after HCT (12) or SOT (7). These patients received EBV-CTLs from their primary transplant donor (7), tabelecleucel from a third-party donor (11) or from both types of donor (1). Patients were treated for either isolated CNS disease (10) or CNS and systemic disease (9). Patients developing EBV-PTLD after HCT were recipients of T Cell Depleted (N=6), cord blood (N=3) and conventional (N=3) transplant. Those developing EBV-PTLD after SOT had undergone renal (N=4) heart (1) liver (1) and heart/liver (1) transplants.All of these patients had received prior therapy including rituximab (N=17), radiation therapy (N=6), and chemotherapy (N=9).Primary donor EBV-CTLs were generated from EBV seropositive hematopoietic transplant donors at the time of or after the patient underwent transplant. Third party tabelecleucel were selected from a bank of 330 lines generated under GMP conditions from normal HCT donors who specifically consented to use of their T cells in patients other than their designated transplant recipient. Selection of third party tabelecleucel lines was made on the basis of HLA restriction by at least one HLA allele shared by the patient's tumor and the HCT donor, and matching for ≥ 2/10 recipient alleles. Patients received 3 weekly infusions of approximately 1-2x106 T cells/kg/infusion. Patients were sequentially evaluated for clinical and radiographic response, and quantifications of EBV DNA by PCR. Patients not achieving a complete response to an initial cycle of EBV directed cellular therapy were eligible to receive subsequent cycles of cells from either the same or a different donor. Responses were assessed 21-35 days after the first of each cycle of EBV-CTLs. Responses were evaluated based on Lugano criteria with CNS disease being assessed by MRI, CSF or thalium scan. Two patients treated for CNS and systemic disease had simultaneous therapy of their CNS disease and achieved responses of both CNS and systemic disease but the CNS specific response could not be attributed to cell therapy alone.Of the 19 patients, 7 achieved complete responses and 5 durable partial responses for an overall response rate of 63%. The one-year overall survival (OS) for this cohort of 19 patients was 60% with responding patients experiencing one year OS of 91.7% and non-responding patients one year OS of just 14.3%. Eight of the 10 patients treated for isolated CNS disease responded to adoptive EBV directed T cell therapy. One-year overall survival for patients treated for isolated CNS disease is 70% and with CNS and systemic disease is 55.6%. Toxicities associated with infusions in this cohort are limited with 8 patients experiencing adverse events of ≥ grade 3 severity with one patient experiencing a possibly related grade 3 eventThis study demonstrates a high response rate among patients with otherwise refractory EBV PTLD affecting the CNS. Adoptive therapy with EBV directed cellular therapy (primary donor or third party derived) can effectively treat this otherwise frequently lethal complication. The availability of 3rd party tabelecleucel enables treatment early in the course of disease and may thereby improve response rates while minimizing toxicity from chemotherapy.Atara Biotherapeutics holds the IND and has a license to develop and commercialize the EBV and CMV cell therapy programs at Memorial Sloan Kettering. MSKCC and several MSK investigators hold a financial interest in Atara Biotherapeutics. DisclosuresProckop:Atara Biotherapeutics: Research Funding; Mesoblast: Research Funding. Doubrovina:Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding. Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite: Consultancy. O'Reilly:Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding.

Risk of recurrence of primary sclerosing cholangitis after liver transplantation is associated with de novo inflammatory bowel disease.

AIMTo evaluate risk factors for primary sclerosing cholangitis (PSC) recurrence (rPSC) after orthotopic liver transplantation (OLT) in patients with well-preserved colons.METHODSWe retrospectively evaluated the medical records of all patients transplanted for PSC in our center between July 1994 and May 2015 and selected 47 with follow-up of at least 60 mo for further analysis based on strict inclusion and exclusion criteria. rPSC was confirmed by magnetic resonance or endoscopic retrograde cholangiopancreatography and liver biopsy. All patients were evaluated by protocolary pre-OLT colonoscopy with randomized mucosal biopsies. Colonoscopy was repeated annually after OLT. Both organ donors and recipients were human leukocyte antigen (HLA) typed by serological and/or DNA methods. All input data were thoroughly analyzed employing relevant statistical methods.RESULTSAltogether, 31 men and 16 women with a median (range) age of 36 (15-68) years at the time of OLT and a median follow-up of 122 (60-249) mo were included. rPSC was confirmed in 21/47 (44.7%) of patients, a median 63 (12-180) mo after transplantation. De novo colitis [rPSC in 11/12, P ≤ 0.05, hazard ratio (HR): 4.02, 95% confidence interval (CI): 1.58-10.98] and history of acute cellular rejection (rPSC in 14/25, P ≤ 0.05; HR: 2.66, 95%CI: 1.03-7.86) showed strong positive associations with rPSC. According to the univariate analysis, overlapping features of autoimmune hepatitis (rPSC in 5/5, P ≤ 0.05) and HLA-DRB1*07 in the donor (rPSC in 10/15, P ≤ 0.05) represent other potential risk factors for rPSC, while the HLA-DRB1*04 (rPSC in 0/6, P ≤ 0.05), HLA-DQB1*03 (rPSC in 1/11, P ≤ 0.05), and HLA-DQB1*07 (rPSC in 0/7, P ≤ 0.05) recipient alleles may have protective roles.CONCLUSIONDe novo colitis and acute cellular rejection are clinical conditions significantly predisposed towards recurrence of PSC after liver transplantation.

Introgression of a functional epigenetic OsSPL14WFP allele into elite indica rice genomes greatly improved panicle traits and grain yield

Rice yield potential has been stagnant since the Green Revolution in the late 1960s, especially in tropical rice cultivars. We evaluated the effect of two major genes that regulate grain number, Gn1a/OsCKX2 and IPA1/WFP/OsSPL14, in elite indica cultivar backgrounds. The yield-positive Gn1a-type 3 and OsSPL14WFP alleles were introgressed respectively through marker-assisted selection (MAS). The grain numbers per panicle (GNPP) were compared between the recipient allele and the donor allele groups using segregating plants in BC3F2 and BC3F3 generations. There was no significant difference in GNPP between the two Gn1a alleles, suggesting that the Gn1a-type 3 allele was not effective in indica cultivars. However, the OsSPL14WFP allele dramatically increased GNPP by 10.6–59.3% in all four different backgrounds across cropping seasons and generations, indicating that this allele provides strong genetic gain to elite indica cultivars. Eventually, five high-yielding breeding lines were bred using the OsSPL14WFP allele by MAS with a conventional breeding approach that showed increased grain yield by 28.4–83.5% (7.87–12.89 t/ha) vis-à-vis the recipient cultivars and exhibited higher yield (~64.7%) than the top-yielding check cultivar, IRRI 156 (7.82 t/ha). We demonstrated a strong possibility to increase the genetic yield potential of indica rice varieties through allele mining and its application.

Adoptive T-Cell Therapy with 3rd Party CMV-pp65-Specific CTLs for CMV Viremia and Disease Arising after Allogeneic Hematopoietic Stem Cell Transplant

▪Adoptive immunotherapy with transplant donor-derived virus-specific T-cells is effective in the treatment of CMV viremia and disease complicating allogeneic hematopoietic stem cell transplant (HCT), but is not available if the donor is seronegative or unavailable to provide lymphocytes. In addition, CMV-specific T-cell lines (CMV-CTLs) from non-identical donors may be restricted by HLA alleles not shared by the patient, rendering them ineffective. This limitation has become more problematic with increased use of haploidentical HCT donors.We treated 50 transplant recipients with third party donor-derived CMVpp65-specific T-cells between 10/14/11 and 11/28/16, evaluable for response assessment as of 6/20/17. Patients had received an unmodified (n=11) or T-cell depleted HCT (n=33) or a cord blood (n=6), transplant. Fifteen were treated for overt CMV disease involving CNS (N=6) GI (N=10) and Lung (N=2) and 35 for CMV viremia persisting despite >2 weeks of induction therapy with 1-3 antiviral agents. Treatment with CMVpp65-CTLs was initiated at a median of 151 (29-4940) days post transplant and 128 (7-564) days after CMV reactivation. One patient was treated for CMV colitis developing more than 10 years after transplant due to immune suppression for chronic graft versus host disease. Patients had received a median of 3 (1-6) prior antiviral treatments.Third party CMVpp65-CTLs were selected from a bank of 186 lines generated under GMP conditions from normal HCT donors who specifically consented to use of their T cells in patients other than their designated transplant recipient. Selection was made on the basis of HLA restriction by at least one HLA allele shared by the patient and HCT donor, and matching for > 2/10 recipient alleles. If such a line was not available, a patient could be treated with a line matched at only one HLA allele as long as the restriction was through that matched allele. Patients received 3 weekly infusions of approximately 1x106 CMVpp65-CTL/kg/infusion. Patients were sequentially evaluated for clinical and radiographic changes, quantifications of CMV DNA by PCR and IFN+ CMVpp65-specific T-cells in the blood. Responses were assessed 28-42 days after the first of each cycle of CMVpp65-CTLs. Response in patients with CMV disease was considered complete (CR) if all sites were cleared of virus by biopsy and blood sampling and partial (PR) if symptoms resolved and viremia met criteria of PR. In patients treated for persistent viremia, responses were complete if CMV DNA was cleared in repeated testing, and partial if the level of CMV fell based on the testing method by >50% (N=2) or by 2log10 (N=12).Of the 50 patients 18 had a complete and 14 a partial response for an overall response rate of 64%. Response rates in patients with disease (5CR+4PR/15) were similar to those of patients with persistent viremia (13CR+10PR/35). In patients treated for viremia alone, survival at 6 months was 65.7% and in those with disease 60.0% (a). More extensively pretreated patients who received CMVpp65 CTLs > 100 days post CMV initial detection fared as well as those treated earlier (62.1% vs. 66.7% OS) (b). Patients who responded to CMVpp65-CTL therapy (CR or PR) had an improved survival with 6 month overall survival of 81.3% (b) and 12 month overall survival of 62.1% (c); only 1 of these 32 patients died of CMV. In contrast 7 of 18 non-responding patients died of CMV; overall survival in this cohort was 33.3% at 6 months. By 12 months, 8 non-responding patients had died of CMV and overall survival had decreased to 22.2%.Toxicities associated with CMVpp65-CTL infusions in this cohort are limited with 5 patients experiencing adverse events of > grade 3 severity deemed possibly related to CMVpp65-CTL therapy. Two of these patients died, one due to sepsis and one due to progression of CMV.This study demonstrates a high response rate among patients with otherwise refractory CMV viremia and disease. The bank of CMVpp65-CTLs can provide an immediate source of HLA partially-matched appropriately restricted T cells for adoptive immunotherapy to treat persistent CMV viremia and CMV disease, including disease isolated to the CNS. The availability of 3rd party CMVpp65-CTLs enables treatment early in the course of disease and may thereby improve response rates while minimizing toxicity from anti-viral therapy. [Display omitted] DisclosuresDoubrovina:Atara: Consultancy, Research Funding. Hasan:Atara Biotherapeutics: Consultancy, Other: During time of this study, Research Funding; Merck: Employment. Kernan:Gentium: Other: Received grants from Gentium during the conduct of the study and research was supported by The National Cancer Institute of the National Institutes of Health under award number P30 CA 008748, Research Funding. Koehne:Atara: Consultancy, Patents & Royalties. O'Reilly:Atara: Patents & Royalties, Research Funding.

P184 Next generation sequencing of small bowel biopsy reveals unresolved donor alleles and eliminates further augmented immunosuppression for antibody mediated rejection

Aim A young pediatric small bowel recipient was transplanted with a donor who was typed only at low resolution by an outside center. For 6 years, the patient received multiple blood transfusions and was monitored by IgG and C1q using LabScreen SAB (One Lambda). High MFI DSA to DQ2 was treated by IVIG desensitization; however, based on the low resolution typing, DQ2 DSA remained. To determine whether this was real DSA or whether the donor had a different DQ2 heterodimer, a fresh frozen biopsy sample was obtained to try to identify the donor HLA alleles by Next Generation Sequencing (NGS). Methods Full length genomic DNA from the patient and biopsy was sequenced for 11 loci (A,B,C,DRB1/3/4/5,DQA1/B1,DPA1/B1) using TruSight HLA v2 Sequencing Panel (Illumina), and analyzed by Assign v.2.1 (Illumina). Two to 3 field resolution was obtained. Results At each locus on the biopsy DNA, the sequence showed discontinuous alignments and much higher numbers of mismatched nucleotide positions than usual due to the mixture of both patient and donor. Individual sequenced reads containing mismatched nucleotides compared with the two predominant alleles were applied to BLAST using both the NCBI and IMGT websites. Once multiple alleles of the first field were obtained, polymorphic positions among exons were compared with other mismatched positions of the sequenced reads to determine the second and third allelic fields. Among all loci, more than two alleles were observed. By eliminating the recipient HLA alleles from the output, the donor’s HLA typing became clear. Typing of the donor by NGS from a biopsy sample revealed that the donor did not have the target heterodimer that had been giving high MFI values by both IgG and C1q. The DQ2 that had been followed was, in fact, not a DSA. Since a prior weak DR53 DSA had already resolved, continued treatment for AMR (Antibody-Medicated Rejection) was not indicated and augmented immunosuppression was discontinued. Conclusions This case decisively demonstrates the power of NGS technology to identify 2–3 field allele level HLA typing from a chimeric specimen using a frozen biopsy specimen. It also highlights the power of the technology to inform clinical decision making.

P070 A post-transplant chimerism study protocol that incoporated both STR and QPCR methods

Aim To monitor post-stem cell transplant chimerism status, most clinical laboratories use commercial kits for short tandem repeat (STR) or quantitative real-time PCR (qPCR). We present a protocol that combines both systems to maximize monitoring performance. Methods PowerPlex Fusion STR kit (Promega) was used to genotype 22 autosomal STR markers. Donor (%) was assigned as an average from five informative loci (Types 1 and 2). Fragment analysis used 3130xl Genetic Analyzer (Applied Biosystems) and GeneMapper software (Thermo Fisher Scientific). KMRType kit (GenDx) was used for genotyping 29 insertion/deletion markers (4-color multiplex) on QuantStudio 12K Flex Real-Time PCR instrument (Thermo Fisher). Two informative loci (insertion in recipient -deletion in donor) selected were used to determine minor allele (%) (one-color) by KMRTrack kit (GenDx) on the Roche LightCycler480 Real-Time PCR instrument (KMRTrack). The average from the two loci was assigned as minor allele (%). Results The average number of STR informative loci for a patient-donor pair was 18.3 (range: 16–21) for unrelated (N = 30) and 9.0 (range: 6–16) for related (N = 30). The number of qPCR informative loci was 6.0 (range: 4–9) for unrelated (N = 28) and 3.5 (range: 3–4) for related (N = 2). In qPCR, sensitivity increases when more DNA is used per reaction and surpasses STR sensitivity of 3–5% when >2 ng DNA is used per reaction. We validated the qPCR method using 183 different donor-recipient-marker combinations and 14 ng whole blood DNA/reaction, which achieved sensitivity of 0.5%. Correlation coefficient (r) of intended % donor (mixed DNA) vs. assay-determined was 0.999 by STR (N = 12 for range: 5–95% donor) and 0.91 by qPCR (range: 0.5–10% minor allele). Average %CV was similar at low minor allele range; STR (29.6% for 5% donor detection) vs. qPCR (average 27.8% for detecting 0.5%–10% minor allele). At high minor allele range, %CV decreased for STR (CV Conclusions The combination of STR and qPCR can improve a post-transplant monitoring protocol. STR (higher precision) is used immediately after transplantation when high recipient (minor) alleles are present. Testing will be switched to qPCR (higher sensitivity) once complete chimerism is achieved.

Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB).

Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC1F1, BC2F1 and BC3F1. The average recipient allele of the selected four BC1F1 plants was 80.75% which were used to produce the BC2F1 generation. BC1-P7 was the best BC1F1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC3F1 was 95.37%. Hsp gene expression analysis was carried out on BC1F1, BC2F1 and BC3F1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.