High Resolution HLA Typing Research Articles

P144 UNOSSM matching system failed to exclude a donor typed as B∗51:02 for a kidney candidate with HLA-B51 avoid

This reports a failure of DonorNet® match run to automatically exclude a B∗51:02 import deceased donor (DD) for candidate with B51 unacceptable antigen (UA) listed in UNetSM. The candidate was 61 y/o female with 100% CPRA re-listed for kidney transplant. The targets of all detectable HLA antibodies were listed as UA in UNet expecting that any DD offered by the DonorNet match run must be compatible i.e., virtual crossmatch (VXM) negative & donor-specific antibodies (DSA) negative. The UA lists 46 class I antigens, including B51 and 10 class II antigens (Fig. 1). It is expected that candidate will not be offered donors carrying any of the listed UA. However, import DD typed as B∗51:02 (Fig. 2) was offered as compatible by the DonorNet on 2/20/2017. The algorithm used by the allocation system did not list B∗51:02 as an equivalent of B51 (Fig. 3). As a consequence, patient was transplanted with DSA to B51 (MFI = 4679). The retrospective pronase crossmatches were weakly positive [MCS: T = 67 (cutoff = 50), B = 128 (cutoff = 120)]. The patient could have suffered a HYPERACUTE REJECTION had the DSA been strong enough to cause a CDC positive crossmatch. UNOS had been notified, but any change in the UNet system needs policy change and committee approvals which may take some time to implement. This event emphasizes the importance of maintaining accurate and updated HLA equivalency values in UNet to protect patient safety in the era of VXM. This incident questions the utility of reporting high resolution HLA types for DDs in DonorNet for match run. Until DD donor typing laboratories produce authentic high resolution HLA typing results with appropriate proficiency testing performed in a manner comparable to, and not more extensive than routine DD samples, only the serological equivalents for HLA-A, B, C, DRB1 and DQB1, first field typing for DQA1 and two field typing for DPB1 should be reported for match runs. UNOS should mandate laboratories to upload detailed typing reports that show single allele/allele string in DonorNet to better interpret the compatibility.Download : Download high-res image (556KB)Download : Download full-size image

P011 Advances in HLA clinical relevant exon novelty detection from over 21,000 donor recruitment samples

Aim The assignment of exon novel alleles is clinically relevant in hematopoietic stem cell transplantation (HSCT), in particular for donor selection. Here we investigate the exon novelty in over 21,000 National Marrow Donor Program (NMDP) registry donors by using next generation sequencing (NGS) approach on buccal swab DNA. Methods Multiplex long-range PCR primers amplified the full-length of HLA class I genes (HLA-A, -B, -C) from promoter to 3’ UTR. Class II genes (-DQB1, -DRB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2 × 250 bp paired-end sequencing on the Illumina MiSeq. Exon novelty was assigned using GenDx NGSengine v2.4.0 and Omixon Twin v1.0.7 software to ensure high confidence in allele calling. Exon novelties were confirmed by Sanger sequencing and 3D protein structure was predicted using Phyre2. Results Using this automated workflow, over 21,000 NMDP registry donors were successfully typed using high-resolution by NGS. NGS-HLA typing identified 94 exon novelties (0.044%) at five loci (HLA-A = 33, -B = 16, -C = 18, -DQB1 = 12, and -DRB1 = 15). We discerned 29 synonymous and 65 non-synonymous mutations. Among them 17 novel alleles (HLA-A = 3, -B = 5, -C = 2, -DQB1 = 4, and -DRB1 = 3) were detected within key exons involved in antigen recognition (exon 2 and 3 for Class I, and exon 2 for Class II). Interestingly, there are three frameshift novelties: A∗33:01:01NEW (1598InsC), A∗66:03NEW (1598delC), and B∗38:01:01NEW (9 bp del 1705-1713). Based on the 3D protein structure, these three proteins are truncated on α 3. Future experiments will determine if these proteins lose function or have lower cell surface expression. Conclusion Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. High resolution HLA typing provides more accurate matching of the HLA-A, -B, -C, -DQB1, and -DRB1 alleles to improve patient survival. This work is the first step to define permissive mismatches in the context of HSCT, and it emphasize the need to improve our knowledge on donor selection.

P233 On the performance characteristics of luminex single antigen bead (LSAB) assay mean fluorescence intensity cutpoint in predicting flow cytometry crossmatch (FCXM) results

Aim LSAB mean fluorescence intensity (MFI) is often used to predict physical FCXM results, and LSAB MFI cutpoints are used to list unacceptable antigens. We therefore determined the accuracy of LSAB assay MFI cutpoint used to score the LSAB assay as positive or negative in foretelling FCXM results. Methods One hundred and forty-six pre-transplant sera from 146 patients with end stage renal disease were tested against potential kidney donor’s T cells and B cells using a 3-color flow cytometry clinical protocol; the same 146 sera were tested using LSAB assay and the cumulative MFI directed at donor HLA class I and/or II antigens were determined. A T cell FCXM with a median channel shift (MCS) ≥ 40 with the patients serum vs. negative control serum was classified as a positive test; a B cell FCXM with MCS ≥ 50 with the patients serum vs. negative control serum was classified as a positive test. In the LSAB assay, MFI > 2000 directed at donor HLA antigens was scored as positive DSA. These cutpoints are our laboratory’s cutpoints for both ASHI proficiency testing and for clinical use. Results Statistical analysis demonstrated a significant association between LSAB results and FXCM results (Table 1). LSAB assay results predicted T Cell FCXM results with a sensitivity of 70% and a specificity of 100% (Fischers Exact Test, P 0.0001, Positive Predictive Value (PPV = 100%, Negative Predictive Value (NPV) = 91%). LSAB assay results also predicted B Cell FCXM results with a sensitivity of 86% and a specificity of 72% (Fischers Exact Test, P 0.0001, PPV = 62%, NPV = 91%) (Table 1). In a subset of 31 patients, both donor FCXM and auto FCXM results were available and 17 of 31 donor positive FCXM were also auto B cell FCXM positive (17/17) and auto T cell FCXM positive (1/17). Conclusions LSAB assay results are significantly associated with FCXM results. Auto B cell FCXM and high resolution HLA typing should further refine the relationship between these two high sensitive assays. Download high-res image (138KB) Download full-size image

P201 Erroneous HLA-B allele typing by sequencing-based typing method that utilizes combinations of Sanger sequencing and rSSO typing

Allele-level HLA matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplantation (HSCT). Sanger sequencing has been the gold standard for allele-level HLA typing since 1996. The typing strategy typically employs the sequencing of exons 2 and 3 for HLA Class I and exon 2 for HLA Class II. These exons encode the antigen recognition domains and sequencing only these exons are considered to be clinically relevant for HSCT. Sanger sequencing results significant number of ambiguities in HLA typing due to partial sequencing of limited exons, inability to phase heterozygous alleles, and growing number of new HLA alleles. Additional typing methods are required to resolve phasing and ambiguity problems. Next-generation sequencing (NGS) permits sequencing of the entire HLA gene and generates unambiguous and phased-resolved HLA typing results up to four field levels. Here we describe an erroneous HLA-B allele typing by Sanger sequencing. Sanger sequencing (Genome Diagnostics B.V.) of an unrelated NMDP donor showed the following possible HLA types: B∗15:01:01,18:14 or B∗15:07:01,18:01:01 or B∗15:07:01,18:17 N or B∗15:07:03,18:01:10. The B∗15:01:01, B∗15:07:01, B∗18:01:01, and B∗18:14 are common and well-documented alleles. The high-definition reverse sequence-specific oligonucleotide (rSSO) typing (One Lambda) ruled out all possible types except B∗15:01:01,18:14. The rSSO bead for B∗15:07:01 showed reactivity below the specified cutoff. We performed NGS typing on this sample, which enabled phasing of polymorphic positions 363 and 485 in exon 3 resulted unambiguous B∗15:07:01,18:01:01 typing. In summary, the strategy of Sanger sequencing plus rSSO typing resulted in incorrect HLA-B assignment for the NMDP donor, which in turn caused the exclusion of matched donor. The incorrect HLA allele assignment can be detrimental to HSCT recipient survival. NGS provides the capability to phase polymorphisms thereby eliminating most ambiguities and provides high-resolution HLA typing without reflexive, time-consuming and expensive additional testing, and therefore NGS is the new gold standard for allele-level HLA typing.

P048 NGS performance on a transplanted liver biopsy sample: A case study

A male pediatric patient was diagnosed with biliary atresia and was transplanted with liver from a deceased donor. The surgery went well, but years later the patient developed antibodies to Cw12, DR14, DQ7 and DQA1∗04:01 and various DP alleles. The need for high resolution typing to determine the identity of de novo DSA and the lack of any remaining pre-transplant sample on a deceased donor led to the use of NGS to determine chimeric typing. Genomic DNA was extracted from a post-transplant liver biopsy from this patient by using Gentra Puregene Tissue kit. We used the Immucor NGS FLEX HLA typing system for amplification and sequencing of HLA genes HLA -A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 followed by analysis with MIA FORA software. The NGS methodology is a clonal sequencing approach which solves the phase ambiguity problems inherent with Sanger-based sequencing when alleles are amplified and sequenced together. The genomic DNA extracted from the transplanted liver biopsy is chimeric, with residual recipient DNA at very low but detectable levels. The presence of chimeric reads is the result of co-amplification of both donor and recipient HLA genes. The majority of sequencing reads with zero mismatches belonged to the donor and were concordant with the deceased donor HLA type provided by UNOS. Unclaimed sequencing reads appeared as grey background and derived from the recipient HLA type (previously obtained with pre-transplant samples). In this case and a few other chimeric cases examined in our laboratory, the MIA FORA NGS software detected the HLA type of donor as the called alleles and showed some unclaimed reads that did not align to the donor type but rather to the HLA type of recipient. We note that if the donor and recipient HLA genes are perfectly matched in one or both alleles then they are indistinguishable. The clonal sequencing nature of the NGS reveals PCR primer specificity by identifying the sequences of all co-amplified genomic regions within the intended target region in the same biopsy sample. This case demonstrates the clinical utility of high resolution HLA typing by NGS technology in the clinical setting to de convolute a solid organ biopsy admixture i.e. the ability to do chimeric typing.

P073 Multiplexing a variety of clinicaaximize capacity of ion torrent next generation sequencing

Aim We have established the Ion-torrent based Next Generation Sequencing (NGS) system in our clinical laboratory. The chief use of the combination Ion-ChefTM and Ion-S5 is for high resolution HLA typing. However, the full sequencing capacity of the system is not always reached with the HLA typing workflow. In this study, we validated the practicality of combining library pools of different clinical tests into a single NGS run. Methods HLA library pools of 24–32 patient samples were generated with NXType™ Ion-Torrent NGS for Class I (HLA-A, -B, and -C) and Class II (HLA-DR, HLA-DQ, and HLA-DP). These libraries were run alone and then separately combined with library pools of other clinical NGS-based assays. These included: (1) our lab-generated custom tests (APOL1 genotyping (single gene, three SNPs) and Cholestasis genotyping (15 genes, multiple SNPs and INDELS), and (2) commercial assays (16S-Metagenomics, Thermo Fisher Scientific, Inc.) and Lymphotrack, T and B cell clonality (Invivoscribe). All library pools were run on Ion-Chef™ and Ion-S5. HLA data was analyzed with TypeStream™ software v1.1.0.11. The concentration of HLA library pools was maintained at 100 pM. Importantly, the concentration of library pools of other tests were adjusted between 5 and 20 pM, depending on the test (number of genes and size of amplicons) and the number of samples in the pool. Exported data of APOL1, Cholestasis and 16S-Metagenomics were analyzed with Ion Reporter™ software. Lymphotrack data was exported and analyzed with Lymphotrack data analysis software. Results The number of reads covering HLA genes were slightly reduced when runs were combined with libraries of other tests, however, the results of HLA library pools run alone and combined were 100% concordant. The coverage and read numbers obtained for the other tests were sufficient or exceeded the minimal required. Conclusions To our knowledge, this is the first report showing that multiplexing different assays in a single run of Ion-S5 was achievable. We demonstrated that mixing sequencing samples of different genotyping tests in a single NGS run generated accurate results of each individual test. The findings of this works have a positive impact reducing turnaround times and sequencing costs.

P156 Allelic and epitope HLA mismatch analysis in lung transplant

Aim The urgent need for donor lungs and a shortage of suitable organs requires transplantation of HLA mismatched donor:recipient pairs. Post-transplant high resolution HLA typing of the donor and recipient, if available, presents a post hoc opportunity to investigate comparative allelic and epitope mismatch strategies to predict post-transplant events and optimize immunosuppressive therapy. Methods Thirty donor-recipient lung transplant pairs with available extracted DNA were studied (De Vlaminck et al. (2015)). High resolution NGS HLA typing required targeted sequencing and was performed according to Moonsamy et al. (2013). Respective two-region HLA types were assigned using Conexio ASSIGN ATF 454 software and compared to the catalog of common and well-documented HLA alleles. Epitope mismatches were determined using HLAMatchmaker. Prior transplant studies noted the importance of HLA-A, HLA-B and DRB1 for short and long term survival and therefore were selected for this study. Results Thirty lung transplant pairs had 3 (n = 1), 4 (5), 5 (10) and 6 (14) HLA-A, HLA-B and HLA DRB1 allelic mismatches. Epitope mismatches ranged from 16–51. The epitope mismatch extremes each had 6 allelic mismatches: 16: HLA-A,-B 9; DRB1 7 and 51: HLA-A,-B 19; DRB1 32. The single 3 allelic mismatch pair had 31 epitope mismatches: HLA-A,-B 10; DRB1 21. DRB1 epitope mismatch contribution to total epitope mismatches ranged from 19% to 63%. Conclusions Allelic (antigenic) mismatches have a more limited range than epitope (immunogenic) mismatches, and mismatch burdens using these two models varied considerably in lung transplants in this study. The relative contribution of epitope mismatches in HLA-A, HLA-B and DRB1 varies in transplant donor recipient pairs. Prognostic estimates of antibody mediated rejection to inform surveillance and increased risk of post-transplant malignancies and infection to inform long term immunosuppressive therapy merit investigation using epitope mismatch burden.

P044 One year experience with HLA typing by next generation sequencing (NGS), achievements and challenges

Aim After a year of implementing HLA typing by NGS, we present our most relevant achievements and challenges. Methods Class I (full HLA-A, -B, and -C genes) and HLA Class II (Exon 2 thru intron 3 of HLA-DR and HLA-DQ, and Exon 2 thru 3’UTR of HLA-DP) amplified with NXType™ Class I and Class II primer sets. Automated template preparation and chip loading were performed in the Ion Chef™ system and run on Ion S5™. Data was analyzed with TypeStream™ v1.1.0.11 software. Results Having typed over 1000 samples by NGS, with 50% from bone marrow programs and 50% from solid organ programs, our workflow has changed significantly: Threefold increase in capacity with simultaneous decrease in turn-around-time (TAT, on average 10 days) for high resolution typing. Our current capacity is up to 96 HLA typings in 3–4 days vs. minimum of 16 days for the same volume with SBT by Sanger. Resolution of most ambiguities (98.7%), minimizing the need for reflex testing.Repetitions due to technical failures and ambiguities decreased from 4.3% with SBT-Sanger to 0.3% with NGS. In spite of positive workflow changes, there remain challenges: Automation of library preparation has not been implemented.No merging of library preparation reagents into a single kit.Smaller runs increase the number of reads, leading to artifacts and high background generated by the software, interfering with analysis and limiting flexibility for STAT sample processing.For solid organ typings, generating high resolution results while reporting Serological Equivalents for UNOS, poses logistical difficulties.TypeStreamTM software not interfaced with Fusion, requiring continuous need to run SSOP for DSA analysis.Unresolved ambiguities (1.3%), due to incomplete exon coverage and phase ambiguities. Resolution may be obtained by increasing gene coverage, length of reads and with additional testing methods. Conclusions We have confirmed that NGS is both a suitable and faster method to obtain high resolution HLA typing. We have seen a significant decrease in TAT along with a significant cost savings due to fewer repetitions, fewer reagents, and most significantly, reduced time from our most valuable asset, our technologists.

P153 Utility of the virtual crossmatch for living donor evaluations

Aim Our institution performs 750–900 flow cytometric crossmatches (FCXM) annually in support of living-donor renal transplantation. The FCXM is costly, time consuming, and has interpretational conundrums (i.e., FCXM + donor specific antibody (DSA) negative or FCXM−/DSA+). By combining high resolution HLA typing with solid-phase antibody (SAB) testing, recipients should be transplantable based solely on a virtual crossmatch (vXM). Given the increased utilization of the vXM, our aim was to assess its utility in living donor evaluations. Methods We reviewed 100 living donor FCXMs and retrospectively performed a vXM for each pair. vXM was performed only on cases with available donor typing and SAB specificity testing. Additionally, ΔMESF values of the FCXM, FlowPRA results, and mean fluorescence intensity (MFI) values of DSA were analyzed. Results Initially, there was a 75% correlation between FCXM and vXM: 60 cases were negative for both FCXM and vXM; 15 cases were positive for both. However, 20 cases were FCXM+ (mostly B-cell) but vXM−, which tended to occur in non-alloimmunized recipients. 3/20 had autoimmune disease and 8/20 were transplanted without development of DSA. Although deemed compatible, the remaining 12 were not transplanted. These 20 cases were considered False Positive FCXMs. 5 cases were FCXM−/vXM+. Of these, 4 had Class I DSAs and 1 had both Class I and II DSAs; however, all 5 had low MFI values. (Table 1). Thus, reclassifying the false positive FCXMs, the final correlation was 95%. Download : Download high-res image (146KB) Download : Download full-size image Conclusions Though the initial FCXM/vXM correlation rate was only 75%, there are key characteristics such as differences in FCXM, ΔMESF, PRA values, and DSA MFI values that can help interpret discrepant result. Notably, there were 4 times more false positive FCXMs then false positive vXMs. Reclassifying the False Positive FCXM cases increased the correlation to 95%. These data support the utility of the vXM as a replacement for a physical FCXM for most living donor evaluations.

P252 Conceptualization to implementation in 90 days of full gene HLA typing using mia fora HLA flex kits at Jeevan stem cell foundation, India

Aim Next Generation Sequencing (NGS) is rapidly becoming the method of choice for HLA typing for Hematopoietic Stem Cell Transplantation. Jeevan Stem Cell Foundation, Chennai, India validated the full gene HLA typing using Immucor’s MIA FORA FLEX kits from conceptualization to implementation in ninety days. This implementation, in line with the Indian Government’s policy of “Make in India”, enabled Jeevan to establish a cost effective high resolution HLA typing to international standards in India to support the transplant physicians and registries in the region and prevent Indian DNA being shipped to other countries. Methods Reference DNA used for validation were previously typed by SBT included samples from UK NEQAS, FRED HUTCHINSON and Jeevan’s registry samples. DNA was extracted using the Qiacube. HLA typing for A, B, C, DRB1 and DQB1 were performed. All workflows were performed according to the supplier’s instructions. NGS sequencing was done using the mid output flow cell kit from Illumina and sequenced on the Illumina MiniSeq platform. Immucor’s proprietary software was used to analyse the data. Results Results were evaluated per allele on the 2nd field consensus compared to the reference, the number of allelic ambiguities, genotype ambiguities and allelic drop outs were measured. The second field resolution was used because higher resolution typing was not available for all samples. MIA FORA NGS software provided high resolution typing for all samples. There results from NGS typing compared to SBT typing are shown in Table 1. Conclusions The implementation of the MIA FORA FLEX HLA typing kit at Jeevan is the first outside USA. With the MIA FORA kits, the turnaround time will depend upon the number of samples. In our experience reports for 48 samples can be delivered within 72 h. There were no ambiguous results and no allelic dropout. The proprietary software of Immucor was easy to use and significantly reduced the analysis time. Download high-res image (213KB) Download full-size image

P258 Evaluation of high-resolution HLA typing of Japanese samples using NXType™ NGS HLA typing kit

Aim The human MHC region has been shown to be associated with a wide range of diseases.The recent advancement in the next generation sequencing (NGS) technology has definitely helped in increasing the resolution (up to 4-field) of HLA genotyping. However, it remains a challenge in interpreting the results from NGS platform due to biases including systematic sequencing error by sequencing platform, difference in the sensitivity and specificity of HLA calling algorithm, ability to resolve HLA alleles ambiguity by incorporating phasing information, and more importantly incompleteness of the current IMGT database. Methods In order to access the sensitivity and specificity of the NXTypeTM NGS HLA typing kit (One Lambda), a total of 105 Japanese samples were sequenced for HLA class I genes using the Ion Torrent Personal Genome Machine (PGM) following the NXType protocol. HLA calling was performed using the HLATypeStream v1.0.0.86 with IMGT/HLA databses of 3.21.0. Concordance rates were evaluated by comparing HLATypeStream results with Luminex-based HLA typing result for up to 2-field result. Results With the default setting of HLATypeStream and without manual interpretation of the results, the allelic concordance is more than 98% for HLA-A, HLA-B and HLA-C. Other commercially available HLA calling software and open-access software were also evaluated. Conclusions Careful inspection of the final results and occasionally manual interpretations of results are necessary especially for rare or novel HLA alleles.

OR22 High-resolution HLA typing and identification of a novel allele with a splice variant using RNA-SEQ

Aim RNA sequencing (RNA-Seq) is a powerful next-generation sequencing method for discovering, profiling and quantifying RNA transcripts. RNA-Seq can also identify splice junctions and single nucleotide polymorphisms (SNP) in coding exons. In this study, we tested if RNA-Seq reads provide sufficient coverage and depth for high-resolution HLA genotyping. Methods RNA-Seq data was available from 24 individuals from an acutely infected HIV cohort. We used two methods to extract HLA reads from the RNA-Seq data: 1) FR-HIT recruited RNA-Seq reads to the IMGT coding sequence reference database; and 2) the SNP-tolerant aligner GSNAP aligned the reads to a constructed GMAP database consisting of cDNA from HLA genes. The resulting HLA-specific FASTQ reads that were generated were analyzed by commercial HLA typing software. Results We obtained 100% sequence coverage and a median sequence read depth of 331, which was sufficient to genotype 15 HLA loci from each of the donors. HLA genotypes for 8 classical HLA class I and II loci matched genotypes generated from the Sanger sequence-based typing method, as well as targeted next-generation sequencing of genomic DNA. We identified a novel HLA-DQB1 allele in the Thai population with a frequency greater than 5% that was identical to the 05:01:01 allele in all exons except exon 1, where it differed by one SNP at position 81. The DQB1 * 05:01:01 allele, like several other DQB1 alleles, has exon 5 spliced out. This deletion however, cannot be confirmed from the reads that are obtained by sequencing genomic DNA. RNA-Seq confirmed that the novel allele does not express exon 5, as the RNA-Seq data showed a complete absence of exon 5 reads compared to reads from targeted genomic sequences. Conclusions We have successfully genotyped 15 HLA loci and confirmed a splice variant in a novel allele using RNA-Seq. This method highlights the importance of mining RNA-Seq data for high-resolution HLA genotyping. Additional analysis methods may be able to identify allele-specific expression of HLA alleles from RNA-Seq data.

Efficacy of two different doses of rabbit anti-T-lymphocyte globulin to prevent graft-versus-host disease in children with haematological malignancies transplanted from an unrelated donor: a multicentre, randomised, open-label, phase 3 trial

Although rabbit anti-T-lymphocyte globulin (ATLG) is largely used for the prevention of immune-mediated complications in patients given allogeneic haemopoietic stem-cell transplantation (HSCT) from an unrelated donor, the optimum dose of this drug in children is still undefined. We aimed to test whether a higher dose of ATLG was superior to a lower dose for prevention of grade II-IV acute graft-versus-host disease (GVHD). We conducted a multicentre, randomised, open-label, phase 3 trial in seven Italian centres comparing two different doses of ATLG (30 mg/kg vs 15 mg/kg, given intravenously over 3 days, from day -4 to -2) in children (aged 0-18 years) with haematological malignancies transplanted from an unrelated donor, selected using high-resolution typing for HLA-class I/II loci. All patients received a myeloablative regimen and cyclosporine-A plus short-term methotrexate as post-transplantation GVHD prophylaxis. Patients were randomly assigned (1:1) to either of the two groups and were stratified by the degree of HLA-compatibility with their donor, the source of haemopoietic stem cells used (bone marrow vs peripheral blood stem cells), and the disease risk category. The randomisation was open label; all investigators were aware of the treatment allocation. The primary endpoint of the study was 100-day cumulative incidence of grade II-IV acute GVHD. Statistical analyses were done according to the per-protocol principle. Other outcomes included cumulative incidence of chronic GVHD, non-relapse mortality, disease recurrence, and probability of overall survival and event-free survival. This study was registered with ClinicalTrials.gov, number NCT00934557. Between Jan 15, 2008, and Sept 25, 2012, 89 patients were randomly assigned to the 30 mg/kg ATLG group and 91 to the 15 mg/kg ATLG group; 84 patients in the 30 mg/kg ATLG group and 88 in the 15 mg/kg ATLG group were included in the analysis. The median follow-up for the whole study population was 3·4 years (IQR 1·7-5·1). The 100-day cumulative incidence of grade II-IV acute GVHD was 36% (95% CI 28-48) in the 15 mg/kg ATLG group and 29% (20-40) in the 30 mg/kg ATLG group (hazard ratio [HR] 0·74, 95% CI 0·44-1·25; p=0·26). The cumulative incidence of non-relapse mortality was 9% (5-18) in the 15 mg/kg ATLG group and 19% (12-30) in the 30 mg/kg ATLG group (HR 2·08, 0·89-4·96; p=0·092). Cumulative incidence of disease recurrence was 15% (12-24): 14% (8-23) in the 15 mg/kg ATLG group versus 20% (13-31) in the 30 mg/kg ATLG group (HR 1·54, 0·74-3·21; p=0·25). The 5-year overall survival probability was 70% (62-77) for the whole study population: 78% (69-87) in the 15 mg/kg ATLG group versus 62% (50-73) in the 30 mg/kg ATLG group (HR 1·80, 1·01-3·20; p=0·045). The 5-year event-free survival was 77% for children in the 15 mg/kg ATLG group versus 61% in the 30 mg/kg ATLG group (HR 1·87, 1·07-3·28; p=0·028). Children with haematological malignancies transplanted from unrelated donors selected through high-resolution HLA-typing benefit from the use of a 15 mg/kg ATLG dose in comparison with a 30 mg/kg ATLG dose. ATLG at 15 mg/kg should thus be regarded as the standard serotherapy regimen for unrelated donor allogeneic HSCT in this patient population. Future randomised studies will continue to aim to optimise patient outcome and strategies to prevent acute GVHD occurrence. Fresenius/Neovii Biotech.

The Case for High Resolution Extended 6-Loci HLA Typing for Identifying Related Donors in the Indian Subcontinent

Three-loci low-resolution (LR) or intermediate-resolution HLA typing is generally considered adequate in the related blood and marrow transplantation (BMT) context. However, a single high-resolution (HR) mismatch may have a similar adverse impact on BMT outcome as an LR one. We sought to determine the frequency of mismatches that may go undetected when standard typing (LR or 3-loci HR) is used compared with 6-loci HR typing for related donor compatibility testing, and to assess its impact on relevant BMT outcomes. We analyzed data from a total of 2554 6-loci (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) HR sequence-based typing (full typing [FT]) from 754 patients, 1011 siblings, and 789 parents done at DKMS Germany (www.DKMS.de) between January 1, 2014, and June 21, 2016. We also studied 38 cases in which the family had undergone 3-loci HLA typing (standard typing [ST]). Patients were from India (70%), Pakistan (22%), and Sri Lanka (8%). The IMGT/HLA database (www.ebi.ac.uk/ipd/imgt/hla) was used to tease out nonpermissive DPB1 mismatches. HLA disparity–related outcomes, such as rejection and graft-versus-host disease (GVHD) were assessed in a retrospective matched-pair cohort of 50 patients (25 with ST and 25 with FT) who underwent BMT for severe thalassemia from compatible related donors. We found fully matched (either 12/12 HR matches or with a single permissive DPB1 mismatch) related donors for 285 patients (38%). Of these donors, 89% were siblings and 11% were parents. The likelihood of matching on an individual locus on LR but not on HR was found to be 5%. A total of 9 donors (3%; 7 siblings and 2 parents) who would have been considered a full match by HR typing on A, B, and DRB1 alone were not a match by extended 6-loci HR typing. Five of these 9 donors had a mismatch on C or DQB1, and 4 had a nonpermissive DPB1 mismatch. In this group, 5 donors (56%) belonged to a consanguineous family, in 2 donors (22%) there was no reported consanguinity, and in 2 donors (22%) consanguinity was unknown. We identified 18 donors (6%; 13 siblings and 5 parents) who would have been considered a 12/12 match by LR HLA typing alone but were found not to match on extended HR typing. In this group, 11 donors (61%) were from consanguineous families, 3 donors (17%) had no reported consanguinity, and in 4 donors (22%) consanguinity was unknown. Outcome analysis showed that the actuarial proportion of patients with GVHD was 4% in the FT group compared with 16% in the ST group, with log-rank P = .1952. The ST group included 2 patients with grade III-IV acute GVHD and 1 patient each with moderate and severe chronic GVHD, whereas the FT group only 1 patient with grade III acute GVHD. We conclude that even in the context of related donors, the use of LR and/or 3-loci (A, B, and DRB1) HR HLA typing might result in a sizable risk of missing a clinically relevant mismatch, which may have an adverse impact on transplantation outcomes. In the Indian subcontinent, this observation is not limited to putatively compatible parents or consanguineous families; we recommend full 6-loci HR HLA typing even for matched related BMTs.

Allogeneic Hematopoietic Stem Cell Transplantation for Adolescents and Young Adults with Acute Myeloid Leukemia

Few reports have focused on adolescent and young adult (AYA) patients with acute myeloid leukemia (AML) treated with hematopoietic stem cell transplantation (HSCT). We performed a retrospective analysis based on data obtained from a Japanese nationwide registration database to compare HSCT outcomes in AYA patients with AML with those in children with AML. An analysis of the 2973 patients with de novo AML who received allogeneic HSCT from 1990 to 2013 showed inferior 5-year overall survival (OS) (54% versus 58%, P < .01) and increased treatment-related mortality (TRM) (16% versus 13%, P = .02) in AYA patients. Multivariate analysis for both OS and TRM showed a significant negative impact on AYAs. However, the negative impact of older age lost its significance in an additional analysis focusing on 1407 recent transplant recipients with high-resolution HLA typing (2000 to 2013). Finally, we analyzed the impact of transplantation center type on HSCT outcomes in 317 adolescent patients (15 to 18 years old) and found no difference in outcomes between patients treated at a pediatric or an adult hospital. Higher age was a strong predictive factor for inferior OS resulting from increased TRM, which can be eliminated with better donor selection using high-resolution HLA typing.

Genotyping Applications for Transplantation and Transfusion Management: The Emory Experience.

Current genotyping methodologies for transplantation and transfusion management employ multiplex systems that allow for simultaneous detection of multiple HLA antigens, human platelet antigens, and red blood cell (RBC) antigens. The development of high-resolution, molecular HLA typing has led to improved outcomes in unrelated hematopoietic stem cell transplants by better identifying compatible alleles of the HLA-A, B, C, DRB1, and DQB1 antigens. In solid organ transplantation, the combination of high-resolution HLA typing with solid-phase antibody identification has proven of value for highly sensitized patients and has significantly reduced incompatible crossmatches at the time of organ allocation. This database-driven, combined HLA antigen/antibody testing has enabled routine implementation of "virtual crossmatching" and may even obviate the need for physical crossmatching. In addition, DNA-based testing for RBC antigens provides an alternative typing method that mitigates many of the limitations of hemagglutination-based phenotyping. Although RBC genotyping has utility in various transfusion settings, it has arguably been most useful for minimizing alloimmunization in the management of transfusion-dependent patients with sickle cell disease or thalassemia. The availability of high-throughput RBC genotyping for both individuals and large populations of donors, along with coordinated informatics systems to compare patients' antigen profiles with available antigen-negative and/or rare blood-typed donors, holds promise for improving the efficiency, reliability, and extent of RBC matching for this population.

Allogeneic hematopoietic cell transplantation in patients with AML not achieving remission: potentially curative therapy.

Patients with acute myeloid leukemia (AML) who fail to achieve complete remission (CR) have a dismal prognosis. Although data suggest that durable remissions can be achieved in approximately 30% of patients with refractory or relapsed AML after allogeneic hematopoietic cell transplantation (HCT), only a small fraction of those patients are offered this therapeutic option. Importantly, patients with primary refractory AML have distinctly better outcomes following allogeneic HCT than those with refractory relapse. Access to suitable donors could be one of the main barriers in these situations. However, with recent developments in the field of allogeneic HCT, such as alternative donor sources, high-resolution HLA-typing, reduced intensity conditioning regimens and improvements in supportive care, this approach has the potential to offer long-term survival for patients with refractory and relapsed AML and should be considered as early after diagnosis as possible. Incorporating novel agents into the conditioning regimen or as post-transplant maintenance therapy could further improve outcomes and render older or medically infirm patients with refractory or relapsed AML eligible for allogeneic HCT. In this review, we summarize existing data on allogeneic HCT in patients with refractory or relapsed AML and explore novel approaches with the potential to improve outcomes in this patient population.

Low Dose Antithymocyte Globulin (ATG) for Graft-Versus-Host Disease (GVHD) Prophylaxis

ATG appears to reduce the incidence of acute and chronic GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Potential risks of this strategy include viral reactivation, delayed immune recovery and increased relapse rates. The ideal dosing of rabbit ATG in this context is largely unknown. We therefore hypothesized that low dose ATG would reduce the incidence of acute and chronic GVHD in matched unrelated donor (MUD) transplants without compromising survival and relapse rate. A retrospective analysis was performed of a cohort of high-risk MUD HSCT recipients treated since year 2013, when our practice changed to include rabbit ATG at 3 mg/Kg for all MUD transplants at the Case Medical Center in Cleveland, Ohio. Herein we present the results of this analysis.Methods. 58 MUD transplants were performed between years 2013 and 2016, with a median follow up of 262 days post-transplant. All donor-recipient pairs were matched by high resolution HLA typing at HLA-A, -B, -C, and DRB1, (8/8 matches) with the exception of 4 pairs (7/8 matches. Median age was 56 years (range, 53-64). Underlying diagnoses were AML (n=26), MDS (13), CML (n=5), NHL (n=9), Hodgkin's lymphoma (n=2), Multiple Myeloma (n=1) and myeloproliferative disorders (n=2). Preparative regimens were ablative in 26 cases (45 %) and of reduced intensity in 31 cases (55 %). Graft source was bone marrow (n=5) and peripheral blood (n=53). All but 4 pts received GVHD prophylaxis with tacrolimus, and mini-methotrexate (5 mg/m2 on days +1, +3, +6 and +11), in addition to rabbit ATG 3 mg/Kg divided in two doses on days -2 and -1 pre HSCT. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Human Herpes Virus (HHV6) PCR were conducted thrice weekly during the first 100 days after HSCT.Results. The 100-day cumulative incidence of grade II-IV acute GVHD was 41% (95% CI: 29-57; Fig 1), while the cumulative incidence of grade III-IV acute GVHD was 18% (95% CI: 9-35; Fig 2). 1-year cumulative incidence of chronic GVHD was 27% (95% CI: 17-42; Fig 3). At 180 days, the incidence of CMV viremia (defined as more than 1,000 copies/mL) was 25% (95% CI: 16-40), while the incidence of EBV and of HHV6 viremia was 35% (95% CI: 24-51) and 14% (95% CI: 8-27), respectively. There was no instance of EBV-related lymphoproliferative disorder.3-year overall survival estimate is 48% (95% CI: 34-62). Cumulative incidence of Non-relapse mortality (NRM) and relapse at 1 year was 21% (95% CI: 12-37) and 44% (95% CI: 29-65), respectively.Conclusion. Our study shows that low dose rabbit ATG appears to reduce chronic GVHD rates without a major effect on acute GVHD incidence. CMV, EBV and HHV6 reactivation did occur, albeit at rates that are somewhat lower than those historically reported, without EBV-driven lymphoproliferative disorder. [Display omitted] [Display omitted] [Display omitted] DisclosuresCaimi:Genentech: Speakers Bureau; Roche: Research Funding; Novartis: Consultancy; Gilead: Consultancy.

Long-Term Results on Outcome of Children with Hematological Malignancies Transplanted from an Unrelated Donor Enrolled in an Open Label, Randomized Trial Comparing Two Different Dosages of Rabbit Anti-Thymocyte Globulin

▪Introduction. Although rabbit anti-thymocyte globulin (ATG) is largely used for preventing immune-mediated complications in patients given allogeneic hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD), the optimal dosage of this drug in children is still undefined. We conducted an academic open-label, multicenter, randomized trial comparing two different dosages of rATG in children with hematological malignancies transplanted with either bone marrow (BM) or peripheral blood stem cell (PBSC) from a MUD selected using high-resolution typing for HLA class I/II (i.e. A, B, C, DRB1) loci. The study aimed at testing whether a higher dose of rATG (30 mg/Kg over 3 days, from day -4 to day -2) was superior to a lower dose (15 mg/Kg over 3 days, from day -4 to day -2) in terms of grade II-IV acute graft-versus-host disease (GvHD) prevention. Secondary end-points included cumulative incidence (CI) of chronic GvHD, non-relapse mortality (NRM), disease recurrence and event-free survival (EFS). The drug was kindly provided by Fresenius/Neovii.Patients and methods. Inclusion criteria were: diagnosis of acute or chronic leukemia in remission/chronic phase, non-Hodgkin lymphoma or myelodysplasia; age at HSCT 0-19 years; availability of an unrelated donor selected using high-resolution molecular typing of HLA-A, B, C and DRB1 loci, completely matched or with a single allelic disparity at one of the HLA loci or with 2 allelic disparities, or with one antigenic disparity; use of G-CSF-mobilized PBSC or BM-derived hematopoietic stem cells.From January 2008 to September 2012, 180 patients were enrolled in the study. Eight patients, 5 randomized to the rATG 30 mg/kg group and 3 to the 15 mg/kg group, did not proceed to transplant due to further relapse before HSCT. The remaining 172 patients, 84 belonging to the 30 mg/kg group and 88 belonging to the 15 mg/kg group, were transplanted and included in this analysis; 94 children had acute lymphoblastic and 42 acute myeloid leukemia. The 2 randomization groups were comparable for all demographic and transplant-related variables evaluated (see Table 1). Patients were stratified according to the stem cell source (BM vs. PBSC), degree of HLA compatibility with the donor (unrelated donor perfectly matched or with 1 allelic disparity vs. unrelated donor with 2 allelic disparities or with 1 antigenic disparity) and disease risk group (standard- vs. high-risk, SR and HR). All patients received a fully myeloablative preparative regimen. All patients received cyclosporine-A and short-term methotrexate as post-transplant GvHD prophylaxis. The trial was registered at ClinicalTrials.gov (NCT00934557). Data were analyzed as of January 31st, 2016.Results. With a median follow-up of 4.5 years (range 3.3-7.6 years), the 100-day CI of grade II-IV acute GvHD in the high and low rATG group was 29% and 36%, respectively (P=NS, Figure 1A); the CI of extensive chronic GvHD in the two groups was 6% and 10%, respectively (P=NS, Figure 1B). The CI of NRM was 19% and 9% in the high and low rATG group, respectively (P=0.09). Children receiving the higher rATG dosage had a greater incidence of viral reactivations as compared to those who received the lower dosage. The difference was statistically significant for EBV reactivation (37% vs. 23%; P = 0.038) and for Adenovirus reactivation (12% vs. 1%; P = 0.004). The overall CI of disease recurrence was 17% and did not differ between high and low dose rATG.The 5-year EFS for the whole cohort of patients was 69%; it was 61% and 77% for children given high and low dose rATG, respectively (P=0.028, Figure 1C). EFS was 78% and 55% in the SR and HR groups, respectively (P=0.001, Figure 1D). EFS of the 136 children with acute leukemia given either high or low dose rATG was 60% and 77%, respectively (P=0.049). In multivariate analysis on EFS, the following variables were associated with an unfavorable outcome: rATG dose of 30 mg/kg (relative risk 1.90; P=0.026), HLA mismatch > 1 allele (relative risk 2.08; P=0.01) and HR disease (relative risk 2.46; P=0.0015).Conclusions. Our data indicate that, in children with hematological malignancies transplanted from a MUD selected through high-resolution HLA typing, the use of low dose rATG results into decreased incidence of NRM and better EFS. Low dose rATG is able to spare life-threatening viral infections, without significantly increasing the incidence of acute and chronic GvHD. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Selection of Unrelated Allogeneic Hematopoietic Cell Donors Based on KIR3DL1 Allotypes Is Feasible and Results in Improved Disease-Free Survival in Transplant Recipients with MDS and AML

Donor natural killer (NK) cells impart a potent anti-leukemia effect after allogeneic hematopoietic cell transplantation (allo HCT). The killer Ig-like receptors (KIR) play a central role in modulating NK effector function. Chief among them, the inhibitory KIR3DL1 exhibits a high degree of allelic polymorphism. We previously demonstrated that highly expressed KIR3DL1 alleles (KIR3DL1 allele groups *001 and *002) bind preferentially to HLA-Bw4 with I80 to confer a high degree of inhibitory signaling. Similarly, poorly expressed KIR3DL1 alleles (KIR3DL1 allele groups *005 and *007) bind to HLA-Bw4 with T80 to confer a strong inhibitory signal. In a retrospective cohort of 299 patients undergoing allo HCT for AML, we demonstrated that these highly inhibitory donor KIR3DL1/recipient HLA-Bw combinations (KIR3DL1-HIGH) resulted in a greater incidence of recipient relapse when compared to other donor/recipient KIR3DL1/HLA-Bw pairings with low or absent inhibitory signal (KIR3DL1-LOW/NO). (Giglio F et al, ASH Annual Meeting Abstracts, 2012) Here, we evaluated whether it was feasible to prospectively type and use KIR3DL1 allotypes in unrelated allo HCT donor selection, and whether this intervention would result in improved outcomes in patients undergoing transplant for AML and MDS. We performed PCR-SSP based intermediate resolution KIR3DL1 allele typing on all unrelated adult donors evaluated for patients with MDS and AML at our center from 2013 to present as previously described. (Boudreau J et al, PLoS One, 2014) KIR3DL1 status was provided to the treating physicians, who made the final donor selection.A total of 941 prospective allo HCT donors underwent high resolution HLA typing and KIR3DL1 allotyping for 252 patients (median per patient = 4, range 1-12). Among all donors evaluated, 27% were found to be KIR3DL1-HIGH when considering the respective recipient HLA-Bw. Having multiple donors evaluated improved the likelihood of avoiding a KIR3DL1-HIGH donor: Among recipients with one donor evaluated, 27% had only KIR3DL1-HIGH donors available compared to 12% in recipients with 2-3 donors evaluated and 4% for recipients with >3 donors evaluated (P < 0.0001). Among all evaluated patients, 41% had both KIR3DL1-HIGH and KIR3DL1-LOW/NO donors available, indicating a potential selection based on KIR3DL1 was possible.Among 252 patients evaluated, 115 proceeded to allo HCT (48 with MDS, 67 with AML). The median age at transplant was 62 years (range 3.6-78). Donors were HLA matched in 105 transplants and were single loci mismatched in 10 transplants. Conditioning was myeloablative in 73 (11 with total body irradiation). GVHD prophylaxis was with ex vivo CD34+ selection in 65 patients and with tacrolimus/methotrexate in the remaining patients. The median follow-up in transplanted patients was 13.1 months (range 0.5-43.3 months). On univariate analysis, the 2-year disease-free survival was 64% (95% confidence interval: 54-76%) in those with KIR3DL1-LOW/NO donors versus 39% (22-68%) in recipients with KIR3DL1-HIGH donors (P = 0.05). The incidence of relapse was similar but favored patients with KIR3DL1-LOW/NO donors (26% [15-37%] versus 35% [13-57%], P = 0.5). 2-year overall survival was similar between those with KIR3DL1-LOW/NO versus KIR3DL1-HIGH donors (75% [65-86%] versus 69% [52-91%], P = 0.43).The time from the initiation of a formalized donor search to transplant did not differ between recipients with KIR3DL1-LOW/NO versus KIR3DL1-HIGH donors (median 81 v. 83 days, respectively, P = 0.97). Recipients with KIR3DL1-LOW/NO donors were CIBMTR Transplant Risk Score low = 53, intermediate = 6, and high = 33; whereas recipients of KIR3DL1-HIGH donors were low = 17, intermediate = 2, and high = 4 (P = 0.15). These results indicate that disease severity and transplant urgency did not influence whether a KIR3DL1-LOW/NO donor was able to be selected.In summary, these prospective data on 115 patients from a single center support improved outcomes in patients with AML and MDS undergoing unrelated donor allo HCT using a donor with low or absent KIR3DL1 inhibition. A multi-center, prospective study to evaluate the prospective use of HLA and KIR genotype based selection of URDs for patients undergoing allo HCT for AML is underway (NCT02450708). [Display omitted] [Display omitted] DisclosuresKernan:Gentium: Research Funding; The National Cancer Institute of the National Institutes of Health: Research Funding.