Postergroup No. 2/2
Stem cell transplantation
Form of presentation:
HLA-DPB1 TCE permissivity as a secondary selection criteria in unrelated stem cell transplantytion (SCT) - 1 year single centre experience
P. Jindra (Pilzen, CZ)
With increasing number and typing quality of unrelated donors (UD) the probability of finding multiple perfectly matched UDs for Caucasian patients is high. Current selection algorithm depends on matching for 5 genes, HLA-A,-B,-C,-DRB1, and -DQB1 (10/10 matched). Recent data has suggested that DPB1 compatibility is also important. In unrelated settings HLA-DPB1 disparity occurs in ~ 80% of pairs matched for 10/10 of the non-DP HLA loci due to low LD between HLA-DP and the other HLA class II loci. An approach to defining ‘non-permissive’, poorly tolerated HLA-DPB1 mismatches on the basis of alloreactive T-cell epitope (TCE) groups was recently introduced and shown to be of clinical relevance. We summarize a 1-year pilot experience with the policy of secondary selection according the TCE in 26 patients for whom 90 UDs (median 4, range 2-6 UD) where six HLA loci were typed and TCE reactivity established. The primary selection consists of standard 5-loci matching, i.e. no additional UD was typed in order to find DPB1 matched/permissive UD. A DPB1 matched donor was found for 6 (23%) patients whereas out of 20 patients with DPB1 mismatched UDs, a permissive UD was identified for 14 (54%) patients. Only for six (23%) patients were non-permissive DP-MM UDs the only ones available. The proportions of DPB1 matched, DPB1-MM-permissive and DPB1-MM-non-permissive donors were 8, 50 and 32, respectively (9%, 56% and 36%). Overall 20 (77%) patients have DPB1-matched/permissive DPB1-MM UDs and thus would have benefitted from this selection. Our data confirm the feasibility of inclusion of HLA-DPB1 in UD selection algorithms without increasing the number of number of confirmatory typings for each patient.
The choice of suitable polymorphisms for cell chimerism monitoring of patients after allogeneic hematopoietic stem cell transplantation
K. Pegova (Prague, CZ)
Allogeneic hematopoietic stem cell transplantation (alloHSCT) is one of the most promising choice of therapy not only for leukemic patients but also for many non-malignant diseases. AlloHSCT leads to phenomenon called cell chimerism, co-existence of genetically different cells in one organism. The goal of cell chimerism monitoring is the early detection of relapse and disease recurrence. Sensitivity of the method and selection of suitable polymorphisms are crucial for correct determination of chimerism status. It is also important to take potential genetic changes into consideration (such as genome instability, loss of heterozygosity, cytogenetics of original disease), which are characteristic for different types of cancer. Among the most common cytogenetic abnormalities are changes at chromosome 5, 7 and 8 (associated with myelodysplastic syndrome or acute myeloid leukemia). In the case of mixed chimerism (MC, detection of recipient and donor genotype together) it is important to quantify portion of autologous haematopoiesis. If there are genetic changes in the recipient cells (for example deletion) it could half the ratio of donor-recipient blood cells and the patient could seem to be in better therapeutic condition, than they really are. The loss of heterozygosity can lead to complete chimerism detection (CC, detection of only donor genotype). Therefore MC can be overlooked. For monitoring of cell chimerism it is essential to precisely detect ratios of autologous hematopoiesis. To prevent incorrect detection, at least two informative markers are used and the association of the most common cytogenetic changes with each diagnosis are taken into account.
Retrospective analysis of stored umbilical cord blood units – ethnicity, quality parameters and HLA
V. Robertson (Newhouse, GB)
Precious Cells International Ltd (Precious Cells) is the first public/private hybrid bank established in the UK. Since May 2014, Precious Cells has been collecting altruistic umbilical cord blood (UCB) donations at six UK hospitals focusing its collections in ethnically diverse areas. To assess this approach, a retrospective analysis of ethnicity data for 457 UCB stem cell units was carried out. For 67% (n=307) of units, both parents were of the same ethnic group; 32% were classified as white northern European, 27% as South Asian, 17% as Sub-Saharan African and 12% eastern European based on Precious Cells’ ethnic group classification. 33% of units were from parents of different ethnicities. Focusing on a subset of 96 units collected during 2015-2016, the mean weight of UCB units at collection was 141 g and the mean CD45+ cell count was 2.3 x10^9. Post-processing, the UCB unit mean CD45+ cell count was 1.13 x10^9, mean CD34+ cell count was 5.64 x10^6 and mean viability was 97.2%. Of these 96 units, 79 met the 6th edition NetCord-FACT requirements for these parameters. These units were ABO typed and HLA typed for HLA-A, -B, -C (exons 2 and 3) and HLA-DRB1/3/4/5, -DQB1 and -DPB1 (exon 2) by NGS. None of the units were homozygous. Using 2013 NMDP US donor registry haplotype reference data, the number of Precious Cells UCBs matching the top five haplotypes at HLA-A, -B, -C, -DRB1 and -DQB1 was determined for African American (AFA), Hispanic (HIS), Caucasoid (CAU), Asian and Pacific Islander (API) groups. For two of the top three AFA haplotypes and for all three HIS and CAU haplotypes there was at least one UCB haplotype match. Two of the HIS and CAU haplotypes were common (HLA-A*01:01, -B*08:01, -C*07:01, -DRB1*03:01, -DQB1*02:01 and HLA-A*03:01, -B*07:02, -C*07:02, -DRB1*15:01, -DQB1*06:02). There were only HLA class I matches for two of three API haplotypes (HLA-A*33:03, -B*44*03, -C*07:01 and HLA-A*33:03, -B*53:01, -C*04:01) but no complete haplotype matches despite 24% of this cohort being defined as Asian. This may be due to the differences in the population making up the API ethnic group in the UK and the USA; further analysis of this cohort is required. However, Precious Cells’ UCB stem cells units are of high quality and carry common AFA, HIS and CAU haplotypes at five loci, potentially meeting the needs of these ethnic groups.
HLA-DPB1 disparities in 10/10 HLA-matched unrelated hematopoietic stem cell transplantation
E. Khamaganova (Moscow, RU)
HLA-A, -B, -C, -DRB1 and -DQB1 matching improves the outcome of hematopoietic stem cell transplantation (HSCT), but the impact of HLA-DPB1 matching is controversial. The aim of our study was to evaluate the impact of T-cell epitope (TCE) HLA-DPB1 mismatches on outcome of patients who underwent 10/10 HLA matched unrelated HSCT. 51 patients treated with allogeneic HSCT in our transplant center from 10/10 HLA-matched unrelated donors were included in the study. High-resolution typing for HLA-A, -B, -C, -DRB1 and -DQB1 was performed by PCR-sequence-based typing (SBT) using Protrans S4 kits. High-resolution typing for HLA-DPB1 was done by PCR with sequence specific primers (SSP) using Olerup typing kits. HLA-DPB1 permissive/non-permissive mismatches were examined according to TCE grouping (http://www.ebi.ac.uk/ipd/imgt/hla/dpb.html). The diagnoses were: acute myelogenous leukemia (AML, n=29), acute lymphoblastic leukemia (ALL, n=17), chronic myelogenous leukemia (CML, n=2), non-Hodgkin lymphoma (n=2), chronic lymphocytic leukemia (n=1). Early-stage disease included AML and ALL in first complete remission and CML in first chronic phase (n=26). Advanced-stage disease included all other patients (n=25). Overall survival (OS), event-free survival (EFS) and grade II–IV acute graft-versus-host disease (aGvHD) were the analyzed clinical end points. OS was defined as survival without lethal event from any cause, EFS was defined as survival in complete remission without lethal event from any cause. Grades of aGVHD were defined by Glucksberg scale. Proportional hazard Cox models were used for comparisons of time-to-event curves for OS, EFS and aGVHD. 10 donor-recipient pairs were HLA-DPB1 matched, 23 pairs had permissive HLA-DPB1 mismatches and 18 pairs had non-permissive mismatches. The factors significantly increasing risk of aGVHD were peripheral blood grafts, advanced-stage disease and male sex of recipients. No significant impact of HLA-DPB1 disparities on OS, EFS, aGVHD was observed.
Presence of KIR2DS1 receptor in donors of allogeneic hematopoietic transplantation due to myeloid malignancies improves overall survival in recipients with HLA-C2 antigens
D. Inotai (Budapest, HU)
By the recognition of HLA-C2 antigen of recipient cells, the activating killer immunoglobulin like receptor, KIR2DS1 on donor natural killer (NK) cell may lead to increased graft versus leukemia effect in patients treated by allogeneic hematopoietic transplantation (HSCT) influencing disease free (DFS) and overall survival (OS). The goal of the present study was to examine the effect of donor KIR status in conjunction with recipient HLA-C type on the outcome of HSCT. 249 consecutive adult patients, after first allogeneic HSCT (HLA-identical sibling n=117 or unrelated donor n=132) for a malignant myeloid condition, at a single center between 2007 and 2013, were retrospectively analyzed. Median follow-up was 36 months (range 6-92 months). Genotyping for the presence of KIR genes was performed by an allele specific multiplex PCR using archived DNA samples. Low resolution HLA-C typing was performed by sequence specific oligonucleotides as part of the routine work-up prior to HSCT. There was no difference in DFS or OS between patient subgroups stratified by the presence or absence of KIR2DS1 or HLA-C1/C2. In the patient subgroup with acute myeloid leukemia (AML) and with KIR2DS1 positive donors (n=58), we found improved OS for patients carrying at least one copy of the HLA-C2 antigen (n=36) compared to those homozygous for the HLA-C1 antigen (n=22). OS at three years for HLA-C2 carriers was 71.7% compared to HLA-C1 homozygotes with 45.7% (p=0.047). This association remained significant even with multivariate analyses by Cox regression considering age, sex, the type of conditioning and type of donor as covariables, (hazard ratio=0.422, 95% confidence interval: 0.179-0.995, p=0.049). Our results indicate that the combination of donor KR2DS1 and recipient HLA-C2 may be a favorable genetic constellation in allogeneic HSCT for AML with respect to overall survival.
Optimizing donor recruitment strategies: a comparison between the HLA genetic composition of Italian patients and the Italian volunteer bone marrow donors enrolled into the Italian Bone Marrow Donor Registry Donor Center RM01
M. Testi (Roma, IT)
HLA incompatibilities represent a major barrier for allogeneic hematopoietic stem cell transplant. Therefore, the knowledge of immunogenetic profiles of enrolled marrow unrelated donors (MUDs) is very relevant for a successful search. A recent publication provides evidence that a 9–10/10 HLA MUD is available for about 75% of Italian patients, a slightly lower percentage respect to other populations. An explanation for such a high degree of genetic heterogeneity might be found in the history of Italy, where several different ancient populations settled down. The aim of the study was to analyze HLA-A, -B, -C and -DRB1 allele and phenotype frequencies in 1,926 Italian volunteer bone marrow donors enrolled into the Italian Bone Marrow Donor Registry Donor Center RM01, to obtain data about common, rare and very rare HLA alleles and phenotypes. Moreover, in order to plan a data-driven strategy for a future registry expansion, we compared the HLA profiles of the local enrolled MUDs with those of 315 Italian patients, coming from different Italian regions, that already carried out a MUD search. Among the 1,926 Italian MUDs we observed a total of 50 HLA-A, 96 -B, 45 -C and 53 -DRB1 alleles. The alleles showing a frequency >1% were 17/50 HLA-A, 28/96 -B, 18/45 -C, and 22/53 –DRB1, counting for more than 90% of the total cumulative frequencies for each locus, with A*02:01, B*18:01, C*04:01 and DRB1*07:01 reported as the most frequent alleles. Moreover we observed 1,699 HLA-A,-B and 1,895 HLA-A,-B,-DRB1 distinguishable phenotypes out of the total 1,926 MUDs, with a percentage of unique phenotypes of 88.2 and 98.4 % respectively, showing a good phenotypic variability of the registry. A comparison of allele frequencies between the group of 315 patients and 1,926 donors enrolled in the DC RM01 did not reveal significant differences, confirming that the enrolled donor population is near to represent a true reflection of the entire Italian population.
HLA DPB1 mismatching in unrelated hematopoietic cell transplantation (MUD-HSCT) in pediatric hematological diseases
K. Spanou (Athens, GR)
The allelic compatibility at HLA-A, -B, -C, -DRB1 and DQB1 loci (10/10) between donor and recipient plays an important role in the outcome of hematopoietic stem cell transplantation (HSCT). Unrelated donors with HLA compatibility 10/10 often have HLA DPB1 mismatches, due to the low LD between DPB1 and other HLA class II loci. Multi-center studies showed significant associations of DPB1 mismatches with the outcome of HSCTs. Classification of DPB1 mismatching, based on T-cell epitope groups, identify mismatches that might be tolerated (permissive) and those that could increase risks (non-permissive). The purpose of this study was to correlate DPB1 mismatches between donor (D) and recipient (R) with the non-engraftment, graft rejection and acute graft versus host disease (GvHD) in allogeneic HSCTs from unrelated donors. We studied two groups of children from the Stem Cell Transplant Unit in "Aghia Sophia" Children's Hospital, 10/10 matched but with at least one DPB1 mismatch. Group A consisted of sixteen patients with hemoglobinopathies (10/16 boys, 13 thalassemia major, 3 sickle cell disease), where the non engraftment/rejection was significantly associated with non-permissive DPB1 mismatches in the HvG direction. All but two patients are alive while 4 continue to receive transfusions. Group B included twenty five patients with hematological disorders (17/25 boys, 11 ALL, 10 AML and 4 MDS). Non permissive DPB1 mismatches were observed in twelve (12/25) D-R pairs of which six (6/12) had GvHD III-IV. Four patients (4/25) are not alive (3/4 with DPB1 non permissive mismatches), while twenty one patients (21/25) are alive. In this group our results do not show significant associations of DPB1 mismatches with the development of severe GvHD. In hemoglobinopathies the avoidance of DPB1 mismatches in HvG direction is favorable for the outcome of MUD-HSCT, confirming DPB1 T-Cell Epitope Algorithm as a useful tool for donor selection.
NGS for HLA typing of new, rare or problematic alleles: new opportunity for the immunogenetics laboratory
M. Andreani (Roma, IT)
In the present study we describe the introduction of NGS technology for high resolution HLA typing in our laboratory. In order to contribute to the validation of the system we typed all the new alleles that we previously discovered in our laboratory using SBT and/or cloning methods. Moreover we confirmed the HLA typing of rare alleles found in individuals belonging to populations not very well known from an immunogenetic point of view, besides null, questionable expression and homozygous alleles. Finally we also typed alleles characterized by intronic polymorphisms. Fifteen DNA samples were analyzed in a workflow using NXType, Ion Torrent S5 platform and TypeStream software (Thermo Fisher - One Lambda, Canoga Park, CA, USA) for the loci HLA-A, -B, -C, -DRB1, -DRB3,4,5, -DQB1 and -DPB1, for a total of 193 alleles. Data showed a near-perfect concordance with results previously obtained at 2 fields for all the 193 alleles studied. Allele HLA-C*02:106, recently added to the nomenclature was identified as C*02:02:02:NEW. It is worthy to note that the characterization of HLA-C*02:106 was performed using a DNA cloning method. Similarly, DRB3*01:16, different from DRB3*01:01:02:01 for a single amino acid substitution in exon 3, was identified as DRB3*01:01:02:NEW, despite the simultaneous presence of DRB3*02:02:01:02. For heterozygous DNA samples, most were resolved, except in 2% of the cases, belonging however to “P” group. In particular, the presence of DRB1*09:01:02/*09:21 was probably due to the challange of investigating exon 1 for DRB1. All other ambiguities found at the DPB1 locus, as follows: DPB1*02:01:02,*04:02:01:01/DPB1*105:01,*416:01; DPB1*04:01:01:01,*04:02:01:02/ DPB1*105:01,*126:01 and DPB1*13:01:01/*107:01. Moreover, our investigation showed that 24 alleles out of the 193 analyzed were characterized by polymorphisms located in intronic regions, lack full-length genomic reference sequences and have not been reported in the IPD-IMGT/HLA Database.
Five-locus high resolution HLA haplotype frequencies in the Bone Marrow Donors Registry of Pavlov First Saint-Petersburg State Medical University, Russian Federation
E. Kuzmich (St.-Petersburg, RU)
In December 2016 the Database of the Bone Marrow Donors Registry of Pavlov First Saint-Petersburg State Medical University included information about HLA phenotypes of 13000 volunteer donors. High resolution HLA typing by monoallelic Sanger-sequencing was carried out for 1000 donors. Data analysis was performed by Arlequin program version 3.5. The expectation–maximization (EM) algorithm was used for the determination of five-locus haplotype frequencies. In the studied group of donors the most common alleles were: HLA-A*02:01 – 23.9%, HLA-A*03:01 – 13.9%, HLA-A*01:01 – 10.9%, A*24:02 – 10.2%, HLA-A*11:01 – 4.6%, HLA-A*25:01 – 4.2%. HLA-B*07:02 – 11.6%, HLA-B*08:01 – 6.7%, HLA-B*18:01 – 5.1%, HLA-B*13:02 – 4.9%, HLA-B*35:01 – 4.9%, HLA-B*44:02 – 4.7%. HLA-C*07:02 – 13.1%, HLA-C*07:01 – 10.7%, HLA-C*04:01 – 9.3%, HLA-C*06:02 – 9.0%, HLA-C*12:03 – 8.3%, HLA-C*02:02 – 5.5%. HLA-DRB1*07:01 – 13.2%, DRB1*15:01 – 11.5%, HLA-DRB1*01:01 – 10.2%, HLA-DRB1*03:01 – 8.2%, HLA- DRB1*13:01 – 5.2%, DRB1*16:01 – 4.4%. HLA-DQB1*03:01 – 14.6%, HLA-DQB1*02:01 – 13.9%, HLA-DQB1*05:01 – 11.5%, HLA- DQB1*06:02 – 10.7%, HLA- DQB1*03:02 – 6.2%, HLA- DQB1*06:03 – 5.8%. The most frequent five-locus high resolution HLA haplotypes were: A*01:01-B*08:01-C*07:01-DRB1*03:01-DQB1*02:01 (3.4%); A*03:01-B*07:02-C*07:02-DRB1*15:01-DQB1*06:02 (3.1%); A*03:01-B*35:01-C*04:01-DRB1*01:01-DQB1*05:01 (1.8%); A*02:01-B*07:02-C*07:02-DRB1*15:01-DQB1*06:02 (1.3%); A*25:01-B*18:01-C*12:03-DRB1*15:01-DQB1*06:02 (1.03%); A*02:01-B*13:02-C*06:02-DRB1*07:01-DQB1*02:01 (1.01%). This study reports the most frequent HLA alleles and five-locus high resolution haplotypes in the donors of Bone Marrow Donors Registry of Pavlov First Saint-Petersburg State Medical University.
Improving genotyping capabilities in a close family cohort by expansion of a qPCR marker set for chimerism monitoring
E. Rozemuller (Utrecht, NL)
Quantitative PCR not only allows quick and easy monitoring of chimeric mixtures after transplantation, but it can also accurately detect the presence of genetic material as low as 0.05%. The detection of adverse transplant events is thereby highly improved in comparison to STR analysis.
Further development of chimerism monitoring by qPCR can be necessary in terms of the genotyping capabilities, especially for family members of the first and second degree due to their similarity in genetic make-up. This can be realized by increasing the number of available markers. We have now expanded our KMRtype®/KMRtrack® marker set to a total of 39 markers. This set is a combination of Celera (AlleleSEQR) and GenDx developed assays. The expanded marker set was tested by screening DNA samples of a close family cohort consisting of 4 grandparents, 2 parents and 12 children (Coriell Institute for Medical Research). The genotyping capabilities of the marker set expansion was assessed by determining the family member combinations that were distinguishable and resulted in informative markers. The KMRtype protocol was applied for genotyping, using a multiplexed qPCR system encompassing 3 markers in a single reaction (GenDx). All experiments were performed using the ViiA7 qPCR system (Thermo Fisher). When testing the close family cohort with the expanded chimerism marker set, each possible combination resulted in at least 1 informative marker. The majority of combinations (96%) resulted in more than 2 informative markers. As compared to the previous set, the expanded set reduced the number of combinations having 1 informative marker by 59% (from 29 to 12). It can be concluded that the genotyping capabilities of KMRtype/KMRtrack significantly improved with the addition of chimerism monitoring markers.
Extended MHC haplotype disparity level is relevant for the patients survival and graft versus host disease in T cell-replate HSCT from unrelated donor
J. Nowak (Warsaw, PL)
Life-threatening risks in unrelated hematopoietic stem cell transplantation (HSCT) including graft versus host disease (GvHD) and mortality are associated with HLA disparity between donor and recipient. The increased risks might be dependent on disparity in not routinely tested multiple polymorphisms in the genetically dense MHC region, organized in two extended MHC haplotypes (Ehp). For modeling we considered that in HLA mismatched donor-recipient pairs increased frequencies of SNP disparities in MHC regions adjacent to mismatched HLA loci were discovered and in extremely strong linkage disequilibrium. Patients (N=889) received T cell-replete HSCT from 2001 to 2012. We assessed the clinical role of Ehp disparity levels in donor-recipient pairs by the in silico detection of HLA allele phase mismatch using PHASE 2.1 algorithm. We compared cumulative incidences of acute (a)GvHD, chronic (c)GvHD, overall survival (OS) and non-relapse mortality (NRM) in patients given HSCT from unrelated donor with 1 or 2 Ehp mismatches. Co-variate adjustments were made in multivariate step-wise analysis with backward elimination. We found a highly significant increment of 100-day aGvHD (58% vs. 41%) and 5-year cGvHD (62% vs. 37%) with increasing Ehp mismatch level, even with the same level of 1 (out of 10) HLA mismatch. In adjusted multivariate models Ehp disparity level remained independent prognostic factor for aGvHD (p=0.0000018, HR=2.70, CI95% 1.75-4.14), cGvHD (p=0.00000085, HR=3.28, CI95% 2.04-5.25) and HLA mismatch level alone has been excluded from these models. Patients with double Ehp disparity had worse 5-year OS (45% vs. 56%, pa=0.014, HR=2.14, CI95% 1.13-4.05) and NRM (40% vs. 31%, p=0.017, HR=3.91, CI95% 1.70-8.97), as compared to patients with single Ehp disparity. We conclude that HLA-linked factors contribute to the high morbidity and mortality in recipients given HLA-mismatched unrelated HSCT.