High resolution allele frequencies of the inventory of the Hellenic Cord Blood bank (HCBB)
A. Dinou (Athens, GR)
Cord blood (CB), is an alternative source of hematopoietic stem cells for allogeneic stem cell transplantation. Although one of its advantages is the lesser degree of compatibility required between graft and recipient, there is increased demand for CB Units typed at high resolution. The Hellenic Cord Blood Bank is routinely using NGS-based typing for the CBUs of its inventory at 7 loci (HLA-A, -B, -C,-DRB1, -DQA1, -DQB1, -DPB1) permitting to obtain high resolution results, with minimal ambiguity, in one pass. The aim of this study was to estimate the distribution of HLA alleles and haplotype frequencies of the CB inventory. For this reason the allelic frequencies of 266 CBUs were obtained for 7 loci at the 2nd field. The most frequent alleles for HLA class I genes were: HLA-A*02:01 (23.9%), HLA-A*24:02 (17.1%); HLA-A*01:01 (10.1%); HLA-B*51:01 (13.8%); HLA-B*18:01 (10.7%); HLA-B*35:01 (9.4%); HLA-B*35:03 (4.5%); HLA-C*04:01 (19.5%), HLA-C*07:01 (13.1%). Regarding class II genes the most frequent alleles were HLA-DRB1*11:04 (19.3%), HLA-DRB1*16:01 (12.3%) and HLA-DRB1*11:01 (8.1%); for DQB1 HLA-DQB1*03:01 (33.6%), HLA-DQB1*05:02 (15.8%) and HLA-DQB1*05:01 (10.7%); for DQA1 HLA-DQA1*05:05 (33.9%), HLA-DQA1*01:02 (18.1%) and HLA-DQA1*01:01 (8.5%); and for DPB1 HLA-DPB1*04:01 (37.4%), HLA-DPB1*04:02 (21.1%) and HLA-DPB1*02:01 (18.4%). The allele frequencies observed are in line with those previously reported for the Greek population. The most common haplotypes estimated by the Arlequin software were HLA-A*02:01~B*18:01~C*07:01~DRB1*11:04~DQB1*03:01~DQA1*05:05~DPB1*04:02 (2.05%) and HLA-A*02:01~B*35:02~C*04:01~DRB1*11:04~DQB1*03:01~HLA-DQA1*05:05~DPB1*04:01 (2.05%). In the future, an in depth and continuous analytical process of the CBUs haplotypes will ensure the dynamic evolution of the HCBB. Activities such as CB collection, planning or inventory renewal will be based on those results in order to achieve an optimal ethnic representation.
Effect of donor KIR genotype on outcomes after haploidentical stem cell transplantation
A. Torío Ruiz (Albacete, ES)
In haploidentical stem cell transplantation (SCT), the selection of the "ideal donor" is not performed in a standardised way according to the KIR genotype expressed by potential donors. We retrospectively analyzed 30 patients and their donors submitted to haploidentical SCT at our centre. All patients were prepared with a conditioning regimen including thiotepa-busulfan-fludarabine with high doses of post-transplant cyclophosphamide (CyPT) and tacrolimus as prophylaxis of graft-versus-host disease (GvHD). HLA and KIR genotyping was performed by PCR-SSO using One Lambda commercial kits. The genotypic variables under study were: donor AA / Bx haplotype, donor B content (classified as neutral, better, best, according to Cooley et al., 2010), KIR inhibitor mismatches, presence of KIR2DS1 and KIR3DS1 in donor, mismatching in KIR ligands in the GVH direction, mismatching in KIR ligands in the HvG direction. We analysed the impact of these KIR genotype variables on overall survival (OS), GvHD free survival, and event-free survival (EFS). Even with our small sample size, statistical significance was found when we analysed the effect of the presence of mismatches on KIR ligands in GvH direction in relation to the diagnosis of chronic GvHD (54% vs. 100%, p = 0.004). Significance was also found when we compared the effect of the presence of AA or Bx haplotype of the donor with the diagnosis of chronic GVHD (50% vs. 86%, p = 0.033). We did not find statistical significance, in spite of the previous data in the literature that relate the existence of mismatches in KIR inhibitors or the presence of certain KIR genotypes (2DS1+, 3DS1+) with post-transplantation evolution regarding OS or EFS. Our results show that KIR genotyping can provide useful information to choose the donor for the haploidentical SCT without T-depletion with high doses of CyPT. Donors with a B haplotype that do not show incompatibilities of KIR ligands in the GvH direction could provide a lower risk of GvHD.
Impact of single nucleotide polymorphisms in the MHC Gamma block on the outcome of unrelated hematopoietic stem cell transplantation
M. Maskalan (Zagreb, HR)
The gold standard in unrelated hematopoietic stem cell transplantation (HSCT) is matching for HLA-A, -B, -C, -DRB1 and -DQB1 alleles. However, this is often not enough to prevent post-transplantation complications. MHC haplotype mismatches (MM) have been associated with higher risk of acute graft versus host disease (aGvHD). The gamma block (GB) is located in the central MHC region, between HLA-B/C and HLA-DRB/DQB blocks. It contains many inflammatory and immune regulatory genes. The single nucleotide polymorphisms (SNPs) within that block can be considered as markers for MHC haplotype matching. The aim of this research was to test whether the MMs in the GB impact the outcome of HSCT. The study group included 30 patients diagnosed with acute myeloid or lymphoblastic leukemia (N=18 and N=12, respectively), transplanted with HLA 10/10 matched unrelated donor (MUD). The pairs were PCR-SSP typed using Gamma Type (GT) assay (Conexio Genomics, Australia) that detects 25 SNPs within the GB. Sixteen out of 30 (53.33%) pairs were GT matched (GT-M) while 14 out of 30 pairs (46.67%) were mismatched (GT-MM). The analysis showed no statistically significant difference in overall survival and relapse occurrence in our tested groups. There were also no significant differences in aGvHD incidence between GT-M and GT-MM patients. Although these preliminary results do not support the hypothesis that GT-MM is associated with higher risk of aGvHD and worse HSCT outcome, due to the small sample size, further investigation on a larger cohort is needed.
Polymorphism and expression of G-CSF receptor and effectiveness of G-CSF induced mobilization in the recipients of autologous peripheral blood progenitor cells
K. Bogunia-Kubik (Wroclaw, PL)
The present study aimed to assess weather G-CSF receptor (CSF3R) expression contributes to G-CSF-mediated mobilization of CD34+ cells for transplantation purposes and could be affected by CSF3R polymorphisms as the rs3917924 wild type CC genotype was previously identified as a factor increasing mobilization efficiency. CSF3R expression on neutrophils and monocytes was investigated by flow cytometry in peripheral blood samples collected before and on the fifth day of G-CSF administration from 33 (F/M=18/15) patients undergoing transplantation of autologous peripheral blood progenitor cells. G-CSF and SDF-1 (CXCL12) serum levels were assessed using a Luminex instrument. Additionally, genotyping for the CSF3R rs3917924, rs3918001, rs3918020, rs3918021 and rs146617729 polymorphisms was performed. Prior to introducing the mobilizing agent, the rs3917924 wild type CC homozygotes were characterized by higher frequency of CSF3R expressing monocytes compared with T allele bearers (mean of 28.266% vs. 13.371% of monocytes; t value of the t-Test for 2 independent means= 2.328; p=0.027). The above relationship vanished by the fifth day of mobilization (t=0.453; p=0.654). Interestingly, at that time SDF-1 level (strongly correlated with the G-CSF level in the serum: Spearman's Rho Correlation Coefficient R=0.573; p<0.0001) as well as changes in CSF3R expression on neutrophils were associated with percentage of CSF3R-positive monocytes (R=0.461; P=0.006 and R=0.488; p=0.004, respectively). In turn, expression of the G-CSF receptor on monocytes corresponded with mobilization yield, expressed by the number of CD34+ cells separated per kg of body weight (R=0.393; p=0.024). To conclude, efficacy of G-CSF induced mobilization is associated with CSF3R expression on monocytes and CSF3R rs3917924 polymorphism.
An evaluation of the GAMMA-TYPE™ Kit
K. Poulton (Manchester, GB)
GAMMA-TYPE™ (Connexio) is a PCR-SSP based kit which allows SNP profiling across the gamma block of the class III region within the MHC. This brief evaluation aimed to ascertain any added value in typing this region in a variety of clinical cases. 15 HSCT donor and recipient pairs were included in the study. Six recipients had second grafts from a different donor after a failed first transplant. Two further recipients had grafts from well-matched unrelated adult donors. Haplotypes were determined either by typing additional family members, or inferred by reference to frequency data. The level of HLA matching for unrelated donors was 9/10 (n=3); 10/10 (n=11); or 9/12 (n=2); 10/12 (n=2); 11/12 (n=7); 12/12 (n=3) including HLA-DPB1. Five unrelated donor pairs were also gamma block matched, and nine pairs were mismatched. The level of HLA matching did not predict identity within the gamma block: Mismatched pairs had HLA matching levels which ranged between 9/12-12/12, while gamma block matched pairs ranged between 9/12-11/12. All unrelated donor pairs with a 12/12 HLA match were mismatched at the gamma block. The population frequency of the HLA haplotype could not be used to predict the likelihood of additional matching within the gamma block in this limited study. Similarly, matching at the gamma block did not predict engraftment, or failure of the first transplant. One recipient had a 9/10 HLA-A mismatched sibling donor, in a family where haplotypes could not be identified by typing additional family members. Gamma block testing was used to assess whether this was most likely to be due to a crossover, or the presence of an additional haplotype in the family. The siblings were matched within the gamma block, indicating a probable cross-over event. This was extremely useful information, and in this case, the kit provided a rapid resolution. In summary, this is an interesting test, but its added value in matching unrelated HSCT donors requires further investigation.
Implication of killer-cell immunoglobulin-like receptors in donor selection for a matched/haploidentical stem cell transplantation
S. D'Silva (Mumbai, IN)
Killer-cell immunoglobulin-like receptors (KIR) have an impact on transplant outcomes. KIR present on Natural Killer cells interact with HLA class I alleles and modulate the response of Natural Killer cells (increased/decreased cytotoxicity). The objective of the present study was to assess the impact of KIR HLA ligands, KIR gene haplotypes and donor characteristics like gender, age etc. on HSCT outcomes in haploidentical related transplants. Thirty three patient-donor pairs undergoing T-cell replete HLA haplo-matched transplant at Tata Memorial Centre, Mumbai were included in this study. HLA typing and KIR genotyping was carried out using PCR-SSP. The median age of patients undergoing haplotransplants was 25 years. In our cohort transplants with donor >30 years of age showed significantly lower chances of relapse (p=0.040) and higher overall survival (p=0.08). Also, transplants using male donors showed lower relapse (p=0.873) and higher overall survival (p=0.711). Transplants involving patient and donor of the male gender resulted in lower relapse (p=0.173). Among patient-donor pairs having KIR ligand mismatches better overall survival and lower relapse rate were observed (p=0.502 and p=0.608 respectively). Patients carrying the C1C1 ligand for HLA-C epitope but did not carry the TI ligand for HLA BW4 epitope had significantly better overall survival (p<0.0005). Transplants from KIR B/X haplotype donors resulted in lower relapse and better overall survival (p=0.435, p=0.852). Also, patients receiving transplants from donors with telomeric BX haplotype showed better overall survival as compared to transplants from donors with centromeric BX haplotype. This benefit was more pronounced when the patient carried the C1C1 KIR ligand (p=0.140). We conclude that although HLA match is of primary importance, knowing the patient KIR ligands and donor KIR haplotype could help in selecting the best donor and minimize transplant related complications. The HLA-C and Bw4/Bw6 epitopes are important determinants of transplant outcomes whereas KIR genotypes of the donor seem to have no impact in haploientical/mismatched HSCT.
Identification of rare HLA alleles in Polish population with the use of next generation sequencing
M. Kolasi (Poznań, PL)
The HLA (human leukocyte antigen) complex plays a significant role in matching for haematopoietic stem cell transplantation because allele-level HLA matching between donors and recipients reduces the likelihood of rejection and graft-versus-host disease (GvHD). The HLA genes are the most polymorphic in the human genome. Therefore, the changes in their sequence affect the specificity of antigen presentation and histocompatibility in transplantation. At present, sequence-based typing methods, in particular next-generation sequencing (NGS), allow the highest possible resolution. Most of the NGS methods are based on traditional PCR of the target regions followed by massive parallel sequencing of the amplicons. The advantage of NGS is a possibility to obtain the increased amount of sequencing reads per sample and locus. This allows for a highly confident allele-determination and verification of rare HLA alleles. The aim of our study was to determine 11 HLA loci using TruSight HLA Panel (Illumina) and rare HLA allelles in Polish population. DNA samples isolated from peripheral blood were obtained from bone morrow donors. Typing of 11 HLA loci (HLA-A, -B, -C, -DRB1/3/4/5, -DQB1, -DPB1, -DPA1, -DQA1) was performed according to the HLA assay manual using the Illumina MiSeq platform. The raw sequencing data were assembled and analyzed by the Conexio software. Our data obtained by HLA NGS typing showed the presence of HLA alleles such as: DRB1*14:87, DRB1*11:69, DRB1*11:28:01, B*51:08:01, DQB1*03:12, A*11:37 and C*05:37. In addition, our study confirms that application of the NGS method gives more precise sequencing results and allows the identifications of rare HLA alleles by the high-quality typing compared to conventional techniques.
CMV-IgG serostatus of 290,438 German and 31,776 Polish DKMS donors
J. Wuchter (Tuebingen, DE)
In the context of hematopoietic stem cell donations, CMV serostatus is becoming more and more important for donor selection. Consequently, DKMS started testing the CMV-IgG serostatus of all newly recruited donors in Germany and Poland by the beginning of 2014. For all donors recruited by collecting a blood sample the CMV-IgG was tested at “Institut für Immunologie und Genetik”, Kaiserslautern or “DKMS Life Science Lab”, Dresden. We report a total of n=290,438 CMV-IgG serostatus for donors recruited in Germany and n=31,776 for donors recruited in Poland. At the age of 18, 24% (n=2334) of the donors tested positive in Germany, while 75% (n=7355) tested negative. For 1% (n=83) of the individuals the results were questionable. In contrast to that for donors at the age of 18 and recruited in Poland, a negative CMV-IgG serostatus was less common. Here, we report seropositive results for 62% (n=1014) of the individuals and seronegative CMV-IgG results for 37% (n=606). Questionable results have been obtained for 1% (n=13) of the individuals. Analyzing the CMV-IgG serostatus of donors of different age groups recruited in Germany, we found that the share of seropositive individuals increases with age. For instance, in case of donors with ages between 18 and 25, a seropositive result is found for 28% (n=18,374) of the donors while for the age group from 51 to 55 the share rises to 47% (n=11,944). A comparable increase is observed for donors recruited in Poland. In the age group between 18 and 25 the seropositive share is at 63% (n=4,236) while the share increases to 88% (n=991) for the age group between 51 and 55. In summary, this report gives an impression on the proportion of CMV-IgG serostatus in different age groups but also different regions of Europe.
Interplay between donor KIR2DS1 and HLA-C in the outcome after HLA-identical sibling hematopoietic stem cell transplantation?
A. Pasi (Pavia, IT)
NK cells are critical for innate immunity and play a pivotal role in HSCT. NK function is controlled by an array of inhibitory and activating signals that are processed by receptors such as KIRs. Mainly inhibitory KIRs recognize the HLA class I ligand groups (HLA-C1, C2 and Bw4) and mediate tolerance to self but also alloreactivity during HSCT. The activating KIR2DS1 is well known to play a role in both NK cell activation and tolerance by means of HLA-C2 recognition. It has been described that activating donors’ KIR2DS1 affect outcome of HSCT in haploidentical settings and, recently, also in stem cell transplantation from matched unrelated donors. The functionally competent KIR2DS1-expressing NK cells from HLA-C1-positive donor could mediate the anti-leukemic effect in the recipient, mainly in patients with HLA-C2/C2. We evaluated the effect of activating KIR2DS1 in HSCT sibling pairs. We analyzed clinical data (in particular relapse and overall survival) of 53 patients (22 AML, ten ALL, 11 LMC, one NHL, four MDS, five myeloma) who received hematopoietic stem-cell transplantation from sibling donors matched for HLA-A,B,C,DR,DQ or with a single mismatch. We performed KIR and HLA genotyping with PCR-SSO/-SPP technique. Relapse was absent in 43 patients and overall mortality was absent in 38 patients. We found a reduced risk of relapse among patients whose donors were KIR2DS1-positive (62.8% vs 60% p=0.8). A similar scenario was evident considering overall survival: we noted a higher frequency of patients with KIR2DS1 positive donors lacking death event (68.4%vs 46.7% p=0.1). Allografts from KIR2DS1-positive donors with HLA-C1/C1 were associated with a decreased rate of mortality (28.5% vs 34.6%). Even if without statistical significance we found, among patients with HLA-C2/C2 whose donors were KIR2DS1 positive, a higher rate of missing relapse (22.2% vs 16.7% p=0.9) and a better overall survival (23% vs 14.8% p=0.7). This is not a large cohort and it is necessary to increase our sample size. Nevertheless, it is possible that donor KIR2DS1 provides protection against relapse and reduces mortality, in an HLA-C-dependent manner, also in HSCT between identical siblings.
Identification of twenty-one novel MICA alleles by sequence-based-typing
C. Neuchel (Ulm, DE)
The major histocompatibility complex class I-related chain A (MICA) proteins are encoded by a highly polymorphic gene on chromosome 6. To date, 106 MICA alleles are registered in the IPD-IMGT/HLA Database with most of the polymorphisms laying in exons 2-5. Whereas exons 2-4 mainly differ by SNPs, exon 5 sequences vary by a number of GCT repeats. 3262 patients and their unrelated donors from hematopoietic stem cell transplantations (HSCT), which were conducted between 2000 and 2011 in Germany, were MICA typed. Sequenced based typing was performed with an in-house kit using generic primers for exons 2-4 and group specific primers for exon 5. The group specific PCRs allowed for separation of specimens who are heterozygous for an indel at the 3’-end of intron 4 (7T/8T). For data analysis Sequence Pilot was used. Sequence overlays caused by the presence of homozygous 7T/7T or 8T/8T SNPs were analysed with an in-house tool using a string of 37 bases within exon 5 sequences. Out of 6524 typings, we found 21 (0.32%) novel MICA alleles. The exon 2-5 sequences of 16 novel alleles were accepted by GenBank and names were assigned by the WHO Nomenclature Committee for Factors of the HLA System. Five alleles are still in the process of submission. All detected polymorphisms were single nucleotide substitutions. Most of them occurred in exons 2-4 (5, 6, and 8 SNPs, respectively). Two SNPs were found in exon 5. Five SNPs resulted in silent mutations, 16 SNPS resulted in amino acid changes. Our results show, that, even in a population of mainly Caucasoid individuals, a significant fraction of novel MICA alleles occurs. MICA is clinically relevant in organ transplantation and in HSCT, is associated with autoimmune diseases and even with some tumours. For good reason, allelic variations of MICA genes are receiving increasing attention.
UGT2B17 copy number variation and graft versus host disease after allogeneic hematopoietic stem cell transplantation in Czech patients
F. Mrázek (Olomouc, CZ)
Despite complete HLA match at allelic level between the recipient and donor in allogeneic hematopoietic stem transplantation (aHSCT), substantial proportion of patients suffers from severe complications including graft versus host disease (GvHD). Development of GvHD in patients with HLA-identical donors may be attributed to minor histocompatibility antigens (mHA). UDP glucuronosyltransferase family 2 member B17 (UGT2B17) copy number variation (CNV) has previously been shown as mHA associated with acute GvHD after aHSCT, but this finding was not unequivocally replicated. The aim of the present study was, therefore, to provide further data on possible relationship between UGT2B17 CNV and aHSCT outcome. 235 patients who underwent aHSCT in four Czech centres and their stem cell donors were genotyped for UGT2B17 gene CNV by the quantitative-PCR TaqMan copy number assay. Distribution of UGT2B17 genotypes complied with Hardy-Weinberg equilibrium and was almost identical among the patients and donors. Concordant with the data from other Caucasian populations, the frequency of homozygous UGT2B17 deletion (d/d) genotype was 13% in the whole study group. 19 pairs (8.1%) mismatched for UGT2B17 in GvH direction (UGT2B17 gene carrying patients with d/d donors) were detected. Importantly, acute GvHD (grade II-IV) occurred in a similar frequency in UGT2B17 mismatched (33%) and matched (34%) patients. Nevertheless, UGT2B17 mismatched patients tended to develop life threatening grade IV acute GvHD more frequently (11% vs. 2% in matched patients, p=NS). In conclusion, the present study could not provide the data further supporting contribution of UGT2B17 CNV to the occurrence of acute GvHD after aHSCT. Whether the patients mismatched for UGT2B17 with their donors are in higher risk of the grade IV acute GvHD deserves further investigation in studies with higher statistical power.