Np Mayor

Diverging effects of HLA–DPB1 matching status on outcome following unrelated donor transplantation depending on disease stage and the degree of matching for other HLA alleles

Disease stage and recipient/donor human leukocyte antigen (HLA) matching are important determinants of outcome in transplantation using volunteer-unrelated donors (VUD). Matching for HLA-A, -B, -C, -DRB1, -DQB1 is beneficial, whereas the importance of DPB1 matching is more controversial. The impact of HLA matching status may differ dependent on disease stage. We investigated the outcome according to the degree of HLA matching at 6 loci, in 488 recipients of predominantly T-cell depleted bone marrow VUD transplants for leukaemia. Survival was significantly better in 12/12-matched transplants in those with early leukaemia (5 years: 63 versus 41% in 10/10 matched, P=0.006), but not late stage disease. Conversely, within the HLA-mismatched group (⩽9/10), there was a significant survival advantage to DPB1 mismatching (5 years: 39 versus 21% in DPB1 matched, P=0.008), particularly in late leukaemia (P=0.01), persisting in multivariate analysis (odds ratio 0.478; 95% confidence interval 0.30, 0.75; P=0.001). These novel findings suggest that the best outcome for patients with early leukaemia, with a 10/10-matched donor, is achieved by matching for DPB1. Conversely, our results suggest that in patients receiving an HLA-mismatched graft, the outcome is significantly better if they are also mismatched for DPB1. We recommend validation of these results in independent datasets.

The degree of matching at HLA-DPB1 predicts for acute graft-versus-host disease and disease relapse following haematopoietic stem cell transplantation

Summary:The importance of matching for HLA-DPB1 in unrelated donor haematopoietic stem cell (HSC) transplantation is little understood. Most transplant centres do not, currently, prospectively match for DPB1, but emerging data show that DPB1 matching does play a role in determining outcome. We studied the impact of HLA-DPB1 matching on outcome in 143 recipients of T-cell depletion transplants, who matched with their respective unrelated donors (allelic level) at HLA-A, -B, -C, -DRB1 and -DQB1. Of those matched at DPB1, 47.2% (17/36) developed acute graft-versus-host disease (aGvHD) as compared to 66.3% (55/83) of those who were mismatched. This led to a 19.1% (95% CI 0.1–38.3%) increase in the chance of developing aGvHD in mismatched patients (P=0.049). Relapse of the original disease occurred in 51 recipients; 23 of 37 (62%) matched at both DPB1 alleles, 28 of 82 (34%) were mismatched at one or two DPB1 alleles. Thus, there was a significantly higher relapse rate (P=0.0011) in transplant recipients who matched at both DPB1 alleles. In conclusion, a donor/recipient DPB1 match was associated with a significantly lower incidence of aGvHD and a significantly higher incidence of disease relapse. This study provides further evidence for an immunogenic role of HLA-DPB1 in HSC transplants.

HLA Typing for the Next Generation

Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.

Single molecule real-time DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines

The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B‐lymphoblastoid cell lines (B‐LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B‐LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences’ Single Molecule Real‐Time (SMRT) DNA sequencing to HLA type 126 B‐LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra‐high resolution. Amplicon sequencing of full‐length HLA class I genes (HLA‐A, ‐B and ‐C) and partial length HLA class II genes (HLA‐DRB1, ‐DQB1 and ‐DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD‐IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four‐field resolution typing of HLA‐B and HLA‐C. By improving and standardising the HLA typing of these B‐LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.

Recipient/donor HLA and CMV matching in recipients of T-cell-depleted unrelated donor haematopoietic cell transplants

Improving haematopoietic cell transplantation outcomes by selection of an HLA-matched unrelated donor is best practice; however, donor selection by secondary characteristics is controversial. We studied 1271 recipients with haematological malignancies who underwent T-cell-depleted allografts and had complete data on HLA-matching status for six loci (HLA-A, -B, -C, -DRB1, -DQB1, -DPB1) and clinical outcome data. Five-year overall survival was 40.6%. HLA mismatching (at HLA-A, -B, -C, -DRB1, -DQB1) relative risk (RR) 1.22, 95% confidence interval (CI) 1.2–1.5, P=0.033 for 1 mismatch and RR 1.46, 95% CI 1.1–1.9, P=0.009 for >1 mismatch) and CMV mismatching (RR 1.37, 95% CI 1.2–1.6, P<0.001) were significantly associated with inferior survival. Donors aged <30 years showed a trend towards better survival. The multivariate model for mortality, combining CMV and HLA-match status, found an RR of 1.36 (95% CI 1.1–1.7, P=0.003) for HLA matched/CMV mismatched, an RR of 1.22 (95% CI 0.99–1.5, P=0.062) for HLA mismatched/CMV matched and an RR of 1.81 (95% CI 1.4–2.3, P=<0.001) for HLA/ CMV mismatched, compared with the HLA/CMV-matched recipients. These data suggest that HLA and CMV matching status should be considered when selecting unrelated donors and that CMV matching may abrogate the effect of an HLA mismatch.

The novel HLA‐B*44 allele, HLA‐B*44:220, identified by Single Molecule Real‐Time DNA sequencing in a British Caucasoid male

The genomic sequence of the novel HLA-B*44:220 allele identified in a British Caucasoid male.

No impact of NOD2/CARD15 on outcome after SCT: a reply.

In a recent issue of Bone Marrow Transplantation, Dr Sairafi et al. reported the results of a retrospective study to investigate the impact of NOD2/CARD15 single nucleotide polymorphisms (SNPs) on the outcome of allogeneic haematopoietic stem cell transplants (HSCT). They found no significant correlation between the presence of a NOD2/ CARD15 SNP and the incidence of acute GVHD, TRM, relapse-free survival or overall survival. We have recently reported the results of a retrospective analysis of the impact of NOD2/CARD15 SNPs on the outcome of 196 unrelated donor HSCT (UD-HSCT) pairs where the recipient was diagnosed with acute leukaemia. We found that the presence of a NOD2/CARD15 SNP correlated with a significant reduction in overall survival because of an increase in disease relapse. Numerous other studies have shown the presence of SNPs to have a detrimental effect on the outcome of HSCT although the actual complication observed varies between groups and is thought to be dependent on specific characteristics of the cohort itself. The authors discussed the possibility that differences in patient characteristics and transplant regimens between their cohort and those previously reported may explain why they failed to see significant effects on outcome where others have. Thus, we looked for any differences between the cohort described by Sairafi et al. and the patients in our study that might explain the divergent results. The most obvious difference is that their study comprised a mixture of related and UD sources, whereas our study focused solely on UD transplants. We found that the effect of NOD2/CARD15 SNPs was seen only in recipients with acute leukaemia and more specifically the effect was more pronounced in the ALL subgroup (Figure 1). This marked effect in acute leukaemia has also been reported in a related donor cohort, although, as previously, the outcome affected was different in the two studies based on differences in the clinical protocols used. As 37% of the cohort studied by Sairafi et al. were recipients whose diagnosis was not acute leukaemia, the lack of effect seen in these recipients may mask any effect of the NOD2/ CARD15 SNPs in this study. The use and method of T-cell depletion was noticeably different when compared with our data, which we and others believe contributes significantly to the effect of any polymorphisms. In addition, the majority of transplants in the Sairafi study occurred when the recipient was considered to be in a lowrisk stage of their disease, which in itself may predict for a reduced risk of disease relapse. Finally, the frequency of NOD2/CARD15 SNPs seen in their study were markedly lower than in our cohort and in other HSCT studies. (The reported overall SNP frequency was 18% as compared with 27–29% in other studies.) There is evidence to suggest that there is geographic variation in the prevalence of NOD2/CARD15 SNPs, which may explain the low frequency seen. Thus, the lack of correlation to transplant outcome may be due to a lower prevalence of SNPs in this cohort. It would be of interest to see if Sairafi et al. were able to see an effect in more homogeneous subgroups of their cohort, particularly in the acute leukaemia recipients and the UD pairs. This study adds much to the discussion that

Caspase-8 polymorphisms result in reduced Alemtuzumab-induced T-cell apoptosis and worse survival after transplantation

Allo-SCT using unrelated donors is a curative treatment for patients with hematological disorders. The best donor is one matched for 10/10 HLA alleles, however studies have shown an additional survival benefit when considering other genetic factors. It has been shown that a six-nucleotide insertion/deletion polymorphism in the CASP8 gene promoter results in reduced susceptibility of T lymphocytes to undergo apoptosis. In 186 SCT recipients, we found a significantly better OS in those who received a transplant from a WT/WT donor compared with donors with a deletion (3 years: 52 vs 34%; P=0.03; multivariate analysis; RR 0.61; 95% CI 0.38–0.98, P=0.04). This was more marked when both the patient and the donor had a deletion (3 years OS: 62% compared with 36%, P=0.01). As the majority of these patients received Alemtuzumab during conditioning, we went on to analyze the in vitro effect of the polymorphism on Alemtuzumab-induced apoptosis. We showed statistically significantly higher percentages of apoptotic naïve CD4 (P<0.0005) and CD8 (P<0.0005) T cells in WT/WT donors in comparison with donors with a deletion. These data imply an unrecognized role for the CASP8 promoter polymorphism on survival following unrelated SCT particularly in the context of T-cell depletion with Alemtuzumab.

HLA-DPB1*64:01N and DPB1*701:01 sequence extensions by Single Molecule Real-Time DNA sequencing

Pacific Biosciences SMRT sequencing was used to confirm and extend HLA‐DPB1*64:01N and 701:01.

Two novel alleles, HLA-A*32:01:01:09 and 32:01:01:10, identified by Pacific Bioscience’s SMRT Sequencing

Pacific Biosciences SMRT DNA sequencing was used to identify two novel intronic variants of HLA‐A*32:01:01:01.