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Background The immune peptidome of OPSCC has not previously been studied. Cancer-antigen specific vaccination may improve clinical outcome and efficacy of immune checkpoint inhibitors such as PD1/PD-L1 antibodies. Methods Mapping of the OPSCC HLA ligandome was performed by mass spectrometry (MS) based analysis of naturally presented HLA ligands isolated from tumour tissue samples (n = 40) using immunoaffinity purification. The cohort included 22 HPV-positive (primarily HPV-16) and 18 HPV-negative samples. A benign reference dataset comprised of the HLA ligandomes of benign haematological and tissue datasets was used to identify tumour-associated antigens. Results MS analysis led to the identification of naturally HLA-presented peptides in OPSCC tumour tissue. In total, 22,769 peptides from 9485 source proteins were detected on HLA class I. For HLA class II, 15,203 peptides from 4634 source proteins were discovered. By comparative profiling against the benign HLA ligandomic datasets, 29 OPSCC-associated HLA class I ligands covering 11 different HLA allotypes and nine HLA class II ligands were selected to create a peptide warehouse. Conclusion Tumour-associated peptides are HLA-presented on the cell surfaces of OPSCCs. The established warehouse of OPSCC-associated peptides can be used for downstream immunogenicity testing and peptide-based immunotherapy in (semi)personalised strategies.

Graft-versus-host disease (GvHD) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is highly influenced by the degree of HLA matching between recipient and donor. The HLA-class Ib molecule HLA-G has been shown to promote tolerogenicity through its interaction with inhibitory receptors found on several immunocompetent cells. We hypothesized that in an allo-HSCT setting, HLA-G mismatches may negatively impact the HLA-G-mediated tolerogenicity either due to inefficient interaction with the inhibitory receptors of the transplanted immune cells or due to direct allorecognition of mismatched HLA-G on host cells by the immune cells of the donor.In order to explore this hypothesis, we investigated the impact of HLA-G mismatching in 2.083 10/10 matched high resolution HLA-typed allo-HSCT transplants.We found that the risk of chronic GvHD was significantly higher in HLA-G-mismatched transplant cases as compared with the HLA-G-matched control group (HR: 1.46, 95%CI = 1.11-1.91, p = 0.006). Sub-analysis of the mismatch vector revealed that this effect was only detectable in the GvH (HR: 1.89, 95%CI 1.39-2.57, p 0.001) but not the HvG direction (HR: 1.01, 95%CI = 0.63-1.63, p = 0.967). In addition, the negative impact of HLA-G mismatching on chronic GvHD was only significant in younger patients (30y HR: 3.02, 95%CI = 1.25-7.28, p = 0.014;29y HR: 1.28, 95%CI = 0.94-1.72, p = 0.113).Our results indicate that HLA-G mismatches may contribute to the onset of chronic GvHD, especially in younger patients and should therefore be avoided when possible.

A common genetic variant within the T cell receptor alpha (TCRA)-T cell receptor delta (TCRD) locus (rs2204985) has been recently found to associate with thymic function. Aim of this study was to investigate the potential impact of donor rs2204985 genotype on patient’s outcome after unrelated hematopoietic stem cell transplantation (uHSCT). 2016 adult patients were retrospectively analyzed. rs2204985 genotyping was performed by next generation sequencing, p < 0.05 was considered significant and donor rs2204985 GG/AG genotypes were set as reference vs. the AA genotype. Multivariate analysis of the combined cohort regarding the impact of donor’s rs2204985 genotype indicated different risk estimates in 10/10 and 9/10 HLA matched transplantations. A subanalysis on account of HLA incompatibility revealed that donor AA genotype in single HLA mismatched cases (n = 624) associated with significantly inferior overall- (HR: 1.48, p = 0.003) and disease-free survival (HR: 1.50, p = 0.001). This effect was driven by a combined higher risk of relapse incidence (HR: 1.40, p = 0.026) and non-relapse mortality (HR: 1.38, p = 0.042). This is the first study to explore the role of rs2204985 in a clinical uHSCT setting. Our data suggest that donor rs2204985 AA genotype in combination with single HLA mismatches may adversely impact post-HSCT outcome and should thus be avoided., publishedVersion

Thrombotic microangiopathy (TMA) is a rare but severe complication of tumors and their chemotherapeutic treatment. We report on two patients with chemotherapy-induced TMA who were successfully treated with a short course of the terminal complement inhibitor eculizumab. Both patients quickly achieved remission of microangiopathic hemolytic anemia and recovery of renal function. After withdrawal of eculizumab, remission was stable over an observation period of 47 months and 15 months, respectively. Our data show that eculizumab is effective in treating chemotherapy-induced TMA. Discontinuation of eculizumab is feasible once the complement-activating condition is controlled and the trigger is eliminated. Additional studies need to determine the optimal duration of complement-directed therapies and validate effective monitoring strategies after discontinuation of such therapy.

Although allogeneic hematopoietic stem-cell transplantation (HSCT) provides high cure rates for children with high-risk acute lymphoblastic leukaemia (ALL), relapses remain the main cause of treatment failure. Whereas donor killer cell immunoglobulin-like receptor (KIR) genotype was shown to impact on relapse incidence in adult myeloid leukaemia similar studies in paediatric ALL are largely missing. Effect of donor KIR genotype on transplant outcome was evaluated in 317 children receiving a first myeloablative HSCT from an HLA-matched unrelated donor or sibling within the prospective ALL-SCT-BFM-2003 trial. Analysis of donor KIR gene polymorphism revealed that centromeric presence and telomeric absence of KIR B haplotypes was associated with reduced relapse risk. A centromeric/telomeric KIR score (ct-KIR score) integrating these observations correlated with relapse risk (hazard ratio (HR) 0.58; P = 0.002) while it had no impact on graft-versus-host disease or non-relapse mortality. In multivariable analyses ct-KIR score was associated with reduced relapse risk (HR 0.58; P = 0.003) and a trend towards improved event-free survival (HR 0.76; P = 0.059). This effect proved independent of MRD level prior to HSCT. Our data suggest that in children with ALL undergoing HSCT after myeloablative conditioning, donor selection based on KIR genotyping holds promise to improve clinical outcome by decreasing relapse risk and prolonged event-free survival.

Background and objectives Mobilization of CD34+ cells by stimulation with G-CSF shows considerable variation across stem cell donors. Upfront prediction of CD34+ cell counts in peripheral blood based on easily available steady-state parameters would be helpful for the planning of apheresis and stem cell transplantation. Commonly accepted steady-state predictors for the mobilization are gender, body mass index and platelet count. The aim of the study was the identification of novel predictors that might influence mobilization efficacy and to create a model for the prediction of stem cell mobilization. Methods A total of 333 healthy stem cell donors who donated peripheral stem cells in our institution were retrospectively analysed. All available data before stem cell mobilization with G-CSF were included in the database. Primary end-point was CD34+ cell count before the first apheresis. Results In this cohort cholinesterase, differential blood cell counts including platelets, gender and body mass index were significantly correlated with CD34+ cell count. G-CSF dose per lean body weight showed a significant correlation with mobilization efficacy in women but not in men. A multivariate analysis identified gender, cholinesterase and platelet and red cell count as main predictors of mobilization. Red cell count showed a strong gender dependence, with higher predictive value in females. Conclusion The counts of eosinophils, platelets, red cells, cholinesterase and gender are the most important predictors of CD34+ cell mobilization in our deduced models. The red cell count as a predictor for mobilization showed a differential gender dependence.

Increasing donor-recipient HLA disparity is associated with negative outcomes of allogeneic hematopoietic stem cell transplantation (HSCT), but its comparative relevance amid non-HLA donor characteristics is not well established. We addressed this question in 3215 HSCTs performed between 2005 and 2013 in Germany for acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Donors were HLA-matched related (MRD; n = 872) or unrelated (10/10 MUD, n = 1553) or HLA-mismatched unrelated (10/10 MMUD, n = 790). Overall survival (OS) was similar after MRD compared with 10/10 MUD HSCT, reflecting opposing hazards of relapse (hazard ratio [HR], 1.32; P.002) and nonrelapse mortality (HR, .63; P.001). After UD HSCT, increasing HLA disparity was associated with inferior OS (HR, 1.21 [P.02] and HR, 1.57 [P.001] for 9/10 and ≤8/10 MMUD, respectively, compared with 10/10 MUD). Among non-HLA donor characteristics, age, sex mismatching (male recipient-female donor), and cytomegalovirus (CMV) mismatching (positive recipient-negative donor) impacted OS. Multivariate subgroup analysis showed that OS was similar after HSCT from the youngest 9/10 MMUD (30 years) compared with the oldest 10/10 MUD (40 years) (HR, 1.18; P = .25) and also in male patients transplanted from female 10/10 MUD compared with male 9/10 MMUD (HR, .89; P = .46). In contrast, OS of CMV-positive patients tended to be better with CMV-negative 10/10 MUDs compared with CMV-positive 9/10 MMUDs (HR, 1.31; P = .04). Because of low patient numbers in subgroups, definite conclusions and establishment of a hierarchy among HLA matching and non-HLA donor characteristics could not be made. Our data suggest that the impact of donor age and sex mismatch but not CMV mismatch on outcome of allogeneic HSCT may be comparable with that of single HLA disparity.

Unrelated haematopoietic stem cell transplantation (HSCT) has evolved from an experimental protocol to a potentially curative first-line treatment in a variety of haematologic malignancies. The continuous refinement of treatment protocols and supportive care paired with ongoing achievements in the technological field of histocompatibility testing enabled this transformation. Without a doubt, HLA matching is still the foremost criterion for donor selection in unrelated HSCT. However, HSCT-related treatment complications still occur frequently, often resulting in patients suffering severely or even dying as a consequence of such complications. Current literature indicates that other immune system modulating factors may play a role in the setting of HSCT. In this review, we discuss the current clinical evidence of a possible influence of nonclassical HLA antigens HLA-E, HLA-F, and HLA-G as well as the HLA-like molecules MICA and MICB, in HSCT.

Minor histocompatibility antigens (mHAs), recipient-derived peptide epitopes presented on the cell surface, are known to mediate graft-versus-host disease (GVHD); however, there are no current methods to associate mHA features with GVHD risk. This deficiency is due in part to the lack of technological means to accurately predict, let alone confirm, the tremendous number of potential mHAs in each individual transplant. Previous studies have shown that different HLA molecules present varying fractions of candidate peptide epitopes; however, the genetic “distance” between HLA-matched donors and recipients is relatively constrained. From these 2 observations, it is possible that the HLA type for a donor-recipient pair (DRP) would provide a surrogate measurement of the number of predicted mHAs, which could be related to GVHD risk. Because different HLA molecules present variable numbers of peptide antigens, a predicted cumulative peptide-binding efficiency can be calculated for individual DRP based on the pair's HLA type. The purpose of this study was to test whether cumulative peptide-binding efficiency is associated with the risk of acute GVHD (aGVHD) or relapse. In this retrospective Center for International Blood and Marrow Transplant Research study, a total of 3242 HLA-matched DRPs were analyzed for predicted cumulative peptide-binding efficiency using their HLA types and were divided into tertiles based on their scores. Univariable and multivariable analyses was performed to test for associations between cumulative peptide-binding efficiency for DRPs, divided into the HLA-matched related donor (MRD) and HLA-matched unrelated donor (MUD) cohorts, and the primary outcomes of aGVHD and relapse. Secondary outcomes investigated included overall survival, disease-free survival, and transplantation-related mortality. Using a computationally generated peptidome as a test dataset, the tested series of HLA class I displayed peptide-binding frequencies ranging from 0.1% to 3.8% of the full peptidome, and HLA class II molecules had peptide-binding frequencies of 12% to 77% across the HLA-DRB1 allotypes. By increasing binding efficiency tertile, the cumulative incidence of aGVHD at 6 months for MUD patients was 41%, 41%, and 45% for HLA class I (P = .336) and 44%, 41%, and 42% for HLA class II (P = .452). The cumulative incidences of relapse at 3 years for MUD transplant recipients were 36%, 38%, and 38% for HLA class I (P = .533) and 37%, 37%, and 38% for HLA class II (P = .896). The findings were similar for MRD transplant recipients. Multivariable analysis did not identify any impact of peptide-binding efficiency on aGVHD or relapse in MUD or MRD transplant recipients. Whereas GVHD is mediated by minor antigen mismatches in the context of HLA-matched allo-HCT, peptide-binding efficiency, which was used as a surrogate measurement for predicted number of binding antigens, did not provide additional clinical information for GVHD risk assessment. The negative result may be due to the limitations of this surrogate marker, or it is possible that GVHD is driven by a subset of immunogenic mHAs. Further research should be directed at direct mHA epitope and immunogenicity prediction.

A dendritic cell sarcoma cell line, U-DCS, was established from a dendritic cell sarcoma in a 53-year-old Caucasian male patient. Since its establishment, U-DCS has maintained stable phenotypic characteristics in vitro and has a doubling time of approximately 2 days under standard culture conditions. U-DCS is growing with typical dendritic cell morphology in tissue and expresses the dendritic cell sarcoma immunophenotypic markers S100 protein, MHCI, MHCII, and vimentin. Expression analysis revealed transcripts for the toll-like receptors TLR3, -4, -9 and DDX58 (RIG-I), but not for TLR2. U-DCS shows functional features of dendritic cells with the ability of phagocytosis and antigen-specific T cell stimulation. Karyotype-, CGH-, and mFISH analysis point to a chromosomal instability and a hypotetraploid karyotype with approximately 130 chromosomes. U-DCS is the first immortalized human dendritic cell sarcoma cell line and has some morphological and functional features of dendritic cells without dependency on growth factors.

The success of hematopoietic stem cell transplantation is determined by multiple factors. Additional complexity is conferred by covariables showing time-dependent effects. We evaluated the effect of predictors on competing-risk outcomes after hematopoietic stem cell transplantation in a time-dependent manner. We analyzed 14951 outcomes of adult patients with hematologic malignancies who underwent a first allogeneic transplant. We extended the combined endpoints of disease-free and overall survival to competing-risk settings: disease-free survival was split into relapse and non-relapse mortality. Overall survival was divided into transplant-related mortality, death from other causes and death from unknown causes. For time-dependent effects we computed estimators before and after a covariable-specific cut-point. Patients treated with reduced intensity conditioning had a constantly higher risk of relapse compared to patients treated with myeloablative conditioning. For non-relapse mortality, patients treated with reduced intensity conditioning had a reduced mortality risk but this effect was only seen in the first 4 months after transplantation (hazard ratio: 0.76, P

We reported previously on the widespread occurrence of anti-HLA alloantibodies of the IgA isotype (anti-HLA IgA) in the sera of solid-organ re-transplantation (re-tx) candidates (Arnold et al., ). Specifically focussing on kidney re-tx patients, we now extended our earlier findings by examining the impact of the presence and donor specificity of anti-HLA IgA on graft survival. We observed frequent concurrence of anti-HLA IgA and anti-HLA IgG in 27% of our multicenter collective of 694 kidney re-tx patients. This subgroup displayed significantly reduced graft survival as evidenced by the median time to first dialysis after transplantation (TTD 77 months) compared to patients carrying either anti-HLA IgG or IgA (TTD 102 and 94 months, respectively). In addition, donor specificity of anti-HLA IgA had a significant negative impact on graft survival (TTD 74 months) in our study. Taken together, our data strongly indicate that presence of anti-HLA IgA, in particular in conjunction with anti-HLA-IgG, in sera of kidney re-tx patients is associated with negative transplantation outcome.

Full-length sequencing of HLA-DRB4*01:14 showed the same splice site mutation as in HLA-DRB4*01:03:01:02N.

Introduction: A common genetic variant within the TCRA-TCRD locus has been recently identified as a predictive factor of thymic function and T cell repertoire diversity (Clave et al., 2018). Specifically it was shown in a mouse model that transplantation of rs2204985 AA human hematopoietic stem cells (HSC) into immunodeficient mice led to lower thymocyte counts and poorer TCR diversity. T cell mediated pathways are known to play a significant role in immunological processes affecting HSCT outcome like GvL, GvH and infection. Aim of this study was to investigate the potential impact of donor rs2204985 genotype on patient's outcome after unrelated HSCT. Methods: The study included 2,016 adult patients with hematologic malignancies who received their first unrelated (10/10 or 9/10 HLA matched) graft between 2000 and 2013 in a German transplant center. Patients with refractory disease at time of transplantation were excluded from the analysis. Both donors and patients were retrospectively genotyped for the TCRA-TCRD rs2204985 polymorphism by next generation sequencing using a validated protocol on an Illumina Miseq platform. Overall survival (OS), disease free survival (DFS), relapse (RI), non-relapse mortality (NRM), acute GvHD (aGvHD) and chronic GvHD (cGvHD) were evaluated; p Results: The rs2204985 genotype frequencies found in both patients and donors were in line with those previously reported for Caucasian populations indicating a codominance of the two alleles (i.e. A and G). Regarding the impact of this genetic variation on outcome, multivariate analysis of the combined cohort indicated different risk estimates in 10/10 and 9/10 HLA matched transplantations, therefore subanalysis on account of HLA incompatibility was performed. Analysis in the subgroup of single HLA mismatched cases (n=624) revealed that donor AA genotype associated with markedly inferior OS (55.1% vs 70.6%, p=0.004, Fig. 1) and DFS (47.6% vs 63.4%, p=0.002, Fig. 2) one year after HSCT as compared to the donor AG/GG genotypes. These results were confirmed in the corresponding multivariate models (OS HR: 1.48, p=0.003; DFS HR: 1.50, p=0.001) which are visually displayed as forest plots in Fig. 3 and Fig 4, respectively. The adverse effect of donor AA genotype on survival appears to be driven by a combined higher risk of RI (1Y after HSCT: 29.3% vs 18.3%, p=0.048; HR: 1.38, p=0.035) and NRM (1Y after HSCT: 28.6% vs 19.9%, p=0.043; HR: 1.38, p=0.042) as shown by both the univariate and multivariate analyses for the two respective endpoints. No association was found between donor rs2204985 genotype and risk of acute or chronic GvHD. The donor rs2204985 genotype had also no significant impact on any outcome endpoint in the 10/10 HLA matched subgroup. Last, no significant interactions were observed between this variable and the other adjusted covariates in the multivariate models. Conclusion: To our knowledge this is the first study to date investigating the potential effect of donor's genotype regarding a common genetic variant within the TCRA-TCRD locus on the outcome of patients receiving unrelated HSC grafts. Our data suggest that donor rs2204985 AA genotype in combination with single HLA mismatches may adversely affect the outcome of HSC transplanted patients and should therefore be avoided. It is of note that one in four unrelated donors of Caucasian origin is expected to carry the AA genotype. A weaker relapse and -presumably- infection control, especially in the early post-transplantation period, due to compromised T cell reconstitution as a result of the unfavorable donor AA genotype may account for these findings. Confirmatory studies in larger independent cohorts are warranted before final conclusions are drawn. Figure 1 Figure 1. Disclosures Platzbecker: Geron: Honoraria; Janssen: Honoraria; Takeda: Honoraria; AbbVie: Honoraria; Novartis: Honoraria; Celgene/BMS: Honoraria. Sala: Celgene/BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Jazz: Consultancy, Honoraria. Wulf: Gilead: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Clinigen: Consultancy, Honoraria. Kroeger: Celgene: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Gilead/Kite: Honoraria; AOP Pharma: Honoraria; Novartis: Honoraria; Jazz: Honoraria, Research Funding; Sanofi: Honoraria; Neovii: Honoraria, Research Funding. Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding. Hertenstein: Novartis: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; BMS: Honoraria. Schrezenmeier: Alexion, AstraZeneca Rare Disease: Honoraria, Other: Travel support, Research Funding; Roche: Honoraria; Novartis: Honoraria; Apellis: Honoraria; Sanofi: Honoraria.

Unrelated hematopoietic stem cell transplantation (HSCT) has evolved from an experimental protocol to a potentially curative first-line treatment in certain disease instances. Factors enabling this transformation were the optimization of treatment protocols and supportive care as well as the availability of a large number of donors worldwide along with the higher quality and reliability of HLA typing. The main criterion for donor selection is HLA compatibility. In this review we discuss the current clinical evidence of HLA matching in unrelated HSCT. In this context, we address methodical aspects of transplantation immunobiology research and discuss the impact of locus and resolution of HLA differences. Furthermore, we address special constellations such as unidirectional mismatches or the presence of nonexpressed alleles as well as HLA alloimmunization and describe the perspective for HLA typing and matching strategies in the future, given the implementation of novel complete or near-complete gene typing approaches using next-generation sequencing short read technology, which are now entering the standard of clinical care.

Sequencing of the human genome has led to the definition of the genes for most of the relevant blood group systems, and the polymorphisms responsible for most of the clinically relevant blood group antigens are characterized. Molecular blood group typing is used in situations where erythrocytes are not available or where serological testing was inconclusive or not possible due to the lack of antisera. Also, molecular testing may be more cost-effective in certain situations. Molecular typing approaches are mostly based on either PCR with specific primers, DNA hybridization, or DNA sequencing. Particularly the transition of sequencing techniques from Sanger-based sequencing to next-generation sequencing (NGS) technologies has led to exciting new possibilities in blood group genotyping. We describe briefly the currently available NGS platforms and their specifications, depict the genetic background of blood group polymorphisms, and discuss applications for NGS approaches in immunohematology. As an example, we delineate a protocol for large-scale donor blood group screening established and in use at our institution. Furthermore, we discuss technical challenges and limitations as well as the prospect for future developments, including long-read sequencing technologies. © In Copyright http://rightsstatements.org/vocab/InC/1.0/, publishedVersion

Previous studies have suggested that HLA-E may have a significant role in the outcome of matched unrelated hematopoietic stem cell transplantation (HSCT), especially for patients with acute leukemia. We used Center for International Blood and Marrow Transplant Research data and samples of 1840 adult patients with acute leukemia and their 10/10 HLA-matched unrelated donors to investigate the impact of HLA-E matching status as well as of donor/recipient (D/R) HLA-E genotype on post-HSCT outcome. Both patients and donors were HLA-E genotyped by next-generation sequencing. All patients received their first transplant in complete remission between 2000 and 2015. Median follow-up time was 90 months. Overall survival, disease-free survival (DFS), transplant-related mortality (TRM), and relapse incidence were primary endpoints with statistical significance set at .01. D/R HLA-E genotype analysis revealed a significant association of donor HLA-E*01:03/01:03 genotype with DFS (hazard ratio [HR] = 1.35, P = .0006) and TRM (HR = 1.41, P = .0058) in patients who received T cell replete (ie, without in vivo T cell depletion) transplants (n = 1297). As for D/R HLA-E matching, we did not identify any significant effect on any of the clinical outcome endpoints. In conclusion, this is the largest study to date reporting an improvement of DFS and TRM after matched unrelated HSCT by avoidance of HLA-E*01:03 homozygous donors in patients transplanted with T cell replete grafts for acute leukemia.

BACKGROUND Biosimilar granulocyte-colony-stimulating factors (G-CSFs) have been available in the European Union since 2008, and Sandoz' biosimilar filgrastim was approved in the United States in March 2015 for all of the reference product's indications except acute radiation syndrome. Biosimilar G-CSFs have been largely embraced by the medical community, except for some reservations about healthy-donor stem cell mobilization, for which use outside of clinical studies was cautioned against by some members of the scientific community. STUDY DESIGN AND METHODS In a two-center safety surveillance study (National Clinical Trial NCT01766934), 245 healthy volunteer stem cell donors were enrolled. Of 244 donors who began mobilization with twice-daily Sandoz biosimilar filgrastim, 242 received a full (n = 241) or partial (n = 1) course of G-CSF and underwent apheresis. Efficacy and safety were assessed and are reported here. RESULTS Biosimilar filgrastim was accompanied by the typical G-CSF class-related adverse effects of expected frequency and severity. Median mobilization for CD34-positive stem cells was 97/µL (range, 20-347/µL); after one apheresis (91%) or two aphereses (9%) from all but three donors (1.2%), cell doses in excess of the typical 4 × 106 CD34-positive cells/kg of the recipient had been collected (range, 3-52 × 106/kg). Biochemical and hematologic alterations were consistent with previous reports; all had normalized by the first follow-up 1 month after mobilization. Stem cell products engrafted with typical probability and kinetics for G-CSF-mobilized stem cell products. CONCLUSION These data support the use of biosimilar filgrastim for healthy-donor stem cell mobilization as safe and effective.

We present a catalog of common and well-documented (CWD) alleles of the German population for the six HLA loci A, B, C, DRB1, DQB1, and DPB1. This study is based on a sample of over 5 million volunteer adult hematopoietic stem cell donors from the 26 German donor centers. To establish the catalog, allele and haplotype frequencies were estimated with a validated implementation of the expectation-maximization algorithm. CWD criteria similar to existing CWD catalogs were applied in order to be able to put our findings into the context of relevant existing references. Overall, 2155 HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 alleles were identified as CWD in the German donor population representing about 20% of the HLA alleles at two-field resolution in the IPD-IMGT/HLA Database release v3.25.0 from July 2016 for these six loci. We found a substantial concordance of CWD alleles between the three catalogs and showed the contribution of the German donor population to the CWD alleles domain. In conclusion, the definition of CWD criteria that allow interoperability, scalability, and flexibility will be crucial for the development of a worldwide CWD catalog.

Introduction: A series of studies suggest that harnessing natural killer (NK) cell reactivity by killer cell immunoglobulin-like receptor (KIR) genotype based unrelated donor selection could further improve outcome after allogeneic hematopoietic cell transplantation (alloHCT). A Receptor-Ligand model has been proposed for donor selection which aims at augmenting NK cell activation while minimizing inhibition. Information on education of KIR2DS1-positive NK cells (Venstrom et al, NEJM 2012) and the predicted Receptor-Ligand interaction of KIR3DL1-positive NK cells is utilized for this algorithm. By combining this information donors can be classified as KIR-advantageous or disadvantageous. Patients with donors, characterized by activating KIR2DS1 and weak/non-inhibiting KIR3DL1, experienced less relapse and improved survival compared to patients with donors, characterized by lacking an activating KIR2DS1 but presence of strong-inhibiting KIR3DL1. This study aimed at validating this predictor in an independent cohort of patients. Methods: Donor samples were retrieved from the Collaborative Biobank (Dresden, Germany) and mapped to patient outcome data extracted from the German Registry for Stem Cell transplantation. KIR typing was performed using a high resolution amplicon-based next generation sequencing method. KIR typing at the allele level was based on sequencing of exons 3, 4, 5, 7, 8, and 9. The patient population was restricted to patients with AML or MDS. Donor and patient mapping was cross-checked by HLA-typing of the donor sample. The impact of the predictor on overall survival was tested in a Cox regression model adjusted for patient age, a modified disease risk index, performance status, donor age, HLA-match, sex match, CMV match, conditioning intensity, type of T-cell depletion and graft type. Results: Clinical data from 2314 patients were analyzed. The median age at alloHCT was 59.4 years (range, 18.1 to 79.6 years). The indication for alloHCT was AML for 80% of patients and MDS for 20% of patients. Disease risk was assessed as low, intermediate, high or very high in 1%, 52%, 42%, and 5%, respectively. Patient and donor were 10/10 matched in 78% of pairs, whereas a one locus mismatch was reported for 21% of pairs. Myeloablative, reduced-intensity and non-myeloablative conditioning regimens were used in 29%, 67%, and 4% of patients, respectively. ATG was administered in 77% and alemtuzumab in 3% of patients. Twenty percent of patients received no T-cell depletion. In total, 535 patients experienced relapse and 945 patients died. This number of events translated into a power of the confirmatory analysis for the predictor of KIR2DS1 and KIR3DL1 of 67%. Two-year overall and event-free survival for the whole cohort was 51% (95%-CI 48% to 53%) and 44% (95%-CI 42% to 47%) and the 2-year incidence of relapse and non-relapse mortality was 28% (95%-CI 26% to 30%) for both endpoints. In univariate analysis, overall survival (54% versus 56%) and the cumulative incidence of relapse of patients with a KIR-advantageous donor were comparable to patients with KIR-disadvantageous donors. The adjusted hazard ratio from the multivariable Cox regression model for the comparison of patients with KIR-advantageous versus KIR-disadvantageous donors was 0.99 (Wald-test, p=0.95) for overall survival and 1.12 (Wald-test, p=0.41) for relapse incidence. When evaluated separately, the two components of the predictor (degree of inhibition by KIR3DL1 & presence of activating KIR2DS1) did not have an impact on overall survival or the incidence of relapse (see Figure). Also, evaluation of the combined predictor in subsets of patients by disease, type of T-cell depletion and HLA-compatibility did not allow prediction of these outcomes. Conclusions: Relapse incidence and overall survival after unrelated donor alloHCT could not be predicted using information on activating KIR2DS1 and inhibiting KIR3DL1 donor genes in an independent cohort of predominantly Caucasian patients. The predictor had been developed in a cohort of patients with AML who were younger and predominantly had received myeloablative conditioning based on total-body irradiation, ATG was administered less often, but donors often were only partially HLA-compatible. The different outcome in the current analysis thus points at potential interactions between NK-cell mediated allo-reactivity and procedural variations of alloHCT. Figure Figure. Disclosures Schetelig: Sanofi: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Roche: Honoraria; Abbvie: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding. Stelljes:Novartis: Honoraria; MSD: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; JAZZ: Honoraria; Amgen: Honoraria. Ayuk:Therakos (Mallinckrodt): Honoraria; Novartis: Honoraria; Celgene: Consultancy; Gilead: Consultancy. Bethge:Neovii GmbH: Honoraria, Research Funding; Miltenyi Biotec GmbH: Consultancy, Honoraria, Research Funding. Bug:Neovii: Other: Travel Grant; Novartis Pharma: Honoraria, Research Funding; Janssen: Other: Travel Grant; Celgene: Honoraria; Amgen: Honoraria; Astellas Pharma: Other: Travel Grant; Jazz Pharmaceuticals: Other: Travel Grant. Kobbe:Roche: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding; Amgen: Honoraria, Research Funding. Beelen:Medac: Consultancy, Other: Travel Support. Fleischhauer:GENDX: Research Funding.

Background Photodynamic therapy (PDT) represents a palliative treatment option for a selected group of patients with head and neck squamous cell carcinoma (HNSCC). PDT induces a local inflammatory reaction with the potential to initiate antitumor immune responses. However, the systemic impact on peripheral immune cells has not been described so far. Methods HNSCC patients (n = 9) were treated with PDT in a palliative setting. All patients had previously undergone several oncologic treatment regimens. Blood samples were taken before, during and after PDT. Age-matched healthy donors served as control group (NC, n = 15). The frequency and absolute number of T- and B-lymphocytes, CD4+CD39+ regulatory T-cells (Treg) and NK-cells were measured by 10-color flow cytometry. Serum concentrations of T cell related cytokine panel, including HMGB1, IL-6, IL-10 and perforin were measured by bead array and ELISA. Results In heavily pretreated HNSCC patients, the number and frequency of Treg and NK-cells were increased as compared to NC. PDT induced a further increase of the frequency of Treg and NK-cells in the peripheral blood. Additionally, the serum concentrations of HMGB1, IL-6 and IL-10 showed a significant elevation after treatment with simultaneously decreased perforin levels. Conclusion Although PDT is a local treatment regimen, a systemic inflammatory response with altered peripheral immune cell populations and cytokine concentrations is visible. The increased Treg and NK cell numbers after PDT support the hypothesis that PDT may successfully be combined with NK cell or T cell activating immune checkpoint modulators in HNSCC patients to improve HNSCC specific immunity.

Natural Killer cells (NK) are lymphocytes with the potential to recognize and lyse cells which escaped T-cell mediated lysis due to their aberrant HLA expression profiles. Killer cell immunoglobulin-like receptors (KIR) influence NK-cell activity by mediation of activating or inhibitory signals upon interaction with HLA-C (C1, C2) ligands. Therefore, absence of ligands for donor inhibitory KIRs following hematopoietic stem cell transplantation (HSCT) may have an influence on its outcome. Previous studies showed that C1 negative patients have a decreased HSCT outcome. Our study, based on a cohort of 200 C1-negative patients, confirmed these findings for the endpoints: overall survival (OS: HR = 1.41, CI = 1.14-1.74, p = 0.0012), disease free survival (DFS: HR = 1.27, CI = 1.05-1.53, p = 0.015), treatment related mortality (TRM: HR = 1.41, CI = 1.01-1.96, p = 0.04), and relapse incidence (RI: HR = 1.33, CI = 1.01-1.75, p = 0.04) all being inferior when compared to C1-positive patients (n = 1246). Subsequent analysis showed that these findings applied for patients with myeloid malignancies but not for patients with lymphoproliferative diseases (OS: myeloid: HR = 1.51, CI = 1.15-1.99, p = 0.003; lymphoblastic: HR = 1.26, CI = 0.91-1.75, p = 0.16; DFS: myeloid: HR = 1.31, CI = 1.01-1.70, p = 0.04; lymphoblastic: HR = 1.21, CI = 0.90-1.61, p = 0.21; RI: myeloid: HR = 1.31, CI = 1.01-1.70, p = 0.04; lymphoblastic: HR = 1.21, CI = 0.90-1.61, p = 0.21). Interestingly, within the C1-negative patient group, transplantation with KIR2DS2 resulted in better OS (9/10 matched: HR = 0.24, CI = 0.08-0.67, p = 0.007) as well as DFS (9/10 matched: HR = 0,26, CI = 0.11-0.60, p = 0.002), and transplantation with KIR2DS1 positive donors was associated with a decreased RI (HR = 0.30, CI = 0.13-0.69, p = 0.005). TRM was increased when the donor was positive for KIR2DS1 (HR = 2.61, CI = 1.26-5.41, p = 0.001). Our findings suggest that inclusion of KIR2DS1/2/5 and KIR3DS1-genotyping in the unrelated donor search algorithm of C1-ligand negative patients with myeloid malignancies may prove to be of clinical relevance. peerReviewed

The immunomodulatory role of human leukocyte antigen (HLA)-E in hematopoietic stem cell transplantation (HSCT) has not been extensively investigated. To this end, we genotyped 509 10/10 HLA unrelated transplant pairs for HLA-E, in order to study the effect of HLA-E as a natural killer (NK)-alloreactivity mediator on HSCT outcome in an acute leukemia (AL) setting. Overall survival (OS), disease free survival (DFS), relapse incidence (RI) and non-relapse mortality (NRM) were set as endpoints. Analysis of our data revealed a significant correlation between HLA-E mismatch and improved HSCT outcome, as shown by both univariate (53% vs. 38%, P=0.002, 5-year OS) and multivariate (hazard ratio (HR)=0.63, confidence interval (CI) 95%=0.48–0.83, P=0.001) analyses. Further subgroup analysis demonstrated that the positive effect of HLA-E mismatch was significant and pronounced in advanced disease patients (n=120) (5-year OS: 50% vs. 18%, P=0.005; HR=0.40, CI 95%=0.22–0.72, P=0.002; results from univariate and multivariate analyses, respectively). The study herein is the first to report an association between HLA-E incompatibility and improved post–transplant prognosis in AL patients who have undergone matched unrelated HSCT. Combined NK and T cell HLA-E-mediated mechanisms may account for the better outcomes observed. Notwithstanding the necessity for in vitro and confirmational studies, our findings highlight the clinical relevance of HLA-E matching and strongly support prospective HLA-E screening upon donor selection for matched AL unrelated HSCTs.

Die Blutstammzelltransplantation hat sich zu einem etablierten Therapieverfahren fur eine Vielzahl von angeborenen oder erworbenen Erkrankungen des hamatopoetischen Systems entwickelt. Dies spiegelt sich in kontinuierlich steigenden Fallzahlen vor allem im Bereich der unverwandten Stammzelltransplantation wider. Begunstigende Faktoren fur diese Entwicklung sind einerseits die zunehmende Zahl an freiwilligen Stammzellspendern weltweit, die es ermoglichen, fur eine kontinuierlich steigende Zahl von Patienten einen geeigneten Spender zu identifizieren. Andererseits machen Verbesserungen im Bereich der Behandlungsverfahren wie z. B. die dosisreduzierte Konditionierung oder die molekulargenetische hoch aufgeloste HLA-Typisierung (HLA: humanes Leukozytenantigen) die Blutstammzelltransplantation sicherer, sodass klinisch die Indikation haufiger gestellt werden kann. Rund 3000 Suchen nach einem unverwandten Stammzellspender werden in Deutschland aktuell pro Jahr durchgefuhrt. Dabei stellt die Patientengruppe der uber 60-Jahrigen die anteilmasig am starksten wachsende Altersgruppe dar. Hauptindikationen sind nach wie vor akute Leukamieformen. Hauptstammzellquelle sind periphere Blutstammzellen mit einem Anteil von uber 85 % gegenuber Knochenmark. Nabelschnurblut spielt in Deutschland nur eine untergeordnete Rolle. Ein wesentlicher Faktor fur den Erfolg der Blutstammzelltransplantation ist die HLA-Kompatibilitat zwischen Patient und Spender, wobei nach aktuellem Konsensus eine Kompatibilitat auf Allelebene an den Genorten HLA-A, -B, -C, -DRB1 und -DQB1 angestrebt wird (10/10-Kompatibilitat). HLA-Inkompatibilitaten fuhren zu einer erhohten transplantationsassoziierten Mortalitat, insbesondere infolge von GvHD-assoziierten (GvHD: Transplantat-gegen-Wirt-Reaktion) Komplikationen. Dies resultiert in reduziertem Gesamtuberleben und uberwiegt definitiv einen moglicherweise gunstigen Einfluss auf ein Rezidiv der Grunderkrankung im Sinne eines GvL-Effekts (GvL: Transplantat vs. Leukamie). Der Einfluss individueller Inkompatibilitaten auf den Erfolg der Stammzelltransplantation hangt von der tatsachlich vorliegenden Allelkombination am respektiven Genort ab. Als gesichert gilt, dass das Risiko zu versterben mit steigender Anzahl an HLA-Inkompatibilitaten kumulativ zunimmt. Daher werden Spender mit mehr als 2 HLA-Inkompatibilitaten fur die unverwandte Blutstammzelltransplantation praktisch nicht akzeptiert. Liegen einzelne Inkompatibilitaten (9/10) vor, sind an den Genorten HLA-A, -B, und -DRB1 Allel- und Antigeninkompatibilitaten als gleichermasen ungunstig zu bewerten. Fur HLA-C sind Antigeninkompatibilitaten mit erhohtem Risiko assoziiert. Dies konnte fur HLA-C-Allel-Inkompatibilitaten bisher nicht gezeigt werden und geht moglicherweise auf das Vorhandensein permissibler Allelkombinationen (gezeigt fur HLA-C*03:03/03:04) zuruck. Auch fur HLA-DQB1-Inkompatibilitaten konnte bisher kein eindeutig signifikanter Einfluss auf das Uberleben nach Stammzelltransplantation gezeigt werden, jedoch gibt es Hinweise aus einer deutschen Kohorte, wonach HLA-DQB1-Antigen-Inkompatibilitaten, nicht jedoch HLA-DQB1-Allel-Inkompatibilitaten moglicherweise mit einem erhohten Risiko assoziiert sind und nach Moglichkeit zu vermeiden waren. Klarheit werden Studien mit groseren Fallzahlen bringen. Weiterhin ist davon auszugehen, dass zusatzliche Loci im erweiterten HLA-Bereich (z. B. HLA-DPB1, HLA-DRB*345, MICA) sowie Spender-KIR-Gene (KIR: Killercell Immunoglobulin-like Receptor) fur die Histokompatibilitatstestung im Rahmen der Spendersuche fur die unverwandte Blutstammzelltransplantation in Zukunft relevant werden.

Der vorliegende 4. deutsche Konsensus zur immungenetischen Spenderauswahl fur die allogene Stammzelltransplantation wurde von der Deutschen Gesellschaft fur Immungenetik (DGI) und der Deutschen Arbeitsgemeinschaft fur Knochenmark- und Blutstammzelltransplantation (DAG-KBT) gemeinsam erarbeitet und verabschiedet. Er stellt den gemeinsamen Nenner von mehr als 60 Transplantationseinheiten und fast 20 immungenetischen Sucheinheiten dar, die mehr als 3000 deutsche Patienten jahrlich betreuen. Wahrend der 1. Konsensus im Jahre 1996 den Anfang der Ablosung von zellularen und serologischen Methoden der HLA-Testung durch molekulargenetische Verfahren darstellte, wird nun die hochaufgeloste Bestimmung der Antigenbindungsstelle der HLA-Molekule der 5 wichtigsten Genorte als Standard definiert. Das vorliegende Dokument behandelt die Suche nach verwandten und nicht-verwandten Spendern und alle infrage kommenden Stammzellquellen. Bei Sachverhalten, fur die eine eindeutig wissenschaftliche Evidenz vorliegt, gibt dieser Konsensus eine klare Richtschnur vor, wahrend es sonst, soweit moglich, die Handlungsoptionen beschreibt.

Next generation sequencing (NGS) denotes novel sequencing technologies that enable the generation of a large number of clonal sequences in a single sequencing run. NGS was initially introduced for whole genome sequencing and for quantitation of viral variants or genetic mutations in tumor tissues; more recently, the potential for high resolution HLA typing and high throughput analyses has been explored. It became clear that the complexity of the HLA system implicates new challenges, especially for bioinformatics. From an economical point of view, NGS is becoming increasingly attractive for HLA typing laboratories currently relying on Sanger based sequencing. Realizing the full potential of NGS will require the development of specifically adapted typing strategies and software algorithms. In the present review, three laboratories that were among the first to perform HLA-typing using different NGS platforms, the Roche 454, the Illumina Miseq and the Ion Torrent system, respectively, give an overview of these applications and point out advantages and limitations.

Long nanopore reads allow phasing of heterozygous positions but show limitations in sequencing of homopolymers.

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) with the corresponding natural killer group 2, member D (NKG2D) receptor triggers cytotoxic effector activity of natural killer cells and certain T-cell subsets and provides a costimulatory signal for cytokine production. Thus, the presence of MICA/B on transformed cells contributes to tumor immunosurveillance. Consequently, the proteolytic cleavage of MICA/B is regarded as an important immune escape mechanism of various cancer cells. To investigate the molecular machinery responsible for the shedding of endogenous MICA/B, we analyzed different human tumor entities including mammary, pancreatic and prostate carcinomas. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that all tested tumor cells constitutively expressed MICA and MICB on the cell surface and also released NKG2D ligands into the supernatant. We demonstrate that the "a disintegrin and metalloproteases" (ADAMs) 10 and 17 are largely responsible for the generation of soluble MICA/B. Pharmacological inhibition of metalloproteases reduced the level of released MICA/B and increased cell surface expression. Studies using RNA interference not only revealed a prominent role of ADAM10 and ADAM17 in NKG2D ligand shedding but also a tumor cell-specific role of ADAM10 and/or ADAM17 in shedding of MICA or MICB. Moreover, we report that in the prostate carcinoma cell line PC-3, MICA was not shed at all but rather was secreted in exosomes. These data indicate that the release of NKG2D ligands from individual tumor entities is by far more complex than suggested in previously reported MICA/B transfection systems.

We investigated a possible interaction between age-associated risk and HLA-mismatch associated risk on prognosis in different age categories of recipients of unrelated hematopoietic stem cell transplants (HSCT) (n=3019). Patients over 55 years of age transplanted with 8/10 donors showed a mortality risk of 2.27 (CI 1.70–3.03, P