Journal articles on the topic 'Immunogenetics (incl. Genetic Immunology)'

Abstract The completion of the genome draft of the Florida manatee (Trichechus manatus latirostris) in 2012 made immunogenetic exploration of the Sirenia order more feasible. The endangered manatees are sentinel species for coastal ecosystems and their evolutionary origins are complex. It is believed that they evolved from a wading terrestrial mammal that became fully aquatic, which suggests distinctive adaptations for transitioning to a new environment. Discovering the genetic details of their immune system will help understand manatee evolution and allow species-specific diagnostic tools to be developed. Due to bottleneck signatures at other loci, the genetic diversity of the major histocompatibility complex (MHC) is expected to be low. This could pose problems for conservation efforts because low diversity can increase the chance of extirpation. The primary focus of this project is to determine the immune robustness of manatees by looking at their antibody repertoire and MHC polymorphism. Two species will be compared: the West Indian manatee (T. manatus) and the Amazonian manatee (T. inunguis). Using next generation sequencing, we will robustly sample the IgH and IgL rearrangements contributing to the antibody repertoire, and identify dominant MHC alleles and estimate their frequencies. The allelic distribution between the species could reveal clues to their evolutionary relationship and aid conservation efforts to help this species become more stable in their environment.

Abstract The development of an outbred immunogenetics model system is needed to understand how genetic variation impacts phenotypic variation of disease states in humans. Threespine stickleback fish (Gasterosteus aculeatus) provide just such a model. Stickleback are genetically tractable laboratory organisms with a well-annotated genome, but with lines drawn from populations inhabiting vastly different habitats. Individuals from different populations show high levels of genetic variation. The onset of the adaptive immune system is currently unknown in stickleback, a significant hindrance in studies of immunodeficiency diseases. To characterize the early development of adaptive immunity, we will analyze the expression of known early indicators of adaptive immunity maturation. These include recombination activating genes, rag1 and rag2, which are essential to the maturation of T and B lymphocytes, and T cell receptor genes, tcr-β and tcr-γ, which are expressed in mature T lymphocytes. To analyze the expression of rag1, rag2, tcr-β, and tcr-γ, we will perform whole mount in situ hybridization throughout a developmental time series to detect when and where the genes are first expressed, with special focus on the head kidney and the thymus, followed by qPCR to quantify the expression of the early adaptive immunity genes. Knowing when adaptive immunity onset occurs in threespine stickleback advances threespine stickleback as an outbred disease model in immunogenetics studies, allowing manipulative studies of immunological disease phenotypes in the context of genetic variation.

Abstract All jawed vertebrates share fundamental components of the adaptive immune system. Cartilaginous fish are the most divergent jawed vertebrate group relative to mammals and use a major histocompatibility complex, B cells, and T cells. RAG mediates the rearrangement of gene segments to create both B and T cell receptors. B cell receptors further undergo diversification after antigen exposure when activated B cells extensively alter their rearranged genes to enhance affinity to antigen. This receptor modification is mediated by the enzyme activation-induced cytidine deaminase (AID). Remarkably, AID also catalyzes receptor modifications in alpha chain of TCR within shark thymus. Thus, the ancestor to modern vertebrates may have used an AID-like enzyme for lymphocyte receptor development, a mechanism lost in later vertebrates because of its potential for breaking down self-tolerance in mature lymphocytes. At the other end of the vertebrate tree, the repertoire of Bos taurus antibodies is characterized by a subset of heavy chains with variable domains VH encoding ultralong complementarity determining region CDR3. These ultralong CDR3 range from forty to over seventy amino acids in length and form a unique beta-ribbon “stalk” and disulfide bonded “knob”. Deep sequence analysis of the B. taurus heavy chain repertoire unveiled ultralong CDR3s were products of rearrangement between the longest VH and DH segments, IGHV1-1 and DH8. Both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual bovine ultralong CDR3 repertoire. These unheralded uses of AID will be discussed in the broader context of immunogenetics evolution.

Drug-induced liver injury (DILI) is a challenging clinical event in medicine, particularly because of its ability to present with a variety of phenotypes including that of autoimmune hepatitis or other immune mediated liver injuries. Limited diagnostic and therapeutic tools are available, mostly because its pathogenesis has remained poorly understood for decades. The recent scientific and technological advancements in genomics and immunology are paving the way for a better understanding of the molecular aspects of DILI. This review provides an updated overview of the genetic predisposition and immunological mechanisms behind the pathogenesis of DILI and presents the state-of-the-art experimental models to study DILI at the pre-clinical level.

Antibiotic resistance determinants are frequently associated with plasmids and other mobile genetic elements, which simplifies their horizontal transmission. Several groups of plasmids (including replicons of the IncL/M incompatibility group) were found to play an important role in the dissemination of resistance genes encodingβ-lactamases. The IncL/M plasmids are large, broad host range, and self-transmissible replicons. We have identified and characterized two novel members of this group: pARM26 (isolated from bacteria inhabiting activated sludge from a wastewater treatment plant) and pIGT15 (originating from a clinical strain ofEscherichia coli). This instigated a detailed comparative analysis of all available sequences of IncL/M plasmids encodingβ-lactamases. The core genome of these plasmids is comprised of 20 genes with conserved synteny. Phylogenetic analyses of these core genes allowed clustering of the plasmids into four separate groups, which reflect their antibiotic resistance profiles. Examination of the biogeography of the IncL/M plasmids revealed that they are most frequently found in bacteria of the family Enterobacteriaceae originating from the Mediterranean region and Western Europe and that they are able to persist in various ecological niches even in the absence of direct antibiotic selection pressure.

Abstract Of the many minor histocompatibility (H) Ags that have been detected in mice, the ability to induce graft vs host disease (GVHD) after bone marrow transplantation is restricted to a limited number of immunodominant Ags. One such murine Ag, B6dom1, is presented by the H2-Db MHC class I molecule. We present biochemical evidence that the natural B6dom1 peptide is indistinguishable from AAPDNRETF, and we show that this peptide can be isolated from a wide array of tissues, with highest levels from the lymphoid organs and lung. Moreover, we employ a novel, somatic cell selection technique involving CTL-mediated immunoselection coupled with classical genetics, to show that B6dom1 is encoded by the H7 minor H locus originally discovered ∼40 years ago. These studies provide a molecular genetic framework for understanding B6dom1, and exemplify the fact that mouse minor H loci that encode immunodominant CTL epitopes can correspond to classical H loci originally identified by their ability to confer strong resistance to tumor transplantation. Additionally, these studies demonstrate the utility of somatic cell selection approaches toward resolving H Ag immunogenetics.

AbstractImmunoglobulin (IG) loci harbor inter-individual allelic variants in many different germline IG variable, diversity and joining genes of the IG heavy (IGH), kappa (IGK) and lambda (IGL) loci, which together form the genetic basis of the highly diverse antigen-specific B-cell receptors. These allelic variants can be shared between or be specific to human populations. The current immunogenetics resources gather the germline alleles, however, lack the population specificity of the alleles which poses limitations for disease-association studies related to immune responses in different human populations. Therefore, we systematically identified germline alleles from 26 different human populations around the world, profiled by “1000 Genomes” data. We identified 409 IGHV, 179 IGKV, and 199 IGLV germline alleles supported by at least seven haplotypes. The diversity of germline alleles is the highest in Africans. Remarkably, the variants in the identified novel alleles show strikingly conserved patterns, the same as found in other IG databases, suggesting over-time evolutionary selection processes. We could relate the genetic variants to population-specific immune responses, e.g. IGHV1-69 for flu in Africans. The population matched IG (pmIG) resource will enhance our understanding of the SHM-related B-cell receptor selection processes in (infectious) diseases and vaccination within and between different human populations.

AbstractCharacterizing the allelic diversity within major histocompatibility complex (MHC) genes is an important way of determining the potential genetic resilience of a population to infectious and ecological pressures. For the koala (Phascolarctos cinereus), endemic diseases, anthropogenic factors and climate change are all placing increased pressure on this vulnerable marsupial. To increase the ability of researchers to study MHC genetics in koalas, this study developed and tested a high-throughput immunogenetic profiling methodology for targeting MHC class I UA and UC genes and MHC class II DAB, DBB, DCB and DMB genes in a population of 82 captive koalas. This approach was validated by comparing the determined allelic profiles from 36 koala family units (18 dam-sire-joey units and 18 parent-joey pairs), finding 96% overall congruence within family profiles. Cancers are a significant cause of morbidity in koalas and the risk factors remain undetermined. Our analysis of this captive population revealed several novel MHC alleles, including a potential link between the DBB*03 allele and a risk of developing cancer. This method offers a reliable, high-throughput protocol for expanded study into koala immunogenetics.

Abstract Acute rheumatic fever (ARF) is an autoimmune condition caused by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Multiple or severe attacks of ARF can cause cardiac damage, known as Rheumatic Heart Disease (RHD). RHD remains a significant cause of morbidity and mortality is rare in developed countries. In New Zealand, the disease burden of ARF and RHD amongst Indigenous Maori and Pasifika communities is one of the highest in the world, usually affecting children and young adults. Previous work carried out in New Zealand sought to explore the immunogenetics of ARF and RHD and involved candidate gene analysis techniques utilizing samples from individuals diagnosed with RHD where a functional variant in the gene encoding the interleukin IL-6 cytokine known as IL-6 rs1800797 was found to be significantly associated with RHD. Current work seeks to explore the genetic susceptibility to ARF/RHD in greater depth using more advanced genome-wide technology. An international genome wide association study (GWAS) is currently underway to further understand the genetics of ARF/RHD using data obtained from global sources. To undertake the proposed GWAS work and amend the original New Zealand study, an Advisory team comprised of Pasifika and Māori representatives comprised of community leaders, researchers, scientists, and academics is providing the appropriate governance, cultural support and guidance essential for the project work.

Background: Syndromic immunodeficiencies are a genetically and pathophysiologically heterogeneous group of inborn errors of immunity. These are characterized by multiple extra immune clinical symptoms and a wide range of immunological phenotypes with increased susceptibility to infections, autoimmune phenomena, immune dysregulation, organ-specificpathology, and malignancy.Objective: To increase the pediatricians’ awareness of this multifaceted group of primary immunodeficiencies in children.Methods: A comprehensive review of genetic background and clinical symptomatology of syndromic immunodeficiencies as well as current diagnostic approach and treatment modalities.Results: From the pediatrician’s perspective, an early-life diagnosis of syndromic immunodeficiencies, which is frequently indispensable for successful life-saving immunocorrection, poses a diagnostic challenge. Increased pediatricians’ awareness to recognize signs and symptoms of these diseases in affected children is of paramount importance. Current advances in molecular biotechnology and immunogenetics, resulting in the implementation of newborn screening and new-generation sequencing, provide informative tools for definitive diagnosis and, in many new disease entities, for their definition and genotype–phenotype delineation and correlation.Conclusions: A broad spectrum of clinical phenotypes in children with syndromic primary immunodeficiencies requires pediatrician’s special attention, that is, individualized multidisciplinary approach under the supervision of a clinical immunologist.

Abstract AIM: Immunity and [self-]tolerance are usually considered orchestrated by protein repertoires upon genetic [Mendelian] and epigenetic [non-Mendelian] variation mechanisms. All is still not fully understood, as evident from transfer factor and LeDouarin phenomena, environmental influences, peptidome complexity and diversity [Adv.Immunol.11,195-266,1969; Int.J.Dev.Biol.49,131-136,2005]. Endogenous ncRNA incl. microRNA mostly escaped consideration. METHODS: Ann.N.Y.Acad.Sci.1137,316-342,2008. RESULTS: Landsteiner-Chase-Lawrence transfer factors showed up as paradigmatic guides in some terra incognita of adaptive immune memory. Original crude preparations were found as paucidisperse solute systems of numerous functional small ncRNA [<200n] in interaction with proteins. Some environment-modified redox- and metalloregulated small hairpin shRNA [<200 bases] were sequenced. By 5'-CUG-3’-hairpin loops, they may address conserved homologous helix-nucleating domains shared in self and foreign proteofactors of tolerated growth, metabolism, vascularization, cancer and epigenome indexing. CONCLUSIONS: Immune cells are not restricted to known protein recognition systems. They have further repertoires of environment-adapted ncRNA as earliest outputs in transcription and translation. These are superior in diversity/specificity [≥10E17] to immune proteins repertoires [~10E13]. All attributes make them competent to integrate information flow on possible molecular shapes into genomic mechanisms.

Parasites threaten all free-living organisms, including molluscs. Understanding the evolution of immune defence traits in natural host populations is crucial for predicting their long-term performance under continuous infection risk. Adaptive trait evolution requires that traits are subject to selection (i.e. contribute to organismal fitness) and that they are heritable. Despite broad interest in the evolutionary ecology of immune activity in animals, the understanding of selection on and evolutionary potential of immune defence traits is far from comprehensive. For instance, empirical observations are only rarely in line with theoretical predictions of immune activity being subject to stabilizing selection. This discrepancy may be because ecoimmunological studies can typically cover only a fraction of the complexity of an animal immune system. Similarly, molecular immunology/immunogenetics studies provide a mechanistic understanding of immunity, but neglect variation that arises from natural genetic differences among individuals and from environmental conditions. Here, we review the current literature on natural selection on and evolutionary potential of immune traits in animals, signal how merging ecological immunology and genomics will strengthen evolutionary ecological research on immunity, and indicate research opportunities for molluscan gastropods for which well-established ecological understanding and/or ‘immune-omics’ resources are already available. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

One of the urgent problems of modern medicine is the search for genetic markers of chronic obstructive pulmonary disease (COPD).The aim of the study was to investigate the relationship between HLA haplotypes and COPD in individuals whose illness developed during the professional work in the workshop of organic-silicon production of PJSC "Khimprom" (Novocheboksarsk, Russia).The study included patients with COPD in remission, belonging to the Chuvash ethnic group of Russia - 36 women and 26 men (mean age 45.4±2.3 years). The work experience averaged 15.2±2.4 years, the duration of COPD - 12.3±2.4 years. Smokers made up 22.0±5.8% of the total number of patients. The comparison group was a group of non-patients with COPD workers of organic-silicon manufacture. This group was positioned as "stable" to the development of COPD. Class I HLA antigens were typed in the standard microlymphocytotoxic test using histotyping anti-HLA sera to 8 HLA-A antigens of the locus A1, A2, A3, A9, A10, A11, A19, A28 and to 18 HLA-B antigens of the locus В5, В7, В8, В12, В13, В14, В15, В16, В17, В18, В21, В22, В27, В35, В40, В41, В42, В53) (CJSC “Interregional Center of Immunogenetics and Histotyping Reagents “Gisans”, St. Petersburg). HLA genotyping of class II alleles was carried out by the multi-primer polymerase chain reaction method in DNA, obtained from nuclear cells of peripheral blood, using reagent sets of the firm “DNA-Technology” (Moscow), according to the reagent manufacturer's method. They were typified by 14 alleles of the DRB1 locus, 8 to the DQA1 alleles and 11 to the DQB1 alleles. The frequencies of two-locus haplotypes were calculated using the computer program "Arlequin v. 3.01 ". The association strength of HLA with COPD was determined by the relative risk (RR) of the formula J. Haldane. The statistical reliability of the difference between RR and 1 was determined by the exact two-sided Fisher test. As a result of the study, the positive association of COPD with haplotypes was established: HLA-A9-DQA1*0501, A10-DQA1*0103, A28-DQA1*0102, B7-DQA1* 0103. These haplotypes can be attributed to genetic markers of predisposition to the development of COPD. The negative association with the disease was established for haplotypic combinations of the alleles HLA-A2-B8, A19-DQB1*0502-04, B12-DQB1*0502-04, B27-DQA1*0103, DRB1*01-DQA1*0101, DRB1*07-DQA1*0201, DRB1*13-DQA1 * 0102. This series of haplotypes can be attributed to the category of protective genetic markers of COPD in conditions of organic-silicon manufacture. In continuation of our studies, further research is needed to identify "marker" HLA haplotypes in COPD in other ethnic populations, as well as under the influence of other aggressive air pollutants. The results of the study indicate the association of COPD with a number of specific HLA-haplotypes.

Abstract Background. The strongest established prognostic factors for follicular lymphoma are age and clinical factors. Biologic studies suggest a major role for the host immunologic environment in follicular lymphomagenesis, which is in part determined by host genetic background. Cytokines are key regulators of immune function and regulation, are highly polymorphic, and have been implicated in lymphoma etiology and prognosis. We evaluated the hypothesis that inherited variability in cytokine and related immune genes impact overall survival in follicular lymphoma. Methods. We genotyped 73 single nucleotide polymorphisms (SNPs) from 44 candidate cytokine and immune genes in 278 follicular lymphoma patients who participated in a population-based case-control study conducted from 1998–2000 in the Detroit, Iowa, Los Angeles and Seattle SEER (Surveillance, Epidemiology and End Results) cancer registries. Baseline clinical data and survival through early 2005 were obtained from cancer registry files. Cox proportional hazards analysis was used to estimate hazard ratios (HR) and corresponding 95% confidence intervals for the association between individual SNPs and overall survival, adjusting for age, demographic and clinical factors. We also used parallel modeling strategies to identify the best summary multi-SNP risk score to predict survival, and applied these results in a time-dependent receiver-operator characteristics (ROC) analysis. Results. The median age at diagnosis was 57 years (range, 25–74), and 59 (21%) of the patients died during follow-up, with a median follow-up of 59 months (range, 27 – 78 months) for surviving patients. In multivariate modeling, SNPs in IL8 (rs4073; HRTT=2.14, 1.26–3.63), IL2 (rs2069762; HRGT/TT=1.80, 1.06–3.05), IL12B (rs3212227; HRAA/CC=1.83, 1.06–3.06), and IL1RN (rs454078; HRAA=1.93, 1.11–3.34) were the strongest and most robust predictors of survival. A summary score of the number of deleterious genotypes from these four genes was strongly associated with survival (p=1.4 × 10−5) after accounting for demographic and clinical variables (HR=2.06 per deleterious genotype, 1.52–2.79). A combination of the four SNPs and the demographic and clinical risk scores was strongly associated with survival (p=1.8 × 10−11); the 5-year Kaplan Meier survival estimates were 96% (93%–100%), 72% (62%–83%) and 58% (48%–72%) for low, intermediate, and high risk groups respectively. In a time-dependent ROC analysis the three risk groups had an area under the curve (AUC) of 0.83 at 72 months of follow-up (95% CI 0.72–0.93). Conclusion: Host genetic variability in immune genes, particularly IL8, IL2, IL12B, and IL1RN, individually and particularly in combination, was associated with overall survival in follicular lymphoma after accounting for demographic and clinical factors. Host immunogenetics is a promising class of prognostic factors in follicular lymphoma that warrants further evaluation.

BackgroundCharacterizing differentially expressed genes (DEGs) of systemic lupus erythematosus (SLE) is necessary to understand immunogenic interactions, and how several key immune genes were closely associated with the susceptibility of SLE.ObjectivesThis study aimed to identify DEGs of SLE using gene expression-based computational methodologies to analyze disease-immune interactions, which affect the development and progression of SLE.MethodsTwenty-six patients with SLE and 46 healthy controls were selected from the Gene Expression Omnibus database. The significantly enriched immune and virus related gene lists were computed and visualized by using the DEGs from the Gene Set Enrichment Analysis. Quantification of 38 immune cells were performed in determining the impact of immune cells on the virus mediated immunity in SLE by using ImmQaunt algorithm.ResultsThirty nine upregulated and 57 downregulated were identified in SLE patient compared to the healthy controls. Upregulated genes were significantly implicated in Gene Ontology gene sets as cytokine mediated signaling, secretion, and exocytosis in immune response pathways. In addition, these genes were enriched in hepatitis C, influenza A, measles, Epstein–Barr virus, and herpes simplex virus 1 infection in Kyoto Encyclopedia of Genes and Genomes pathways. Especially, FCGR1A, IRF7, OAS2, CAMP, MX1, OAS3, OAS1, DEFA3, ISG15, and RSAD2 were involved in virus mediated SLE mechanism, and the expression for OAS1, OAS2, and IRF7 was closely associated with the quantities of colony forming unit-monocyte and colony forming unit-granulocyte.ConclusionIdentifying virus mediated SLE genes and quantifies of immune cells were used to understand the pathological process and perform early diagnosis of SLE, and will lead to clinical tools for treating SLEs in patients.References[1]Ghodke-Puranik Y and Niewold TB. Immunogenetics of systemic lupus erythematosus: A comprehensive review. J Autoimmun 2015; 64: 125-136.[2]Teruel M and Alarcón-Riquelme ME. The genetic basis of systemic lupus erythematosus: What are the risk factors and what have we learned. J Autoimmun 2016; 74: 161-175.[3]Lauwerys BR, Hachulla E, Spertini F, et al. Down-regulation of interferon signature in systemic lupus erythematosus patients by active immunization with interferon alpha-kinoid. Arthritis Rheum 2013; 65: 447-456.[4]Frishberg A, Brodt A, Steuerman Y and Gat-Viks I. ImmQuant: a user-friendly tool for inferring immune cell-type composition from gene-expression data. Bioinformatics 2016; 32: 3842-3843.Disclosure of InterestsNone declared.

Abstract Abstract 28 Identical twins have provided substantial insight into the natural history, developmental timing and sequence of genetic events in ALL. Much of these data are focussed on ALL with ETV6-RUNX1 fusions. More limited data exists for the most prevalent subset of childhood ALL with hyperdiploidy though retrospective screening from cord blood (Maia et al. Genes Chromosomes and Cancer, 2004) provide evidence that hyperdiploidy itself can be an early, possibly initiating and pre-natal event. We have now explored the genetic complexity of hyperdiploid ALL using four pairs of monozygotic twins, three concordant and one discordant for ALL. In parallel with our prior studies on ETV6-RUNX1 ALL (Bateman et al. Blood, 2010), we hypothesized that hyperdiploidy itself would be a consistent pre-natal event and all other potential ‘driver’ mutations would be secondary and probably post-natal. If correct, this twin pair should share identical hyperdiploid karyotypes but have different copy number alterations (CNA) or other secondary mutations (e.g. in FLT3 or RAS). We used Affymetrix SNP 6.0 mapping arrays to identify CNA using the Genome Orientated Laboratory File (GOLF) v3.2 software package and dCHIP. The SNP array was performed using leukaemic DNA compared to matched remission DNA for all cases. The mutation screen included the activating D835 FLT3, NRAS exon 2 (codons 12 and 13) exon 3 (codon 61), KRAS exon 2 (codons 12 and 13) and exon 3 (codon 61), PTPN11 (exon 3 and exon 13). In the discordant twin pair, Fluorescence In-Situ Hybridisation (FISH) was used to interrogate the pre-leukaemic cells of the unaffected twin for the CNA found in the corresponding affected twin. In the identical twin pairs concordant for hyperdiploid ALL, the whole chromosome gains were identical within each twin pair apart from an additional but sub-clonal gain of chromosome 10 in one twin of twin pair set 2. The median number of whole chromosome gains was 56 (Range 55 to 59). The total number of CNA found was 6 (Range 0 to 4 per case) (excluding T cell receptor and immunoglobulin rearrangements). These included genes previously found to be recurrently altered in hyperdiploid ALL such as PAX5, CDKN2A/2B and ADD3. All the CNA abnormalities detected were different within each twin pair. The mutation screen revealed an NRAS exon 2 codon 13, GGT to GAT (glycine to aspartic acid) mutation in one twin of twin pair set 1, which was absent in leukaemic DNA of the co-twin. In the twin set discordant for ALL, the CNA at 19p13.3 (incl TCF3 gene) found in the leukaemic DNA of twin 1 were absent from the hyperdiploid pre-leukaemic cells of the unaffected twin 2. These pre-leukaemic cells were found at a consistent frequency between 0.0012% and 0.002% (over a period of 13 months). These data accord with the view that hyperdiploidy is a pre-natal and probably initiating event in childhood B cell precursor ALL and that the set of additional ‘driver’ genetic changes including CNA and sequence based mutations are all secondary. As all of the latter are single twin restricted, it is likely, though not formally proven, that they occur post-natally and perhaps more proximal to disease diagnosis. These data extend our understanding of the history of B cell precursor ALL. Whole genome sequencing is required to complete the picture of essential genetic events and their timing. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: The current routine genetic work-up in hematological malignancies includes chromosome banding analysis (CBA) to detect complete or partial chromosomal deletions and fusions, and the identification of point mutations and small deletions or insertions by sequencing panels (max. length ~50 bp). Deletions of individual genes (e.g. IKZF1 in ALL) are only detected by specifically designed molecular tools. Therefore, those microdeletions might be overlooked by the current gold standard despite their clinical relevance. We established a bioinformatic pipeline to screen for microdeletions in whole genome sequencing (WGS) data of myeloid malignancies. Aim: (1) Screen for recurrent microdeletions in myeloid malignancies with a normal karyotype, and (2) characterize a patient specific profile of microdeletions in genes with known clinical and/or prognostic relevance. Patients and Methods: We analyzed 1356 cases (M/F: 778/578) of myeloid malignancies with a normal karyotype according to CBA (aCML: n=47; AML: n=251; CMML: n=165, mastocytosis: n=90; MDS: n=415, MDS/MPN-RS-T: n=69; MDS/MPN-U: n=42; MPN: n=250; PNH: n=27) using WGS. Median age was 71 [20-94] years. Amplification-free WGS was performed on the NovaSeq or HiSeq system with a median coverage of 103x (Illumina, San Diego, CA). Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and somatic copy number variant (CNV) discovery was performed with GATK (v 4.0.2.1), following best practice guidelines. Only gene overlapping CNV calls were considered for analysis (gene coordinates biomaRt (v 2.42.1), GRCh37 Ensembl). Results: On average, 38 genes per patient were partially or completely deleted and the size of the deletions ranged from 0.9 kb to 32 Mb (median 399 kb). The microdeletions affected a broad list of genes, but no gene was present in >5% of myeloid malignancies. As technical validation, we used 36 B-ALL samples (normal karyotype) and identified the known deletions of IKZF1 (42%); PAX5 (25%) and CDKN2A/CDKN2B (22%) with expected incidences. We focused on a patient-by-patient analysis of genes (n=47) with known clinical relevance in myeloid malignancies. We identified deleted genes in 46 out of 1356 patients (3.4%). In aCML 13% of patients had one of the above-mentioned genes deleted (6/47), in mastocytosis only 1% (1/90). The most frequently deleted genes were TET2 (20/1356, 1.5%) and RUNX1 (9/1356, 0.7%). Other deletions also affected transcription factors (e.g. GATA2) or epigenetic regulators (e.g. DNMT3A, figure 1). No deletion of splicing factors, RAS genes or cohesion complex regulators was observed. We found only two deletions of kinases, which are predominantly affected by activating mutations (both FLT3). Instead, the deletions in 41 patients involved genes with a known loss-of-function mutation profile in myeloid malignancies. This corresponds to 89% (41/46) of patients with microdeletions or 3% (41/1356) of all analyzed patients with myeloid malignancies. Microdeletions are thus another genetic element that can lead to loss of gene activity. Deletions and mutations are either alternative genetic mechanisms or co-operate as double hits to affect the same gene. We found additional mutations present in 18 of the 46 patients with microdeletions (39%, figure 1). The majority of these (n=14) involved TET2. TET2 mutations had a median variant allele frequency of 82% [9-100%] indicative of a mutation on the non-deleted allele. For the remaining genes (incl. RUNX1), deletions are predominantly an alternative genetic mechanism to mutations. For validation of WGS results we applied interphase FISH and identified 6/9 RUNX1 deletions. The remaining three microdeletions were only detectable by WGS and too small to be identified by FISH. Conclusions: (1) WGS data unrevealed a plethora of microdeletions, which can be an alternative genetic mechanism to mutations, but are not detected with today's standard diagnostic tools. (2) In the light of increasingly personalized therapy and diagnostics, all genetic mechanisms should be considered, which impact the function of clinically relevant genes. (3) Bioinformatic pipelines for WGS as a potential diagnostic tool in the near future should address microdeletions in genes with relevance for patients' diagnosis, prognosis and hopefully targeted treatment. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Background: AML with myelodysplasia related changes (AML-MRC) is as specific WHO category with poor prognosis. It requires ≥ 20% of blasts, and (1) the history of MDS or MDS/MPN, or (2) "MDS related cytogenetic abnormalities", or (3) multilineage dysplasia. Drugs such as Vyxeos® have been approved by FDA and EMA only for treatment of t-AML or AML-MRC. However, counting blasts or grading dysplasia in clinical routine is hampered by limited reproducibility due to different levels of expertise and small phenotypic alterations, challenging upfront treatment decisions. Cytogenetics is not available in all cases and has 5-10 days of turnaround time (TAT). In contrast, next-generation sequencing (NGS) panels for AML are now broadly available at faster TAT. Aim: (1) Use machine learning to define a molecular AML-MRC signature; (2) compare the impact of conventional WHO definitions and molecular factors on classification and outcome. Patients and Methods: Gold standard routine AML diagnosis was performed on 739 cases. Overall survival (OS) data was available for 619 patients. Amplification-free whole genome sequencing was performed on HiSeqX and NovaSeq with median coverage of 106x. Gender-matched reference DNA was used for unmatched normal variant calling with Strelka2. Pindel was used for FLT3-ITD. For variant classification, we applied a GnomAD cutoff of 0.0005 and filtered on protein-truncating and (likely) pathogenic variants from databases. Results: According to WHO standards 165/739 (22%) cases fulfilled MRC criteria (96 male; 69 female). The non-MRC cohort (n=574) represents a heterogeneous AML population incl. the WHO defined recurrent cytogenetic abnormalities or t-AML (301 male, 273 female). Median age was higher in the MRC cohort (73 [22-90] vs. 64 [18- 93] years, p<.001) and OS was significantly shorter (median 6 vs. 23 months, p<.001). Mutation analysis was limited to 73 frequently mutated genes, in order to allow application of our model on prospective diagnostic cases analyzed by common routine panels. In the MRC group, up to seven mutations were found per patient and an average of 2.7 genes per patient were mutated. The most frequently mutated gene in AML-MRC was TP53 (62/165, 38%) as expected by the inclusion of complex karyotypes. TP53 mutations were associated with shorter OS in the MRC cohort (median: 3 vs. 11 months, p =.001). We used machine learning (ML) approaches to identify with LASSO regression and 10-fold cross-validation the most informative features to distinguish between MRC and patients without MRC. The dataset was randomly divided into a training (90%) and test set (10%) and the procedure was repeated 500 times to cover all the variance in the dataset and to extract the most reliable factors. Factors with the highest weight on AML-MRC prediction were mutations in TP53, RUNX1, SETBP1, splicing factors and epigenetic regulators, and absence of mutations in NPM1, CEBPA and others (s. figure). In order to allow our model to be used in a routine diagnostic workflow, we also used the genes identified by ML but classified mutations by a simpler point system (≥2 points as cutoff for MRC, s. figure). This allowed us to identify 83% (137/165 by ML) and 70% (116/165 by points) of cases currently defined as MRC solely by molecular genetics. Including cytogenetic data and patient's history in an informed genetic model results in 99% (164/165 by ML) and 96% (159/165 by points) of true positive MRC definition. However, the molecular models classified 112 (ML) and 80 (points) of the 574 non-MRC cases, as being AML-MRC. Even after excluding AML with recurrent cytogenetic abnormalities and t-AML, 14% (82/574 DL) or 11% (63/574 points) show a MRC-like molecular profile. In both models MRC-like patients had dismal outcome analogous to AML-MRC (median OS: 6 months for both) and significantly inferior to remaining non-MRC patients (6 vs. 35 months, s. figure). Conclusions: (1) Using patients' history and genetic information instead of morphology allow to identify 96-99% of AML-MRC as defined in WHO today. In the future, extended NGS panels (e.g. incl. fusion gene detection) will allow fast and standardized AML-MRC classification even without chromosome banding analysis. (2) The molecular MRC-like pattern can be found in >10% of patients currently not classified as AML-MRC but with comparably poor OS. This suggests considering MRC treatment strategies for patients with MRC-like molecular profile. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract Background: In hematopoietic stem cell transplantation (HSCT) for hematologic malignancies, natural killer (NK) cells contribute to tumor eradication such that leukemia patients lacking the HLA class I ligand for the donor NK inhibitory killer Ig-like receptors (KIR) have lower relapse rates and longer survival. Since myeloablative chemotherapy followed by autologous HSCT (ASCT) improves survival for children with high risk neuroblastoma (a tumor sensitive to NK killing) we hypothesize that NK cells may be active in this setting and that KIR-HLA combinations where the patient lacks HLA class I ligands for autologous KIR may be associated with improved clinical outcomes. Methods: 155 children with high risk neuroblastoma received myeloablative chemotherapy followed by ASCT between 1992 and 2004. Most patients received anti-GD2 antibody 3F8 and 13-cis-retinoic acid following ASCT. HLA and KIR genotyping was performed. Patients were segregated according to those with or without HLA class I ligand for autologous inhibitory KIR. We examined the 3 inhibitory KIR groups with identified class I ligands: KIR2DL2/2DL3, which recognize HLA-CAsn80(HLA-C1 group), KIR2DL1 recognizing HLA-C Lys80(HLA-C2 group), and KIR3DL1 recognizing HLA-Bw4; as well as 6 activating KIR and 2 KIR haplotype groups. Overall survival and progression-free survival were estimated by Kaplan-Meier method and hazard ratios by Cox regression. No adjustments were made for multiple comparisons. Comparisons of each end point were based on the log-rank statistics. Results: 66% of the 155 children lacked at least 1 HLA ligand for his/ her inhibitory KIR. With median followup of 66.8 months, patients lacking a KIR ligand (n=103) had a 45% lower risk of death compared with patients with all HLA ligands present (n=52) (HR 0.55; 95% CI 0.33–0.90; P=0.015). Similarly, for progression-free survival, the risk of relapse or death was 39% lower for patients lacking an HLA ligand for inhibitory KIR (HR 0.61; 95% CI 0.39–0.97; P=0.035). In particular, patients lacking the HLA-C1 ligand for KIR2DL2/2DL3 experienced an overall survival benefit (HR 0.34; 95% CI 0.11–1.09; P=0.060). Activating KIR and KIR haplotypes were not associated with survival. Conclusion: Among children with high risk neuroblastoma undergoing ASCT, improved overall and progression-free survival is associated with the absence of one or more HLA class I ligands for the patient’s NK cell inhibitory KIR receptor. KIRHLA immunogenetics may therefore be a novel genetic indicator of prognosis for patients undergoing ASCT. Mechanistically, these findings imply that NK tolerance is modified after ASCT, and that KIR-HLA genotypes may also play a role in antibodybased immunotherapy, since most of these patients received 3F8 antibody. These findings require confirmation in a larger prospective study. Figure Figure

Abstract Background: Clonal chromosome aberrations in Philadelphia chromosome-negative metaphases (CCA/Ph-) occur in a subset of chronic myeloid leukemia (CML) patients. A shorter survival was reported for "non -Y" CCA/Ph- cases (Issa et al. Blood 2017). Besides -Y, the most frequent CCA/Ph- is +8, but a broad spectrum of other abnormalities can be found, including 7q-/-7, which is a typical aberration of myelodysplastic syndromes (MDS). In previous analyses we had shown an increased number of molecular genetic aberrations in CCA/Ph- compared to non-CCA/Ph- patients (Schnittger et al. ASH 2013, 2014). However, the clinical impact and the evolution of the mutation pattern is largely unknown. Here we extended follow-up and genetic characterization of the initial CCA/Ph- cohort. Aim: To determine the pattern of molecular mutations and their evolution To determine if mutations are part of the Ph+ or Ph- clone Patients and Methods: We included 52 CCA/Ph- patients (female: 25; male 27), with a median age of 58 [33-81] years, and a median BCR-ABL1/ABL1 ratio of 4.322% [0-58.088%] (N.A. for 4 pts.) at the time of initial CCA/Ph- detection. The following CCA/Ph- were present: trisomy 8 (n=26), other trisomies (n=4), -Y (n=7), del(7q)/-7 (n=4), others (n=7), two CCA/Ph- (n=4). We performed sequencing of myeloid gene panels on follow-up samples (1 to 3 per patient) on Illumina's MiSeq and NextSeq instruments (library preparation: 29-gene panel Thunderstorm RainDance [Lexington, MA] or 28-gene panel TruSeq [Illumina, San Diego, CA]). Data was analyzed with SeqNext (JSI Medical Systems, Kippenheim, Germany). Detected mutations were monitored on additional time points to determine variant allele frequency (VAF: mutated/all reads) development. A reference cohort of 47 patients with no sign of CCA/Ph- after MMR achievement was presented as part of our initial study (Schnittger et al. ASH 2014). Results: Cytogenetic monitoring was available over a median period of 31 [0-126] months for the CCA/Ph- and 26 [12-85] months for the reference cohort. Of the CCA/Ph- patients, 5/52 (10%) acquired additional typical aberrations as CCA/Ph- clone (incl. one -7), while in the reference cohort only one of 47 (2%) patients developed a -Y clone (n.s.). On the molecular level, we conducted a median follow-up of 72 [9-150] months for the CCA/Ph- cohort (mutations and BCR-ABL1/ABL1 ratio). Following the CCA/Ph- detection, somatic mutations were found in 30/52 (58%) patients (up to 4 per patient): ASXL1 (n=13), DNMT3A (n=10), TET2 (n=6), NRAS (n=3), RUNX1 (n=3), non-recurrent (n=8). The VAF of 7 mutations was strongly correlated to the BCR-ABL1 ratio and thus most likely present in the Ph+ clone. Mutations in ASXL1 were present in the Ph+ clone in five patients, of whom four never reached MMR, while 6/8 patients with ASXL1 mutations in Ph-independent clones achieved MMR under first- or second-line TKI therapy. Molecular genetic aberrations in Ph- cells were found in 23/52 (42%) CCA/Ph- patients, but only in 2 of 47 (4%) cases of the non-CCA/Ph- cohort (p<0.001). Importantly, in the reference cohort only TET2 and DNMT3A mutations were identified, which is the typical pattern in age related clonal hematopoiesis (ARCH) and also found in older individuals without a hematological malignancy. In the CCA/Ph- cohort, eight of 23 (35%) showed only TET2 or DNMT3A mutations. However, a highly predictive mutation signature for development of a myeloid malignancy (according to Malcovati et al. Blood 2017) was found in 8/23 (35%). In addition, of three NRAS positive CCA/Ph- cases, one was diagnosed with MDS/MPN overlap (also CALR positive) and two developed s-AML during the follow-up period and the NRAS VAF had increased to ≥35% at our last monitoring time point. Conclusions: ASXL1 mutations can occur in the Ph- as well as in the Ph+ clone and are associated with a poor TKI response, if present in the Ph+ clone. Molecular mutations are significantly more frequent in CCA/Ph- than in non-CCA/Ph-. While the mutation pattern in non-CCA/Ph- resembles ARCH, the spectrum in CCA/Ph- includes a higher risk pattern for development of a myeloid malignancy. Further prospective studies are required to evaluate the clinical impact of mutations acquired during the course of CML in order to determine which mutation pattern is related to a higher incidence of secondary myeloid malignancies. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract Undeniably, sepsis is still a profoundly damaging and life-threatening condition for many individuals. With multiple changes in sepsis patients it is difficult to precisely classify an individual’s response in sepsis as proinflammatory or immunosuppressed. The aim of this study was to investigate genetically determined predisposition to developed sepsis by analysis of distribution of human leukocyte antigen (HLA) class II genes. Samples from patients with sepsis were collected at Pauls Stradiņš Clinical University Hospital, Latvia, in an intensive care unit between October 2016 and May 2017. The study group included 62 patients with sepsis, who were genotyped for HLA-DR; DQ using real time polymerase chain reaction – sequence specific primer (RT PCR-SSP). As a control group, data of 100 individuals were taken from the genetic bank of RSU Joint Laboratory of Clinical Immunology and Immunogenetics. The summarised results showed that the frequency of alleles DRB1*04:01 (OR = 5.54; 95% CI = 1.88–16.29); DRB1*07:01 (OR = 19.03; 95% CI = 2/37–152.82); DQA1*05:01 (OR = 14.17; 95% CI = 5.67–35.4); and DQB1*02:01 (OR = 50.00; 95% CI = 2.90–861.81) were significantly increased in patients with sepsis compared to the control group patients. The frequency of DRB1*16:01 (OR = 0.17, 95% CI = 0.04–0.59); DRB1*17:01 (OR = 0.04; 95% CI = 0.00–0.69); DQA1*01:01 (OR = 0.04; 95% CI = 0.00–0.31); DQA1*01:02 (OR = 0.03; 95% CI = 0.00–0.23); DQB1*02:02 (OR = 0.12; 95% CI = 0.03–0.42) alleles was lower in sepsis patients than in control subjects. The most frequent HLA-DRB1/DQA1/DQB1 haplotypes that was significantly increased in patients with sepsis were: DRB1*01:01/DQA1*05:01/DQB1*03:01 (OR = 12.6; 95% CI = 1.51–105.0; p < 0.003). Sepsis patients with pneumonia and alleles and DRB1 04:01; 07:01, DQB1 02:01 had the highest mortality rate. Undoubtedly, our preliminary data showed that development of sepsis can be associated with alleles and haplotypes of HLA class II genes. For more precise conclusion the research should be continued to include a larger patient group.

BackgroundDespite the recent progress in our understanding of the genetic predisposition to systemic lupus erythematosus (SLE) its clinical and functional significance is not fully clarified yet and needs to be implemented in personalized care [1].ObjectivesTo estimate the association between some single nucleotide polymorphisms (SNPs) of 9 non-HLA genes-candidates as STAT4 rs7574865, PTPN2 rs2542151, PTPN22 rs2476601, AGER rs1035798, TRAF1/C5 rs3761847, SLC7A11 rs13128867, RUNX1 rs9979383, IL6 rs1800795, IL6R rs2228145, IL6R rs4845618 and susceptibility to SLE in Belarusian women for the following predictive model development.MethodsWe examined 316 women: among them 59 SLE patients (mean age 39.84, CI95% 36.62-43.06) classified according to the 1997 American College of Rheumatology (ACR) revised classification criteria and 257 age-matched healthy controls (blood donors, mean age 38.12, 95% confidence interval (CI95%) 36.77-39.46). Deoxyribonucleic acid was extracted from peripheral blood samples by phenol-chloroform method. Genotyping was performed by real-time PCR with fluorescent probes. Descriptive analysis, test for Hardy–Weinberg equilibrium, multiple inheritance models (co-dominant, dominant, recessive, over-dominant and log-additive) for single SNPs, Akaike information criteria (AIC) and Bayesian information criteria (BIC) were analyzed using SNPStats web tool [2]. Pearson χ 2 (χ 2), two-way Fisher exact test (F, p 2-t), odds ratio (OR), likelihood ratio of positive (LR +) and negative (LR –) tests with corresponding CI95% were also calculated.ResultsExact test for all genotype frequencies distribution of all studied SNPs didn’t reveal significant differences with Hardy-Weinberg equilibrium in all controls and SLE groups. We noted significant increase of minor ТТ genotype frequency of STAT4 rs7574865 in SLE vs healthy women with recessive inheritance model as the best-fitting one according to its less AIC and BIC values (OR=3.78 (CI95% 1.35-10.62); p=0.016; LR + =3.45 (CI95% 1.37-8.60); LR – =0.91 (CI95% 0.84-0.98)). We revealed protection of minor A allele of AGER rs1035798 carriership with log-additive model of inheritance as the best-fitting one according to AIC and BIC values against SLE development in women (OR=0.52 (CI95% 0.33-0.83); p=0.004; LR + =0.70 (CI95% 0.50-0.93); LR – =1.47 (CI95% 1.10-1.86)). We also noted significant increase of minor allele G frequency of TRAF1/C5 rs3761847 in SLE vs healthy women with dominant inheritance model (OR=3.61 (CI95% 1.05-12.38); p=0.019; LR + =1.30 (CI95% 1.03-1.43); LR – =0.36 (CI95% 0.12-0.92)) and increase of AA genotype frequency in healthy women (p 2-t =0.041). We revealed that minor allele G of PTPN2 rs2542151 is more frequent in SLE women vs healthy controls and has overdominant model of inheritance (OR=1.98 (CI95% 1.09-3.59); p=0.026; LR + =1.57 (CI95% 1.07-2.20); LR – =0.79 (CI95% 0.62-0.97)).There were no significant differences in genotypes and alleles distribution for PTPN22 rs2476601, RUNX1 rs9979383, SLC7A11 rs13128867 and IL6 rs1800795 in studied population and we noted only non-significant tendency in minor SNP genotypes distribution of IL6R rs4845618 and IL6R rs2228145 between healthy controls and women with SLE.ConclusionOur data suggest the susceptibility to SLE in women with ТТ genotype of STAT4 rs7574865 polymorphism and allele G carriers of both TRAF1/C5 rs3761847 and PTPN2 rs2542151 as well as protective role of AGER rs1035798 A allele carriership againt SLE development in women of Belarusian population.References[1]Ghodke-Puranik Y, Niewold TB. Immunogenetics of systemic lupus erythematosus: A comprehensive review. J Autoimmun. 2015;64:125-36.[2]Solé X, Guinó E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics 2006;22(15):1928–29.AcknowledgementsThis research was supported by The Research Technical Program “DNA Identification” (2017–2021), project number: 6.4Disclosure of InterestsNone declared

Abstract Background: Genome instability is a hallmark of cancer. Mutations in DNA repair pathway genes are frequent in a number of solid tumors. Defects in DNA repair or damage response can weaken response to conventional chemotherapy and are frequently regarded as poor prognostic markers. However, a high tumor mutation burden (TMB, number of somatic mutations per mega base) was recently found to correlate with better response to immune checkpoint inhibitors e.g. in colon cancer. Patients with defects in the DNA mismatch repair (MMR) pathway in solid tumors are among the cases with the highest TMB. Hematological malignancies are generally expected on the lower end of the TMB spectrum. We used whole genome sequencing (WGS) for 3256 patients with hematological malignancies (lymphatic and myeloid) to determine factors of genetic instability across all entities. Aim: To determine the number of known mutations in genes from the DNA repair pathway To estimate TMB using WGS and identify cases with high TMB in hematologic malignancies Methods: We investigated a cohort of 3256 patients with hematological malignancies, who were analyzed according to WHO diagnostic gold standards for routine purposes (incl. 584 acute myeloid leukemia [AML] and 635 myelodysplastic syndromes [MDS] samples). We performed amplification-free library preparation and sequencing on HiseqX and NovaSeq 6000 with a median coverage of 106x. Mapping and variant calling was performed with standard pipelines via BaseSpace (all Illumina, San Diego, CA). A pool of gender-matched genomic DNA (Promega, Madison, WI) was used for a tumor-unmatched normal variant calling. (a) In detail we evaluated 180 genes involved in DNA repair. We filtered on (likely) pathogenic variants from ClinVar and for TP53 on protein-truncating variants and (likely, possibly) pathogenic variants from the IARC database. (b) For TBM calculation we determined protein-altering changes and then subtracted all gnomAD listed variants in order to eliminate most germline variants. Results: We found 479 of 3256 (15%) patients with at least one pathogenic variant according to current database annotations in DNA repair or damage response genes. Most pathogenic variants were found in TP53 (330/3256; 10%) and ATM (25/3256, 1%), however, this is probably the effect of the already available systematic database annotation for both genes. For routine diagnostic purposes TP53 mutation status had been analyzed for 1184 patients with Sanger sequencing (7%) or amplicon next-generation sequencing (93%). A 98% and 99% concordance of the pathogenic and non-pathogenic TP53 status was found in comparison to WGS. Mutations in genes from the DNA double-strand break repair (and/or homologous DNA pairing and strand exchange) pathway were found in 93 patients (3%). Pathogenic and potentially germline MMR gene mutations were found in only 3 patients (0.1%, 2 MLH1, 1 MSH6), which equals the expected frequency in the Western population (0.05-0.3%). Next, we calculated TMB. The average was 2.4 [range: 0.4-39.2]. Only samples above the 95th percentile were defined as "TMBhi" (TMB ≥5). TMB was lowest in chronic myeloid leukemia (CML) and essential thrombocythemia (ET) (< 2) and no ET or CML patient was found among the TMBhi. We then focused on MDS, which is our largest subcohort: 56 of 635 (9%) patients were in TMBhi. Furthermore, among MDS patients, a significantly higher TMB was found in MDS-EB-2 (average 3.3 vs. 2.3 for non EB-2, p<.001) and a significantly lower TMB in MDS with isolated del(5q) (1.7 vs. 2.6 in all other MDS, p<.001). There was no association of high TMB and annotated DNA repair gene mutations. The three MMR deficient patients had an average TMB (1.8-2.2) in the hematologic sample. However, TMB was positively correlated with age (Spearman's rank correlation, p<.001) and average TMB was lower in patients under 60 years (2.3 vs. 2.5, p=0.007, t-test). Conclusions: High TMB is rare in hematological neoplasms, and our data across all entities suggest that the acquisition of mutations over age should be a major contributing factor. While TP53 and a few other factors are well studied, using genome-wide datasets in the near future will allow to deeply understand the broad patterns or signatures of genome stability (incl. copy number or structural variants) and their prognostic or predictive value. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract Advanced tumor-stage mycosis fungoides (MF) and Sézary syndrome (SS) are cutaneous T-cell lymphomas (CTCL) characterized by very poor prognosis. Allogeneic haematopoietic stem cell transplantation (allo-SCT) represents an experimental treatment which has been shown to be very effective in achieving long lasting complete remission, possibly leading to cure in selected patients. However, high transplant-related mortality (TRM) limit its feasibility in the vast majority of patients with CTCL. Reduced-intensity conditioning regimens have been demonstrated to decrease TRM, allowing gradual establishment of full donor chimerism and possible graft-versus-lymphoma effect. In this setting, evaluation of minimal residual disease (MRD) is particularly useful to guide post-transplant strategies such as donor lymphocyte infusion (DLI). Detection of TCRgamma-chain gene rearrangements, owing to the relatively limited complexity of its genetic elements, is routinely used to detect MRD in T-cell malignancies. Nonetheless, due to the extensive combinatorial repertoire and the large hypervariable regions, TCRbeta represents the best target for MRD monitoring, allowing more sensitive detection of patient-specific rearrangements. With this study, we aimed to identify patient-specific TCRbeta rearrangements to monitor MRD in patients enrolled in a clinical phase II trial of reduced intensity allo-SCT for advanced stage refractory MF/SS. Skin biopsy and peripheral blood samples at diagnosis and at different time points after transplant were obtained from 6 out of 9 evaluable patients, all having achieved clinical complete remission (still enduring in 5). The BIOMED-2 multiplex TCRbeta PCR heteroduplex assay (InVivoScribe Technologies, USA) was used for identification of monoclonal TCRbeta rearrangements, clonal PCR products were directly sequenced in both directions using the complete set of V or J primers, and V, D, J segments identified using the ImMunoGeneTics database (http://imgt.cines.fr). Then, clonespecific PCR assays were performed on samples collected from every single patient and specificity tested by parallel amplification of normal polyclonal DNA samples. In all patients a monoclonal TCRbeta rearrangement has been sequenced allowing to obtain clone-specific primers for PCR assays. In 2 patients we identified the presence of a MRD at the early controls after transplant (+2 and +3 months, respectively) when both were polyclonal by standard TCRgamma rearrangement; clone-specific PCR assays for TCRbeta became negative afterwards, in concomitance with the achievement of full donor chimerism. In 3 patients polyclonality of TCRbeta was observed in all post-transplant controls. One developed chronic cutaneous GvHD and skin biopsies verified the absence of clone-specific T-lymphocytes. In another patient, all post-transplant assays performed by the standard TCRgamma-rearrangement were persistently positive, suggesting the presence of a non disease-specific monoclonality. The last of the 6 patients relapsed 52 months after transplant. Retrospective clone-specific analysis of TCRbeta rearrangements in DNA from skin biopsies unveiled the presence of molecular relapse already 24 months before, whereas standard TCRgamma assays became positive only after clinical relapse. Altogether, we observed good stability of monoclonal TCRbeta rearrangements over time. Our results suggest that the analysis of TCRbeta is more sensitive and more specific than the analysis of standard TCRgamma for detection of MRD in CTCLs, allowing earlier identification of relapses and adopatiention of pre-empatientive treatment such as DLI. With this method, we also demonstrated disappearance of MRD in concomitance with the achievement of full donor chimerism after allo-SCT. Finally, disease clone-specific TCRbeta rearrangement detection might be helpful in distinguishing cutaneous manifestations of acute GvHD from early relapse of MF. TCRbeta analyses of samples from the remaining 3 evaluable patients are ongoing and will be included in the final presentation.

Abstract Abstract 3051 INTRODUCTION The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+, Tregs) which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation (allo-SCT), Tregs are known to mitigate graft versus host disease (GVHD) while maintaining a graft versus leukemia effect (GVL). Allele (GT)15 for the functional (GT)n polymorphism in the promoter/enhancer of the FOXP3 gene is associated with a higher expression of FOXP3 and production of a greater amount of Tregs. However, its impact in the allo-SCT setting has not been analyzed. OBJECTIVE To analyze the impact of the (GT)n polymorphism in the promoter/enhancer of the FOXP3 gene on the development of complications and ultimately on the success of conventional HLA-identical allo-SCT. MATERIALS AND METHODS The study includes 33 patients with hematological malignancies, treated with myeloablative HLA-identical peripheral blood allo-SCT (Table 1). Diagnosis, classification and grading of GVHD were made by clinical criteria and confirmed when necessary by pathological examination of histological samples from gut, skin, liver or lung, according to international consensus criteria. Donor and recipient genomic DNA was purified from EDTA anticoagulated peripheral blood before allo-SCT and using QIAamp Blood DNA extraction kit (Qiagen). Genotyping of the (GT)n microsatellite polymorphism in the FOXP3 gene was performed by a fluorescence-based short tandem repeat-polymerase chain reaction (STR-PCR) method (GeneAmp 7900; Applied Biosystems) and sized by capillary electrophoresis (POP7 - ABI PRISM 3130 xL Genetic Analyzer; Applied Biosystems) followed by fragment analysis (GeneMapper 4.0 Software; Applied Biosystems) as previously described [Bassuny WM, et al. Immunogenetics. 2003;55 :149–56]. RESULTS The median follow-up time for the cohort was 34 months (range 9.5–110). Allelic frequencies observed were similar to those previously reported (50.5% (GT)15, 41% (GT)16 and 7% (GT)17; no (GT)14 or (GT)18 alleles were found). Patients transplanted from donors harboring allele (GT)15 showed a lower incidence of grades II-IV acute GVHD (29% vs 67%; p =0.049). These patients also showed a trend to a lower incidence of severe (grades III-IV) GVHD (12% vs 33%; p =0.167) as well as chronic GVHD (75% vs 100%; p =0.143; Table 1, Figure 1). No statistically differences were found between patients transplanted from (GT)15 and non-(GT)15 donors in terms of relapse rate (38% vs 33%; p =0.825; Table 1) or cumulative incidence of relapse (CIR at 2 years 35.3% vs 37.5%, Figure 2). Finally, survival analysis did not show statistically significant differences between the two groups of patients in terms of median event (relapse) free survival (EFS, 15.6 months vs 4.5 months, p =0.686) or overall survival (OS, 29 months vs not reached, p =0.610). CONCLUSIONS Tregs are known to modulate the allotolerance-alloreactivity balance between donor and recipient in the allo-SCT setting, mitigating GVHD while preserving the anti-tumor effect (GVL) of the donor graft. In the present study, the presence of allele (GT)15 in the donor, which promotes a higher expression of FOXP3 and greater amount of Tregs, affected allo-SCT outcome by decreasing grades II-IV acute GVHD and chronic GVHD, without affecting GVL (no differences in CIR and OS). Analysis of this polymorphism can help in appropriate donor selection and, more importantly, drive a tailored management of patients submitted to allo-SCT. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1382 Introduction: Infections remain a significant challenge in treatment of childhood leukemia. Inherited genetic variants may influence the immune-inflammatory response in patients with compromised immune functions and may involve multiple signalling molecules reflecting the interplay of multiple genes. Using an extended candidate gene approach including pathway-analysis and protein-protein interactions we expanded our earlier study on mannose-binding lectin (MBL) polymorphisms to include 30 000 target SNPs within 900 candidate genes with possible relevance for childhood ALL. The candidate genes covered the following domains: i) pharmacogenetics, ii) immunogenetics, iii) apoptosis, iv) neurobiology, v) toxicity, vi) thrombosis, vii) cell cycle control genes, and viii) DNA repair and mitosis. We explored differences in host genomic patterns between patients with and without an infectious event during induction therapy. Material and methods: Included in our study were 72 patients aged 1.0 to 14.9 years diagnosed and treated for non-B ALL at the University Hospital Rigshospitalet, Copenhagen, Denmark, from January 1, 1992, to December 31, 2000. The 7 weeks induction period (NOPHO-ALL 92 protocol) included oral prednisone 60mg/m2/d day 1–36/45, weekly i.v. vincristine 2mg/m2 (6 doses), i.v. doxorubicin 40mg/m2 (3 doses; 4 doses for the high risk groups), l-asparaginase 30 000IE/m2 daily day 36–45, and age adjusted i.t. methotrexate (4 doses). Main endpoints in our analysis were “infectious event” and “infectious event with positive blood culture”. Additional covariates included age, sex, risk group, immunophenotype, white blood cell count, and microbiological data. DNA library preparation was performed according to the SureSelect Target Enrichment System. Illumina GAIIx Genome Analyzer was used for sequencing. Associations were analysed by Fisher's exact test implemented in PLINK. Only SNPs with observed minor allele frequency (MAF) above 5% and at least 50% of non-missing genotypes above 10 times the sequencing depth were used in the analyses. The obtained p-values were adjusted for multiple testing by means of adaptive permutation. The empirical p-values were generated by 1 million permutations of the test and were then corrected using false discovery rate (FDR) for multiple testing. A set of high confidence protein-protein complexes was found. For association of protein-protein complexes similar methods were applied requiring at least 2 SNPs with observed MAF above 1% in at least 2 different genes of the complex. CART analyses were performed using R package applying 3-fold cross-validation using the genotypes of the SNPs associated with risk of infection and positive culture together with the clinical data. Only the SNPs with permutation corrected P-values below 0.01 were used in the analysis. Results: Of the 69 patients with appropriate DNA quantity, 48 (70%) patients experienced an infectious event. Of these, 23 (33%) patients had at least 1 positive blood culture during an infectious event which, among others, included 4 Pseudomonas, 4 Escherichia coli and 3 Candida infections. After correcting for multiple testing by means of adaptive permutation, a total of 103 and 94 SNPs were found to be associated with infectious event and positive culture, respectively (p<0.05). The QQ plots for the single SNP association analysis followed the null distribution and showed a few SNPs above the expected distributions, suggesting true biological signals. Manhattan plots for both analyses indicate that several loci were associated to both risk of having an infection and positive culture. CART analysis demonstrated rs12666401, rs2275287, rs10841795 and rs1136410 to be highly predictive of outcome characterizing 42 of 48 (88%) infectious event patients with > 89% accuracy. Discussion/conclusion: Our data indicate that host genomic profiles can predict the risk of infectious events during induction therapy in children with ALL. Such knowledge might be helpful in adapting more individualised supportive care and treatment protocols. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction: Factor (F)VIII immunogenicity is the main obstacle to both successfully treating hemophilia and receiving FDA approval for new therapeutics. Since immune responses to allogeneically distinct proteins require T-cell activation and proliferation, at least one foreign peptide must be liberated from FVIII that can bind with high affinity to one or more of a patient's HLA-class-II (HLA-II) isomers. Due to the complex pathogenesis of inhibitor development, which involves numerous genetic variations and both treatment and product related variables, our ability to predict which patients will become alloimmunized to FVIII remains suboptimal. The role of FVIII glycosylation in inhibitor development appears to be an important, yet not well characterized, modifying influence; defining the mechanism has been complicated by the fact that some FVIII products are highly glycosylated with thousands of potential glycoforms. Here we describe studies of the spectrum of FVIII peptides eluted from HLA-II complexes and the impact of glycosylation on the proteolysis and/or binding of these peptides to a patient's individual HLA-II isomers, i.e. to further incorporate personalized medicine in hemophilia care. Methods: Immature monocyte-derived dendritic cells (DCs) from 12 unrelated donors were generated in vitro and matured in the presence of an equimolar pool of a full length (FL) and 4 B-domain deleted (BDD) rFVIII proteins free of VWF and other proteins. Following harvest and lysis, HLA-DP, -DQ, and -DR molecules were recovered using a specific immunoaffinity step. Peptides were then eluted from these complexes and sequenced by high resolution mass spectrometry (LC/MS/MS). The bound peptides were mapped to the FVIII reference sequence. The estimated binding affinity of FVIII peptides to the HLA-II alleles found in these donors were obtained from NetMHCIIpan and compared to the peptides observed to be bound. Results: The 12 DC donors express 29 distinct HLA-II isomers (6 DP, 11 DQ, 12 DR) from an overall HLA-II gene repertoire having the following alleles: DP: 01:03/02:01, 01:03/03:01, 01:03/04:01, 01:03/04:02, 02:01/01:01, 02:02/05:01 DQ: 01:01/05:03, 01:02/06:02, 01:02/06:09, 01:03/05:01, 01:03/06:03, 01:03/06:04, 02:01/02:01, 02:01/02:02, 03:01/03:02, 03:02/03:03, 05:01/03:01 DR: 01:01, 03:01, 04:01, 04:04, 07:01, 09:01, 11:01, 11:04, 13:01, 13:02, 14:01, 15:01 These 29 HLA-II isomers were found to have 77 bound peptides that covered 1,202 of the 2,332 total amino acid residues in the NCBI reference FL-FVIII protein, whose domain structure and consensus N-linked glycosylation (NLG) sites are shown in panel A of the Figure. The HLA-II bound peptides were mapped to the FVIII regions from which they derive (panel B). One of the 20 NLG sites known to be glycosylated and 3 of the 4 known non-glycosylated NLG sites were found to be in a bound peptide. A NetMHCIIpan analysis predicted that no FVIII nonamer containing a consensus NLG site binds strongly to any of the 12 DR isomers; but, it also clearly showed that more peptides bind with weak or strong affinity as peptide length increases (data not shown). Conclusion: Despite finding 3 of FVIII's 4 non-glycosylated consensus NLG sites in the tightly bound HLA-II/peptide complexes, only 1 of the 20 glycosylated NLG sites was found among these bound peptides. The near complete absence of N-linked glycans in the presented HLA-II bound peptides is compelling, but incomplete, evidence that glycosylation influences the immunogenicity of FVIII. Potential mechanisms may include effects on internalization, proteolysis and/or HLA-II binding. Further direct studies are required to determine if these post-translational modifications affect proteolysis and/or HLA-II binding; core peptides that can be proteolyzed and/or bound in the absence of glycosylation or with alternative glycan conformations may provide more evidence that glycosylation can modulate immunogenicity. We hypothesize that HLA-II bound and unbound FVIII peptides constitute a novel immune-response-related biomarker that will improve the accuracy of inhibitor risk prediction by allowing pertinent patient-specific pharmacogenomic inputs to be analyzed in a more biologically relevant manner. References: Karosiene et al. NetMHCIIpan-3.0, a pan-specific MHC-II prediction method including all 3 human MHC-II isotypes: HLA-DR, -DP and -DQ. Immunogenetics, 2013. Disclosures Hofmann: CSL Behring: Employment. Zollner:CSL Behring: Employment. Powell:CSL Behring: Employment. Maraskovsky:CSL Behring: Employment. Howard:Baxter: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Haplomics, Inc.: Patents & Royalties; CSL Behring: Consultancy, Honoraria, Research Funding.

Abstract Most CLL is diagnosed with a low tumor burden with no indication for therapy. Biomarkers, such as unmutated IGHV genes, TP53 loss/mutation and raised β2M predict short time to first treatment and overall survival; however there remain patients with good risk biomarkers who nevertheless develop progressive disease. Advances in genomics and immunogenetics have lead to the discovery of new biomarkers and their integration with cytogenetic data refines outcome prediction. However these novel markers are predominantly found in IGHV unmutated cases (U-CLL). To identify novel genetic mechanisms that may contribute to progression, we have studied 13 patients (pts) presenting with cMBL (n=3) or Stage Binet A/Rai 0 disease and good risk markers: IGHV-mutated, excluding poor risk stereotypes (n=13), no 17p or 11q deletion by FISH, (n=13), sole del13q14 (n=8), low CD38 expression (n=13) who all developed lymphocytosis (n=13), between two untreated timepoints (TP1 & TP2), 10 of whom subsequently required treatment. Copy number analysis (SNP6), whole exome sequencing (WES; Agilent SureSelect & Illumina sequencing) of tumour-germline pairs and targeted deep sequencing (TDS; Haloplex, Agilent) of the the WES-identified variants and the 22 most frequently mutated genes in CLL, to a mean depth of 3681 fold, were performed at TP1 and TP2. TP1 was close to diagnosis (median of 1 yr, range 0.11-7.33) with a median time to TP2 of 4.5 yrs (0.2-8.9). In addition, TDS was undertaken at later time points in one patient described in point 4), who relapsed and ultimately transformed. Our analysis shows the following potential mechanisms: 1. Our germline WES data revealed 5 heterozygous missense/frameshift variants in 5 genes in 5 pts, also known to be targeted by somatic mutation in CLL (eg: FBXW7, POT1, SAMHD1. Fig1). 2. We then established the somatically-acquired mutation profile of each patient. We validated 72% (224/312) of the mutations discovered by WES using TDS and identified clinically relevant mutations earlier on in disease, supporting the hypothesis that sub-clonal mutations in genes in addition to TP53 may drive a progressive clinical course. At diagnosis (TP1) by WES/TDS, 5/13 pts had mutations in CLL driver genes (ATM, NOTCH1, SF3B1, TP53) and 2/13 pts had mutations in genes of undetermined clinical significance (CHD2, NFKBIE, ZMYM3). One patient was MYD88 mutated at TP1 and remains untreated after follow up of 12 yrs. In total, the following 9 genes (ATM, CHD2, DDX3X, MYD88, NOTCH1, NFKBIE, SF3B1, TP53 & ZMYM3) were mutated in 62% (8/13) pts at TP1. 3. Of the remaining 5/13 pts lacking a detectable mutation in any of the established CLL genes, we observed on average 7 mutations/patient in genes involved in cancer and each patient harboured one or more mutated genes with a role in haematological malignancy (eg. ITGA6, KLHL6, LTF, TNFAIP3). 4. One patient exhibited a remarkable temporal shift in copy number changes and mutations. At TP2, SNP6 analysis could not detect the del13q observed at TP1, and a clonal trisomy 12 had emerged, along with several mutations associated with progressive disease (BIRC3, IRF4, NOTCH1), that predominate in U-CLL. As a consequence we re-analysed the IGHV mutational status at TP2, and showed that rather than the IGHV3-48 with 92% germline identity identified at diagnosis, our patient exhibited an additional and dominant IGHV5-10-1*01 (100% identity) clone at TP2, 8 yrs after TP1. Additional analysis of intermediate samples detected the unmutated clone as far back as 4 yrs post diagnosis, and TDS analysis showed the NOTCH1 mutation was a minor subclone at diagnosis (0.06% VAF). Ultimately, this patient developed Richters syndrome with expansion of the NOTCH1 mutation (27% VAF). Retrospective sequential immunogenetic analysis of the other 12 cases yielded no other example of this phenomenon. In summary, IGHV-mutated cMBL/early stage CLL with a progressive outcome can be associated with, the presence of germline or subclonal gene mutations of known or putative importance in CLL, or the emergence of a IGHV-unmutated clone. Our data supports deep sequencing in the clinical setting for earlier detection of pathogenetic mutations and emerging immunogenetically distinct subclones in patients with early stage 'good risk' disease. Figure 1: Heatmap representation of the cohorts clinical features and DNA mutation. Patient 287 haboured the IGHV-unmutated clone at TP2-5. Figure 1:. Heatmap representation of the cohorts clinical features and DNA mutation. Patient 287 haboured the IGHV-unmutated clone at TP2-5. Disclosures No relevant conflicts of interest to declare.

Abstract Private germline mutations affecting hematopoiesis can cause progressive myelodysplasia and thus constitute pre-leukemic states. These can remain undetected, progressively transform and reveal themselves as infant leukemias, which can be linked to translocations involving the mixed lineage-leukemia (MLL) gene. Septin proteins play key roles in mammalian cell division and cytokinesis and are found as fusion partners of MLL in infant and early childhood acute myeloid leukemia (AML). We identified and describe a human germline disorder of septins in a newborn with myelodysplasia who required early hematopoietic stem cell transplant (HSCT) to prevent progressive disease. Materials & Methods: A Caucasian newborn male with no birth defects/malformations or suspicious family history presented with severe progressive neutropenia and was found to have bone marrow (BM) dysplasia with tetraploidy of myeloid progenitors. The patient developed unfavorable clonal aberrations (trisomy 7,8,9) and increased tetraploidy. Due to his progressive cytopenias and concern about risk of leukemic transformation, he underwent an allogeneic busulfan-cyclophosphamide/ATG conditioned DQ-mismatched unrelated HSCT at age 1 yo. He is currently 8 years post-HSCT with normal trilineage hematopoiesis (full donor chimerism), no graft versus host disease or any other non-hematological phenotype. To investigate the genetic etiology of this unique phenotype, we performed family trio germline exome/whole genome next-generation sequencing (NGS), and somatic pre-HSCT BM NGS for the index case. An established algorithm filtered for significant candidates following a de novo germline model. Immunohistochemical (IHC) staining of the pre- and post-HSCT BM biopsies for the candidate protein was performed. To validate the germline origin of the candidate mutation, we generated patient and control skin fibroblasts and induced pluripotent stem cells (iPSCs) that underwent fidelity testing by murine injection teratoma assays and 16-marker immunofluorescence (IF) staining. We then studied hematopoietic progenitor cells (HPCs) derived from iPSC-embryoid bodies (EB) in methylcellulose assays. To further determine the pathogenic nature of the mutation, we generated CRISPR/Cas9 knock-out of the human erythroleukemic cell line (TF-1) and studies these cells by cytomorphology, DNA and cell cycle assays. In-silico protein analysis of the candidate mutation and its effects on septin filament formation was performed. Results: Family trio and disease-tissue NGS identified a novel, germline C-terminal mutation in SEPT6, which was acquired de novo in the patient, and was not found in any database of common polymorphisms. IHC of pre-HSCT patient BM showed reduced Septin-6 staining in megakaryocyte and granulocyte precursors compared to post-HSCT and controls. Patient-derived iPSCs carried the mutation, were cytogenetically normal and bona-fide pluripotent by IF and teratoma assays. EB-derived HPCs from these cells recapitulated the patient's phenotype as they differentially failed to produce granulocyte vs erythroid colonies (fold-reductions CFU-M:8, CFU-G:36, CFU-GM:46, BFU-E:6, see Figure). Despite multiple approaches, SEPT6 CRISPR/Cas9 knock-out/in of the patient's mutation was not tolerated in iPSCs and human myeloid (granulo-/myelocytic) cell lines (HL-60, Molm-13, K562), and only tolerated in erythroid TF-1 cells. Analysis of SEPT6 knock-out TF-1 single-clone lines revealed a propensity to poly-nuclearity/lobation, as observed in the patient's BM. SEPT6 knock-in of C-terminal mutations caused cell death, consistent with existing literature. In silico protein analysis (incl. previously published crystallographic data) suggests that the mutation a) most likely modulates the key role of the coiled-coil SEPT6 domain in septin filament stabilization/bundling/bending, and thus deleteriously impacts cytokinesis, and b) perturbs the equilibrium of splice variants, possibly conferring tissue specificity. Conclusions: Mutation of the C-terminus of human SEPT6 causes aberrant cytokinesis in HPCs leading to a severe congenital neutropenia with tetraploidy and progressive myelodysplasia and cytogenetic aberrations. This report implicates a human germline disorder of SEPT6, and further investigations are required to elucidate the role septins in normal and disordered myelopoiesis. Figure. Figure. Disclosures Williams: Bluebird Bio: Research Funding.

Background: In cancer evolution, development of telomere maintenance mechanisms represents a critical feature to achieve replicative immortality and prevent cell death after shortening below a critical threshold. Several studies have indicated that excessive telomere shortening or aberrant telomeric repeats are present in cancer genomes, however, their precise role in myeloid malignancies remains largely unexplored. We stipulated that immature leukemia-initiating cells (LIC) would display higher telomerase activity and thus longer telomeres while in mature LIC lower telomerase activity would be associated with shorter telomeres due to excessive division rate. Additional lesions may also lead to disturbed differentiation programs resulting in altered expression of telomerase. Taking advantage of a vast cohort (N=1648) of patients with myeloid neoplasia analyzed by whole genome sequencing (WGS, mean. coverage 100x) we determined telomere length (TL). Aim: We set out to hypothesize that WGS data can be used to reliably measure the TL and that if systematically applied to well defined patient cohorts, we could test whether preservation of TL or its excessive shortening are prognostically or biologically relevant to clinical outcome. Methods: Our cohort included patients with AML (N=732), MDS (N=701) and MPN (N=102 incl. CML, ET, PMF, PV). Healthy subjects (N=11) and patients with the clonal non-malignant HSC disorder PNH (N=102) served as controls. To retrieve TL and telomere repeat heterogeneity via singleton analysis from WGS data we used two independent bioinformatic tools TelomereHunter and Telseq (1A). Results: First, using bidirectional cross-confirmation with independent bioinformatics pipelines, reliability of WGS-based TL measurements (1B) was confirmed and, as anticipated, age-dependency of TL in healthy controls was reproduced (1C). Next we investigated age as a possible confounder of TL shortening in patients. While in PNH age had an expected impact, patients with AML or MDS exhibited TL decoupled from its inherent age-dependence (r=.00034, P=.72). In general, we noted TL shortening as an overarching feature of myeloid proliferation irrespective of entity (1D). Furthermore, mutations in the promotor region of TERT were not correlated with TL. In AML or MDS, neither morphological nor genetic subclassification where associated with differences in TL (1E), with the exception of a small subset of AML and MDS patients (n=103, n=97), harboring abnormally long telomeres compared to healthy subjects. These were significantly associated with mutations in TP53 (AML P=1.91x10-6, mean TL 558.4; MDS P=.55x10-7, mean TL 414.5) but not with any other common mutation. This is surprising, since TP53 mutations are thought to protect cells from telomeric catastrophe following excessive shortening. Moreover, comparing the VAF of TP53mut AML samples with the corresponding measured TL, revealed a positive correlation (r=.2281, P=.021), irrespective of the blast count by cytomorphology (1F). Singleton analysis from all 1648 patients revealed that in AML and MDS patients, alternative telomere repeat- variants (chiefly TTTGGG) were increased pointing towards a possible implication of alternative telomere extension (1G). Lastly, outcome analysis revealed that in AML, the presence of longer telomeres (compared to controls) was associated with worse prognosis (P=.0035) (1H). Multivariate analysis, which included age, karyotype, TP53 status and gender, revealed that this was due to the strong association with TP53 mutations (n=630 vs n=103). The implications of our study extend beyond the realm of telomere biology and leukemia pathophysiology. Therapies targeting the telomere machinery, e.g. imetelstat, may offer an opportunity for application beyond MPN, its current indication. Ongoing studies are currently investigating its use in MDS. Based on our findings we argue that treatment may be rationally evaluated in TP53mut AML. Conclusion: Our findings provide a rationale for (1) the use of WGS to investigate TL, (2) the fundamental discovery that TL shortening is a common principle in myeloid neoplasms, (3) TP53 mutations being accompanied with increased TL and (4) the exploitation of TL to identify subgroups eligible for treatment with imetelstat. Moreover, WGS-based TL analysis in leukemias can contribute to our understanding of TL maintenance mechanisms in leukemic genome. Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

Introduction: The recently developed DLBCL90 NanoString assay robustly distinguishes primary mediastinal large B-cell lymphoma (PMBCL) from diffuse large B-cell lymphoma (DLBCL), as well as cell-of-origin (COO) subtypes of DLBCL (ABC, GCB, unclassified) and cases with a Double-Hit (DHIT) signature (Ennishi D., JCO 2019). When this assay was applied to biopsies from 343 patients with de novo DLBCL uniformly treated with R-CHOP, nineteen of these cases had a molecular PMBCL signature (mPMBCL), despite the fact that they were diagnosed as DLBCL based on their morphology, immunophenotype and clinical features. Here, we aimed to comprehensively characterize the molecular and clinicopathologic features of these mPMBCL cases. Methods: Survival estimates were calculated using Kaplan-Meier analysis, using time to progression (TTP) and disease specific survival (DSS) as endpoints. We applied whole-exome sequencing, copy number analysis (SNP6.0) and RNAseq to identify somatic mutations, copy number aberrations and differentially expressed genes, respectively. FISH was applied to assess the presence of rearrangements affecting MYC, BCL2 and BCL6. We used data previously obtained within our centre from a PMBCL cohort (n=73) to compare mPMBCL with "bona fide" PMBCL (PMBCL) tumors (Mottok et al, Blood 2019). Results: Median age at diagnosis was significantly higher for mPMBCL compared to PMBCL (62 vs 37 years, p&lt;.001). Only 3 out of 19 mPMBCL presented with an anterior mediastinal mass, a clinical hallmark of PMBCL. Importantly, these cases demonstrated extensive extrathoracic disease, which is unusual for PMBCL. Pleural and/or pericardial effusion was seen in only 11% of mPMBCL compared to 50% of PMBCL (p&lt;.001). Bone marrow involvement was seen in 21% of mPMBCL and not observed in any PMBCL (p&lt;.001). The mPMBCL had a trend toward worse outcomes compared to R-CHOP-treated PMBCL (n=61) (2 year TTP: 62% vs 77%, log-rank p=.12; DSS: 74% vs 84%, p=.10). By COO assignment of the DLBCL90 assay, 16 out of 19 mPMBCL were categorized as GCB-DLBCL. Compared to the other GCB-DLBCL in our DLBCL cohort (excluding DHIT-signature-positive cases), the mPMBCL had inferior outcomes (2-year TTP: 62% vs 87%, log-rank p=.03; DSS: 72% vs 89%, p=.02). There were no significant differences in baseline clinical characteristics between mPMBCL and GCB-DLBCL. Comparison of the mutational landscape of mPMBCL to PMBCL demonstrated perturbations in the central hallmarks of PMBCL pathogenesis: JAK/STAT signaling, NF-ĸB pathway activation and immune evasion. Genomic aberrations affecting JAK-STAT signaling were shared between mPMBCL and PMBCL, with SNVs or indels affecting IL4R, STAT6 and SOCS1 found in 37%, 37%, and 89% of mPMBCL and 36%, 40% and 69% of PMBCL, respectively. Moreover, copy number analysis revealed JAK2 amplifications in 44% of mPMBCL (71% of PMBCL) and differential gene expression analysis showed increased levels of CD274 (PDL1), PDCD1LG2 (PDL2) and genes belonging to the JAK-STAT-signaling network in mPMBCL. In contrast, these genetic aberrations were rarely observed in a recent whole-exome sequencing study on 304 primary DLBCL tumors (Chapuy B., Nat.Med. 2018). Mutations were also observed in NF-ĸB pathways but the patterns of mutations were distinct between mPMBCL (BIRC3 and BTK) and PMBCL (NKBIE) suggesting convergent biology with alternative mechanisms of pathway dysregulation. Similarly, mPMBCL harbored different mutations (CD83) implicated in immune evasion compared with PMBCL (B2M). Finally, we compared the mutational landscape of mPMBCL with recently described genetically-defined subgroups of DLBCL. Interestingly, a large majority of mPMBCL harbored at least one of the mutations characteristic of "Cluster 4" (incl. CD83, HIST1H1E, SGK1), a subset of DLBCLs defined by Chapuy et al that predominantly includes GCB-DLBCLs. Conclusion: We have identified and characterized a subgroup of DLBCL that expresses the PMBCL gene expression signature. Similar to bona fide PMBCL, these tumors are characterized by genomic aberrations that affect JAK-STAT, NF-ĸB signaling and immune response. However, our data suggest that dysregulation of the latter two pathways is established through distinct evolutionary modes that are reflected in differential mutation patterns and anatomical and clinical presentations. Our findings provide potential novel therapeutic avenues for this subset of lymphoma. Disclosures Sarkozy: Takeda: Research Funding. Savage:BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding. Scott:Celgene: Consultancy; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Juno Therapeutics: Consultancy; Tioma: Research Funding; Roche: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy.

BackgroundHyperprogressive disease (HPD) is a distinct pattern of rapid tumor progression observed in patients with cancer who are undergoing immune checkpoint inhibitor therapy. Despite the growing evidence, a universal definition of HPD remains to be established, and incidence rates vary based on the defining criteria. Therefore, a refinement of currently existing criteria is warranted to better characterize this phenomenon and evaluate its incidence.MethodsTwo independent investigators performed a systematic literature search in EMBASE and MEDLINE using keywords selected in Park et al.1: checkpoint, immunotherapy, pd1, pdl1, ctla4, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab and hyperprogress. Studies published from March 3, 2020 to April 20, 2020 that included the incidence and definition of HPD in patients receiving immunotherapy were included for analysis. Selected studies were then combined with those included in the meta-analysis by Park et al.1 Duplicates were removed, and the study with a larger cohort was selected in instances of overlap between two cohorts. In total, 50 studies were included for meta-analysis.2–51 Pooled incidence rates of HPD and prespecified subgroup analyses based on four categories defining HPD (tumor growth rate ratio, tumor growth kinetics ratio, early tumor burden increase, and combination) were obtained with 95% confidence intervals (CI) using a random effects model performed on R.ResultsA total of 6009 patients from 50 studies were included in the meta-analysis. Incidences varied from 0.0% to 43.1% (figure 1), and the overall pooled incidence of HPD was 12.9% (95%CI, 11.1%–14.7%). Significant heterogeneity was observed (I2= 77%; p<0.01). Studies were also grouped into one of 4 categories (table 1) based on the definition of HPD used to calculate the tumor growth acceleration: tumor growth rate ratio (pooled incidence of HPD 10.5%; 95% CI, 7.9%–13.0%), tumor growth kinetics ratio (pooled incidence, 14.8%; 95% CI, 12.0%–17.5%), early tumor burden increase (pooled incidence, 17.2%; 95% CI, 9.7%–24.7%), and combinations of the above (pooled incidence, 12.2%; 95% CI, 9.2%–15.2%).Abstract 238 Table 1Subgroup analyses based on definitions of HPDAbbreviationTGR, tumor growth rate; TGK, tumor growth kinetics.Abstact 238 Figure 1Overall pooled incidence of HPD. The overall pooled incidence of HPD was 12.9% (95% CI, 11.1%–14.7%). Significant heterogeneity was observed (I2 = 77%; p<0.01).ConclusionsThe overall incidence of HPD from 50 studies was 12.9% (95%CI, 11.1%–14.7%). HPD incidence varied from 0% to 43.1% depending on the definition each investigator chose. 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Primary Biliary Cholangitis (PBC) is a rare autoimmune cholangiopathy. Genetic studies have shown that the strongest statistical association with PBC has been mapped in the human leukocyte antigen (HLA) locus, a highly polymorphic area that mostly contribute to the genetic variance of the disease. Furthermore, PBC presents high variability throughout different population groups, which may explain the different geoepidemiology of the disease. A major role in defining HLA genetic contribution has been given by genome-wide association studies (GWAS) studies; more recently, new technologies have been developed to allow a deeper understanding. The study of the altered peptides transcribed by genetic alterations also allowed the development of novel therapeutic strategies in the context of immunotolerance. This review summarizes what is known about the immunogenetics of PBC with a focus on the HLA locus, the different distribution of HLA alleles worldwide, and how HLA modifications are associated with the pathogenesis of PBC. Novel therapeutic strategies are also outlined.

Uveitis is a generic term for inflammation of the uvea, which includes the iris, ciliary body, and choroid. Prevalence of underlying non-infectious uveitis varies by race and region and is a major cause of legal blindness in developed countries. Although the etiology remains unclear, the involvement of both genetic and environmental factors is considered important for the onset of many forms of non-infectious uveitis. Major histocompatibility complex (MHC) genes, which play a major role in human immune response, have been reported to be strongly associated as genetic risk factors in several forms of non-infectious uveitis. Behçet’s disease, acute anterior uveitis (AAU), and chorioretinopathy are strongly correlated with MHC class I-specific alleles. Moreover, sarcoidosis and Vogt-Koyanagi-Harada (VKH) disease are associated with MHC class II-specific alleles. These correlations can help immunogenetically classify the immune pathway involved in each form of non-infectious uveitis. Genetic studies, including recent genome-wide association studies, have identified several susceptibility genes apart from those in the MHC region. These genetic findings help define the common or specific pathogenesis of ocular inflammatory diseases by comparing the susceptibility genes of each form of non-infectious uveitis. Interestingly, genome-wide association of the interleukin (IL)23R region has been identified in many of the major forms of non-infectious uveitis, such as Behçet’s disease, ocular sarcoidosis, VKH disease, and AAU. The interleukin-23 (IL-23) receptor, encoded by IL23R, is expressed on the cell surface of Th17 cells. IL-23 is involved in the homeostasis of Th17 cells and the production of IL-17, which is an inflammatory cytokine, indicating that a Th17 immune response is a common key in the pathogenesis of non-infectious uveitis. Based on the findings from the immunogenetics of non-infectious uveitis, a personalized treatment approach based on the patient’s genetic make-up is expected.

The emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious medical problem worldwide. Acquired OXA-48-like carbapenemases encoded by plasmids are important causes of carbapenem resistance in K. pneumoniae. To explore the links between plasmids and blaOXA-48-like genes in K. pneumoniae, we systematically analyzed the variants of blaOXA-48-like plasmid replicon types, phylogenetic patterns, geographic distribution, conjugative transfer regions, and the genetic environments surrounding blaOXA-48-like of 191 blaOXA-48-like-harboring plasmids, which were identified from 4451 plasmids of K. pneumoniae downloaded from GenBank. Our results showed that seven different variants of blaOXA-48-like genes were identified from the 191 blaOXA-48-like-harboring plasmids in K. pneumoniae, with blaOXA-48, blaOXA-232, and blaOXA-181 being highly prevalent. In K. pneumoniae, blaOXA-48 was mainly carried by the composite transposon Tn1999.2 located on IncL/M-type conjugative plasmids, which were mainly geographically distributed in Switzerland, Germany, and China. In K. pneumoniae, the blaOXA-232 gene was mainly carried by 6.1-kb ColKP3-type mobilizable plasmids, which were mainly isolated in India. In K. pneumoniae, blaOXA-181 was mainly carried by a group of 50-kb ColKP3-IncX3 hybrid conjugative plasmids and a group of small ColKP3-type mobilizable plasmids with lengths of 5.9–9.3 kb, the former was sporadically discovered in China, South Korea, India, and Czech Republic, while the latter was almost all isolated in India. In addition, five blaOXA-245-harboring 65.9-kb IncL plasmids of K. pneumoniae isolated in Spain were found to have the genetic context of blaOXA-245 more complicated than that of blaOXA-48-harboring IncL/M-type plasmids, with two copies of IS1R inserted both upstream and downstream of blaOXA-245-lysR. These findings enhance our understanding of the genetic diversity of blaOXA-48-like-harboring plasmids in K. pneumoniae.

AbstractThe aim of this study was to determine the frequency of carbapenem resistant Klebsiella pneumoniae (CRKP) sequence types (STs) in Iran. Samples were collected from three university hospitals in Sanandaj, Iran, from December 2016 to March 2018. Antibiotic susceptibility testing, phenotypic and genotypic detection of carbapenemases were performed. Common K. pneumoniae capsular types were sought for all isolates. The genetic relatedness of isolates was investigated by multilocus sequence typing (MLST). Plasmids were detected by PCR-based Replicon Typing (PBRT). During the study, 67 K. pneumoniae isolates were identified. Of which, 18 (26.9%) isolates were detected as carbapenem-resistant. The most effective antibacterial agent was tigecycline (97%, 65 isolates) followed by imipenem and ertapenem (73.13%, 49 isolates). PCR showed that 13 isolates (19.4%) had blaNDM-1 gene and 5 (7.5%) harbored blaOXA-48. Examination of common capsular types showed that 2 isolates had K2 and 2 others had K54. REP-PCR revealed 10 clones and 11 singleton strains. MLST analysis of CRKP found ST15 as the most common type (13 isolates, 72.2%), but other STs were also detected namely, ST19, ST117, ST1390, and ST1594. ColE1 and IncL/M plasmids were the carriers of blaNDM-1 and blaOXA-48, respectively. The results showed that CRKP spread in our health centers. Our results, therefore, indicate a worrying trend of resistance to carbapenems in K. pneumoniae.