ALBERT

Description: Key Points Functional reversion of a germline CARD11 mutation in T cells is associated with the development of Omenn syndrome. Defective thymic T-cell development and peripheral lymphopenia are no prerequisite for the development of Omenn syndrome.

Description: BACKGROUND: Complement-mediated thrombotic microangiopathies (TMAs) are a subset of TMAs in which the thrombocytopenia, microangiopathic hemolytic anemia, and end organ damage are caused by mutations in genes encoding the alternative complement pathway. Numerous complement genes have been implicated in complement-mediated TMAs including complement factor H (CFH), CD46 (MCP), complement factor I (CFI), complement component 3 (C3), complement factor B (CFB), factor H related 5 (CFHR5), and thrombomodulin (THBD) among others. Genetic analysis typically yields a mutation in 60% of patients whose TMA is presumed to be mediated by complement dysregulation. However, the description of novel disease-associated variants may increase this proportion. METHODS: A retrospective study of patients with TMAs diagnosed between 2000 and 2014 was performed. TMA diagnosis was made based on thrombocytopenia and evidence of microangiopathic hemolytic anemia. Analysis was performed with Alamut¨ software with additional in silico prediction tools (SIFT, MutationTaster, and Polyphen) for classification of gene variants. Variants of unknown significance (VUS) and likely pathogenic variants were further assessed using several mutation databases, including HGMD, ClinVar, and Factor H database RESULTS: Of patients diagnosed with a TMA, genetic analysis was performed in only a 10% of patients. Of the 29 patients with genetic studies performed, mutations were identified in 18 patients (62%). The majority of the mutations had been described previously in the literature, but four novel variants were identified: three missense and one splice-site. The table below summarizes these variants as well as laboratory findings on presentation. These were two variants of CFH, one variant of CFHR5, and one variant of CFI. In silico modeling of these variants revealed two polymorphisms likely to be pathogenic, one polymorphism likely benign given the lack of predicted splicing changes, and one VUS. Table 1. Protein Mutation Classification Age Sex Hemoglobin (g/dL) Platelets (thousands) Creatinine (mg/dL) CFH c.245-10_245-9dup Likely benign 61 F 11.6 51 6.7 CFH c.476G〉A, p.Ser159Asn Likely pathogenic 43 F 9.1 101 9.1 CFHR5 c.1412G〉A p.Gly471Glu Likely pathogenic 30 F 9.8 129 4.9 CFI c.1190T〉A p.Val397Glu Unknown significance 51 M 9.4 125 6.1 DISCUSSION: With therapy available to target the alternative complement pathway, genetic analysis to identify genetic variants capable of causing complement mediated TMAs is an essential part of the evaluation. This genetic data must be interpreted and correlated with functional analysis and clinical phenotype. The reporting of novel variants in clinical databases, with inclusion of relevant clinical findings, is necessary to accurately classify and verify variants as pathologic mutations or benign polymorphisms. The full understanding of this diverse disease requires a more complete understanding of its genetics. While complement pathway-directed therapies are available, their rational use requires thorough interpretation of laboratory data, including genetic analysis. Disclosures Murray: Mayo Clinic: Patents & Royalties: Patent Application Filed.

Description: Key Points B-cell malignancies were efficiently recognized by T cells expressing high-affinity alloHLA-restricted TCRs specific for CD79b. Aberrant expression of CD79b in non–B cells caused unwanted reactivity, rendering CD79b unsuitable for TCR-based immunotherapies.

Description: Introduction: Chronic antigenic stimulation may play an important role in the pathogenesis of malignant lymphomas. Although most MCL cases are believed to have an antigen-naive B cell as cell of origin, overrepresentation of certain VH genes has been reported. Therefore we screened BCRs from MCLs for possible antigens. Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen MCL specimens and established MCL cell lines. The purified BCR-Fabs were checked for binding to proteins expressed on macroarrays of human cDNA expression libraries and on bacterial lysates. In addition, sera from patients with MCL were screened for antibodies against respective BCR antigens. Results: The recombinant MCL-BCR derived Fabs from 9 patients and of four established MCL cell lines were tested on protein arrays. Recombinant lymphoma-BCR-derived Fabs from 4/9 patients and from 1/4 MCL cell lines reacted with human low density lipoprotein receptor-related protein associated protein 1 (LRPAP1). Specific secondary modifications of LRPAP1 explaining its autoimmunogenicity were not found. 8/30 patients with MCL had anti-LRPAP1-antibodies in their serum, which was the case in only 1/200 healthy controls. Finally, LRPAP1 specifically induced proliferation of Maver1 cells that express a BCR with specificity for LRPAP1. Conclusions: LRPAP1 is the first molecularly defined antigenic target of MCL-BCRs. The high frequency of LRPAP1-reactive BCRs in MCL suggests a role of LRPAP1 in the pathogenesis of MCL, even in cases with unmutated VH genes. The prevalence of LRPAP1-antibodies in MCL patients and healthy controls identifies LRPAP1-antibody as the first serologic risk factor for MCL (odds ratio: 72.36) Supported by Wilhelm-Sander-Stiftung Disclosures No relevant conflicts of interest to declare.

Description: Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. In contrast to mAbs and CARs, T cell receptors (TCRs) recognize antigen-derived peptides that are bound to human leukocyte antigen (HLA) molecules on the cell surface. Since HLA molecules constantly sample the entire endogenous proteome of a cell, extracellular and intracellular antigens are presented and can thus be recognized by a TCR. Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to TCRs and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a HLA-A2/B7-negative healthy individual we isolated T cell clone 4G11 demonstrating high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. No harmful toxicities of clone 4G11 were observed against a wide panel of Bob1-negative stimulator cells including HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Furthermore, stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. Clone 4G11 demonstrated clinical applicability by efficiently recognizing HLA-B7+ primary ALL, CLL and MCL. Furthermore, reproducible strong recognition of purified primary HLA-B7+ MM could be demonstrated. Therefore, the TCR of clone 4G11 may be used for immunotherapy by administering TCR-transduced T cells to patients suffering from B cell malignancies including multiple myeloma. Retroviral gene transfer of TCR 4G11 led to efficient cell surface expression demonstrated by binding of TCR-transduced CD8+ T cells to pMHC-tetramer composed of peptide Bob144 bound to HLA-B7. TCR-modified CD8+ T cells strongly recognized Bob1-expressing HLA-B7+ multiple myeloma cell lines U266 and UM9, and ALL cell lines. TCR-modified T cells efficiently lysed HLA-B7+ primary ALL, CLL and MCL at very low effector-to-target ratios. In addition, highly purified primary multiple myeloma samples were also readily lysed. Furthermore, TCR-transduced T cells strongly proliferated in an antigen-specific manner when stimulated with primary malignant cell samples including ALL, CLL, and MCL or MM cell lines. As expected, TCR-transduced T cells also lysed autologous primary and CD40L-stimulated B cells since these targets cells also express Bob1. In contrast, no lysis of Bob1-negative autologous primary and activated T cells, or monocytes was observed when co-cultured with TCR-transduced T cells. In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. Gene transfer of TCR of clone 4G11 installed Bob1-reactivity and specificity onto recipient T cells shown here by cytolytic capacity and proliferation upon antigen encounter. TCR gene transfer approaches using this Bob1-specific TCR can bring novel treatment modalities and possibly curative therapy to patients with B cell malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Description: Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal syndrome, resulting from autoantibodies against the metalloprotease ADAMTS13. Autoantibodies bind and inhibit plasma ADAMTS13 activity, leading to exaggerated platelet agglutination and thrombus formation in small arteries and capillaries. However, plasma ADAMTS13 deficiency or the presence of anti-ADAMTS13 autoantibodies is not sufficient to cause the disease. Infection or inflammation often precedes the acute onset of TTP. We hypothesize that neutrophil activation or complement activation vial alternative pathway may be the trigger for TTP. In this study, plasma samples were collected prior to plasma exchange from 58 adult patients with acute TTP between 2006 and 2015 at the University of Alabama at Birmingham Medical Center. All patients were diagnosed having TTP on the basis of severe thrombocytopenia, microangiopathic hemolytic anemia, and severe deficiency of plasma ADAMTS13 activity (less than 10%) and the presence of ADAMTS13 inhibitors. All patients were treated by plasma exchange. Thirty plasma samples from healthy individuals were collected for controls. Plasma levels of human neutrophil peptides (HNP-1, -2, and -3) released from activated neutrophils and alternative pathway complement activation markers (iC3b, C4d, C5b-9, and Bb) were determined by enzyme-linked immunosorbent assays (ELISAs). We showed that plasma levels of HNP-1, -2, and -3 in TTP patients were elevated on average by 10-fold when compared with those in the healthy controls (44.12 ± 39.53 ng/ml vs. 3.54 ± 2.97 ng/ml, means ± SD) (p

Description: Introduction: The major task of a B-cell receptor is the binding and internalization of its antigenic target, and its processing for antigen presentation to T-cells. Chronic antigenic stimulation has been discussed to play a role in the pathogenesis of malignant B-cell lymphomas. We therefore systematically searched for the antigenic targets of BCRs from various B-cell neoplasms. Methods: Recombinant BCRs were expressed as recombinant Fabs (rFabs) based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA lymphoma biopsies. Whenever possible, "natural" Fabs (nFabs) were also obtained by papain digestion of fresh or cultured lymphoma cells. Both nFab and rFab were used to screen for binding to proteins expressed on macroarrays derived from human cDNA expression libraries and identical binding pattern of nFabs and rFabs was demonstrated by an antigen competition assay. Results: Two antigens (paratarg-7 and sumoylated HSP-90 which are hyperphosphorylated and sumoylated, respectively, in patients compared to healthy controls) are the targets of paraproteins from (depending on ethnicity) 30-50% of all multiple myeloma patients; the BCR from 67% of patients with primary CNS lymphoma target hyperglycosylated neurabin, 26% of the BCR from ABC-type DLBCL target hypophosphorylated ARS2 and 45% of all mantle cell BCR target LRPAP1; optineurin is the BCR target of 12% follicular lymphomas and various autoantigens have been identified as the targets of roughly 30% of all CLL cases. For all autoantigens binding to its specific BCR, rapid internalization and induction of proliferation was demonstrated, indicating partial dependence on antigenic stimulation even in cell lines that had been in culture for years. Most importantly, BCR-specific cytotoxicity of recombinant pseudomonas-exotoxin conjugated ARS2 against an ABC-DLBCL cell line with BCR specific for ARS2 (OCI-Ly3) was demonstrated in vitro and in vivo after establishment of OCI-Ly3 lymphomas in SCID beige mice. Conclusions: Assuming that only a minority of BCR targets have been identified to date, the prevalence of posttranslationally modified autoantigens strongly supports a role of chronic antigenic stimulation in many B-cell neoplasms. Due to the predominance of a single or few BCR antigens in each malignant B-cell entity studied, BARs represent an attractive and novel therapeutic concept for a broad spectrum of B-cell neoplasms and are the first therapeutic approach in oncology that targets exclusively the malignant cells. BARs can be used for conjugation with toxins, radionuclides and small molecules as well as for bispecific constructs (e. g. with CD3 or CD16) and CAR T-cells, the toxicity of which should be drastically reduced due to the ultimate specificity of BARs that spares normal B-cells. Supported by Wilhelm-Sander-Stiftung Disclosures Pfreundschuh: Roche, Janssen, Celgene: Honoraria, Research Funding.

Description: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease characterized by multiple low-frequency alterations including somatic mutations, copy number alterations (CNAs) and chromosomal rearrangements. We sought to identify previously unrecognized low-frequency genetic events, integrate recurrent alterations into comprehensive signatures and associate these signatures with clinical parameters. For these reasons, our multi-institutional international group assembled a cohort of 304 primary DLBCLs from newly diagnosed patients, 87% of whom were uniformly treated with state-of-the-art therapy (rituximab-containing CHOP regimen) and had long term followup. Tumors were subjected to whole exome sequencing with an extended bait set that included custom probes designed to capture recurrent chromosomal rearrangements. In this cohort, 47% of samples had available transcriptional profiling and assignment to associated disease subtypes. Analytical pipelines developed at the Broad Institute were used to detect mutations (MuTect), CNAs (Recapseq+Allelic Capseq) and chromosomal rearrangements (dRanger+Breakpointer) and assess clonality (Absolute). To analyze formalin-fixed paraffin-embedded tumors without paired normals we developed a method which utilized 8334 unrelated normal samples to stringently filter recurrent germline events and artifacts. Significant mutational drivers were identified using the MutSig2CV algorithm and recurrent CNAs were assessed with GISTIC2.0. In addition, we utilized a recently developed algorithm, CLUMPS2, to prioritize somatic mutations which cluster in 3-dimensional protein structure. With this approach, we identified 〉 90 recurrently mutated genes, 34 focal amplifications and 41 focal deletions, 20 arm-level events and 〉 200 chromosomal rearrangements in the DLBCL series. Of note, 33% of the mutational drivers were also perturbed by chromosomal rearrangements or CNAs, underscoring the importance of a comprehensive genetic analysis. In the large DLBCL series, we identified several previously unrecognized but potentially targetable alterations including mutations in NOTCH2 (8%) and TET2 (5%). The majority of identified chromosomal rearrangements involved translocations of potent regulatory regions to intact gene coding sequences. The most frequently rearrangements involved Ig regulatory elements which were translocated to BCL2, MYC, BCL6 and several additional genes with known roles in germinal center B-cell biology. After identifying recurrent somatic mutations, CNAs and chromosomal rearrangements, we performed hierarchical clustering and identified subsets of DLBCLs with comprehensive signatures comprised of specific alterations. A large subset of tumors shared recurrent alterations previously associated with follicular lymphoma including mutations of chromatin modifiers such as CREBBP, MLL2, and EZH2 in association with alterations of TNFRSF14 and GNA13 and translocations of BCL2. This cluster was enriched in GCB-type DLBCLs and contained a subset with select genetic alterations associated with an unfavorable outcome. An additional cohort of tumors was characterized by alterations perturbing B-cell differentiation including recurrent BCL6 translocations or alterations of PRDM1. A subset of these DLBCLs had alterations of NOTCH2 and additional pathway components or mutations of MYD88 in association with TNFAIP3, CD70 and EBF1, a master regulator of B-cell differentiation. An additional group of DLBCLs exhibited frequent MYD88 mutations in association with alterations of CD79B, PIM1, TBL1XR1 and ETV6 and BCL2 copy gain; these tumors were highly enriched for ABC-type DLBCLs. This coordinate signature and additional alterations of p53 pathway components were associated with outcome. We explored bases for the identified genetic alterations in DLBCL by performing an in silico mutational signature analysis. The most frequent mutational signatures were those of spontaneous deamination (aging) and AID with rare cases of microsatellite instability. We also assessed the clonality of identified genetic features to define cancer cell fraction and establish the timing of specific genetic events. The comprehensive genetic signatures of clinically annotated DLBCLs provide new insights regarding approaches to targeted therapy. Disclosures Link: Kite Pharma: Research Funding; Genentech: Consultancy, Research Funding. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Pfreundschuh:Boehringer Ingelheim, Celegene, Roche, Spectrum: Other: Advisory board; Roche: Honoraria; Amgen, Roche, Spectrum: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Key Points CLEC-2 can be downregulated from circulating platelets by anti–CLEC-2 antibodies through Src-family kinase-dependent internalization. Platelet-specific Syk deficiency abrogates anti–CLEC-2 antibodies-induced thrombocytopenia, but not CLEC-2 internalization.

Description: Background: Obesity occurs in more than 20% of patients (pts) above 65 years. In solid tumors e.g. breast or colorectal cancer, outcome of obese patients is inferior when compared to normal weight patients. In lymphoma patients data so far are controversial, while recently published data showed that obesity in lymphoma patients was not associated with inferior outcome. Methods: 576 patients with aggressive B-cell lymphomas included in the RICOVER60 study ( 6 or 8 cycles CHOP-14 + 8 doses of Rituximab) were analysed as training set. 151 patients from the RICOVER-noRTh study were used as validation set. We analysed EFS, PFS and OS with regard to body mass index (BMI ≥ 30 vs. 〈 30) and gender. No significant changes in body weight/BMI occurred during treatment, therefore BMI at cycle 1 was used for analysis. In Cox regression models BMI was adjusted for known prognostic factors. Results: All pts. were 〉 60 years of age (36% 〉 70 years), 46% female., 1% pts. low weight (BMI 70 years, radiotherapy (yes/no) and relative dose. The findings could be validated in an independent validation set of 151 patients from the RICOVER-noRTh study. The HR for EFS/PFS/OS are: all patients - 1.4/1.4/1.7; male patients - 0.7/0.9/1.5; female patients - 2.6/2.0/1.8. Due to small sample size only the HR for female patients for the EFS is significant (p=0.031). Conclusion: Obesity is a risk factor in elderly female pts. with aggressive B-cell lymphoma treated with R-CHOP with regard to EFS, PFS and OS, adjusted for IPI factors. Sex and weight independently affects rituximab clearance. Females are reported to have slower rituximab clearance with better outcome in the elderly and the rituximab clearance increases with body weight. Therefore worse outcome for obese female patients might be the result of faster rituximab clearance in obese females. Table 1: Multivariate analysis adjusting for IPI risk factors: HR for Obesity (BMI ≥ 30) All pts. male pts. female pts. EFS HR 1.4 [95%CI 1.0-1.9; p= 0.067] HR 1.2 [95%CI 0.7-2.0; p=0.473] HR 1.7 [95%CI 1.0-2.7; p=0.032] PSF HR 1.4 [95%CI 1.0-2.0; p= 0.080] HR 1.2 [95%CI 0.7-2.1; p=0.558] HR 1.9 [95%CI 1.1-3.2; p=0.022] OS HR 1.4 [95%CI 0.9-2.1; p= 0.114] HR 1.0 [95%CI 0.5-2.0; p=0.956] HR 2.0 [95%CI 1.1-3.4; p=0.017] Disclosures Hohloch: Spectrum: Research Funding; Spectrum, Roche: Other: Advisory board. Pfreundschuh:Roche: Honoraria; Boehringer Ingelheim, Celegene, Roche, Spectrum: Other: Advisory board; Amgen, Roche, Spectrum: Research Funding. Schmitz:Roche, Takeda, Gillead, Riemser und ctilifesciences: Other: Advisory board, Speakers Bureau. Truemper:Sandoz, Celgene, AMGEN, Nordic Nanovector: Other: Advisory board; Amgen, roche, Mundipharma: Research Funding.