ALBERT

Description: The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21–derived heavy chains (HCs) with IGLV3-21–derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110–expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR–BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01–expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110–expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

Description: Background Primary mediastinal large B-cell lymphoma (PM LBCL) is putatively derived from thymic B cells. PM LBCL is clinically and morphologically distinct from diffuse large B-cell lymphoma (DLBCL), but shares certain phenotypic characteristics with nodular sclerosing classical Hodgkin's lymphoma. The expression and function of an intact B-cell receptor (BCR) in PM LBCL is controversial. In addition, the pathological mechanisms that drive lymphomagenesis in PM DLBCL are largely unknown. Here, we investigate the expression of functional BCR in PM LBCL and explore the hypothesis that autonomous signaling of the BCR as observed in CLL (Dühren-von Minden, Nature 2012) plays a key role in PM DLBCL pathogenesis. Methods The PM LBCL cell lines Farage, MedB-1, Karpas 1106P, and U2940 were tested in vitro by comparing calcium flux with and without inhibition of BCR signaling by blockade of Syk with the tyrosine kinase inhibitor R406. BCR transcripts were identified from archived fresh-frozen biopsies from two histologically confirmed PM LBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased amplification of BCR transcripts facilitated by template switching (Koning, BJH 2016). Clonal full-length BCR sequences were identified by PacBio next-generation sequencing. To test for autonomous BCR signaling, murine triple KO (TKO) cells were transduced with functional BCR of PM LBCL (Dühren-von Minden, Nature 2012). TKO cells lack the rag2 and lambda5 genes and are therefore developmentally arrested at the pre-B-cell stage. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65 variant. When transduced with a functional BCR, autonomous or antigen-induced BCR signaling can be measured upon induction with tamoxifen as calcium flux by flow cytometry or as robust cellular proliferation. Results All four PM-LBCL lines had higher calcium flux in the absence of R406, suggesting autonomous BCR signaling activity without antigenic stimulation or artificial BCR crosslinking. Functional BCR transcripts could be readily identified in both primary PM LBCL biopsies by ARTISAN PCR. In accordance with our hypothesis, TKO cells transduced with the BCR of both primary PM LBCL cases showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. To test whether this autonomous BCR signal would represent a druggable therapeutic target, the PM DLBCL cell lines were cultured in the presence of increasing doses of the second generation, highly BTK-specific kinase inhibitor acalabrutinib. The ABC DLBCL cell lines OCI-Ly10, TMD8, and U2932, and the GCB cell lines Karpas 422, OCI-Ly7, and OCI-Ly19 were tested in parallel as controls. Cell viability decreased in all cell lines with increasing concentrations of acalabrutinib (Figure). Consistent with their known dependency on active BCR signalling (Young, PNAS 2015; Koning, ASH 2016), ABC DLBCL cell lines were very sensitive to acalabrutinib. As suggested by the experimental evidence for autonomous BCR signalling, PM LBCL cell lines also responded to acalabrutinib, albeit to a lesser degree than ABC DLBCL. Growth inhibition of GCB DLBCL cell lines generally required excessively high doses of acalabrutinib, consistent with the notion that this DLBCL subtype depends on tonic BCR signaling only. Conclusion Consistent with our hypothesis, our data demonstrate expression of an intact BCR with autonomous signaling activity in all investigated cases of PM LBCL. These results point to an oncogenic role of structurally normal BCR in PM LBCL akin to CLL. Similarly to CLL and ABC DLBCL, PM DLBCL cell lines respond to BTK inhibitors, albeit less strongly than the ABC DLBCL cell lines tested. The results identify BCR signaling as a novel therapeutic target in PM LBCL and suggest the initiation of clinical trials with BTK inhibitors in PM LBCL. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: Peptides of the B-cell receptor (BCR) may be presented in HLA molecules and therefore be recognized as epitopes by T cells. Bioinformatic evidence indicates that follicular lymphoma cells are selected against expression of a clonal BCR with a high cumulative predicted binding of BCR-derived peptides to the respective patient's HLA complex (Strothmeyer, Blood 2010). This observation suggests T-cell-mediated immunosurveillance against outgrowth of follicular lymphoma cells according to BCR HLA binding strength. Here, we investigate whether this phenomenon pertains to peripheral B cells in 6 healthy donors: 2 donors homozygous for HLA A01*01 / B08*01, 2 homozygous for HLA A02*01 / B7*02, and 2 donors heterozygous for these alleles. Unbiased representation of full-length V(D)J sequences was considered essential for correct data interpretation. PCR primers annealing to conserved motifs of BCR variable regions (e.g. BIOMED-2 protocol) fail to amplify a fraction of BCR, particularly those modified by somatic hypermutation. Therefore, we developed an improved anchored PCR strategy: cDNA was synthesized from poly(A)-RNA from peripheral blood with primers that anneal to specific Ig constant regions. In the same reaction, the 3' cDNA end is extended by switching to an oligonucleotide template containing an anchor sequence (SMART technology; Clontech). Anchor-tagged cDNA was amplified with a primer annealing to the anchor in combination with a nested constant region-specific reverse primer. Dumbbell adapters were added to the termini of 250 ng of purified PCR products. Circular consensus sequencing of single molecules was performed on the PacBio platform (Pacific Biosciences). Using one SMRT PacBio cell per amplicon, separate sequence libraries were created for μ, γ, κ, and λ BCR transcripts. Sequences covered by at least five reads were selected with SMRT Portal software to obtain 〉95% of sequences without sequence errors as demonstrated on multiple B-cell lines. Selected sequences were analysed by HighV-QUEST software (Alamyar, Immunome Research 2012). After exclusion of non-BCR sequences and duplicate BCR transcripts, a median of 5318 (range: 670-8752) individual BCR sequences was obtained per library. Binding affinity of nonamers in in-silico-translated BCR were calculated for the 4 HLA alleles by the NetMHC 3.4 algorithm. The fractions of BCR lacking any weak HLA binding peptide (NetMHC IC50

Description: T cells recognizing solid tumors and Hodgkin's lymphoma can be released from anergy by immune checkpoint inhibitors. Its therapeutic success seems to be associated with the abundance of neoepitopes within the tumor mutanome. In neoplastic B cells, VDJ recombination and somatic hypermutation (SHM) generate unique immunoglobulin (Ig) peptide sequences that predictably contribute to the lymphoma mutanome. Efficient presentation of a neoepitope by an individual's HLA complex is an essential requirement for neoepitope-directed T-cell immunity. Presentation of Ig-derived peptides in HLA class I has hitherto been demonstrated only indirectly, and there is as yet no direct evidence for presentation of Ig-derived neoepitopes of primary human lymphoma cells. We analyzed HLA-presented Ig-derived peptides in 9 clonal B-cell populations: 3 chronic lymphocytic leukemias (CLL), 1 hairy cell leukemia, 1 follicular lymphoma (FL), 3 EBV-transformed lymphoblastoid cell lines, and the U299 myeloma cell line. Full-length B-cell receptor (BCR) VDJ and VJ sequences were obtained by unbiased ARTISAN PCR and Pacific Biosciences next generation sequencing. 1067 unique Ig-derived nonamers were predicted to bind the HLA class I alleles expressed by the respective B-cell clone by the NetCTLpan bioinformatics tool. 650 candidate epitopes (60.9%) were derived from constant (C) regions; 417 (39.1%) from variable (V) regions. 146 predicted peptides from V regions (35.0%) contained at least one amino acid change due to SHM or at least one amino acid from CDR3 and were therefore considered potential neoepitopes. To investigate experimentally which epitopes are processed intracellularly and presented by HLA class I, HLA class I-peptide complexes were immunoaffinity purified by the monoclonal antibody W6.23 and analyzed by liquid chromatography and tandem mass spectrometry. In total, 53,663 unique peptides (8-15mers) were identified by Uniprot matching with a Mascot Ion Score of 〉20. HLA ligandome sizes of individual B-cell populations ranged from 600 to 14,091 unique peptides per case. Within any HLA ligandome, 6 to 81 peptides were annotated as BCR-derived epitopes if they matched the individual BCR sequences. Of the total of 276 eluted BCR peptides, 203 (73.5%) where derived from C regions and 73 (26.5%) from V regions. 25 eluted peptides (range 0-10 per case) were derived from CDR3 regions or contained SHM-induced amino acid changes, thus fulfilling the definition of neoepitopes. Neoepitopes were detected in all cases with more than 109 cells as input material. Up to date, 4 neoepitopes have been synthesized and their mass spectra confirmed. Confirmation of all remaining neoepitopes is ongoing. No BCR-derived neoepitopes could be detected in an IGHV-unmutated CLL and the FL. These two cases had the lowest number of input cells and small overall ligandome sizes of only 600 and 1015 unique UniProt-matched peptides, respectively. We demonstrate for the first time that primary neoplastic B cells process and present BCR-derived neoepitopes in the context of HLA class I. Despite their origin from only two polypeptides, BCR epitopes and neoepitopes represent app. 0.5% and 0.05% of the total HLA class I ligandome, respectively. The challenging identification of BCR neoepitopes was possible by unbiased identification of BCR transcripts combined with next generation sequencing and targeted search for HLA class I-bound peptides. Matching fragmentation patterns of native and synthetic peptides suggest high specificity of this strategy. With respect to sensitivity, the ability to identify low frequency peptides appears to be strongly dependent on the amount of input cells. Our data close a gap in the mechanism underlying the evidence that highly immunogenic formulations of an idiotype vaccine are able to induce MHC class I-restricted cytotoxic T-cell responses. While phase III trials of idiotype vaccination aiming to induce anti-Ig antibodies failed to demonstrate convincing prolongation of clinical remissions achieved by chemotherapy, our data lend support for exploring idiotype-specific T-cell immunity against B-cell lymphomas. With recent advances in peptide synthesis, adjuvant formulations, and the availability of check point inhibitors to surpass regulatory activity, active immunotherapy targeting the lymphoma idiotype may regain appeal as truly personalized immune therapy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction & Objectives: Primary Cutaneous Follicle Center Lymphoma (PCFCL) is a very indolent mature B-cell lymphoma that shares germinal center morphology with follicular lymphoma (FL) but lacks the characteristic t(14;18). Unlike FL, immunohistochemistry fails to detect expression of BCL2, CD10, and immunoglobulin in PCFCL. Therefore, we investigated expression of B-cell receptor (BCR) transcripts to gain insight into the immunobiology of PCFCL. Materials & Methods: Full-length heavy and light chain BCR transcripts of 13 histologically confirmed PCFCL were amplified using ARTISAN PCR and sequenced on the PacBio RSII system. BCR from 4 cases were sequenced to a depth of 〉2000 sequences per BCR transcript; the remaining cases to a median depth of 1663 sequences (range: 626-5301). BCR from 51 cases of FL and from peripheral B cells of 12 healthy donors were used as controls. Whole genome sequencing (WGS) and RNAseq were performed on 5 PCFCL on the Illumina HiSeq platform. Results: No PCFCL case carried a t(14;18). In addition to previously described CD79B mutations, an L265P mutation in MYD88 was identified in one case, and two PCFCL carried amplifications in chromosome 2 involving the proto-oncogene REL. ARTISAN PCR demonstrated expression of potentially functional VDJ and VJ genes with heavily mutated V regions (VDJ: 5.9-24.0%; VJ: 4.7-17.9%) in all PCFCL cases, which could be confirmed by RNAseq-based de novo BCR assembly. One PCFCL case expressed IgM, another IgA, and the remaining ten cases expressed IgG. PCFCL VDJ carried relatively long heavy chain CDR3 regions with a median of 19 amino acids (versus 17 in healthy donor PBMCs). In contrast to FL, only minimal intraclonal sequence variation (comparable to the known error rate of the used sequencing method) was observed in PCFCL VDJ and VJ sequences, indicating absence of ongoing somatic hypermutation (SHM). VDJ and/or VJ of 11 PCFCL (85%) carried at least one acquired N-linked glycosylation motifs, six PCFCL (46%) at least two, and one case four such motifs. 75% of acquired N-linked glycosylation motifs were found in different positions than the N-linked glycosylation motifs found in FL BCR (Figure). In contrast, only 17.5% and

Description: Background The activated B-cell (ABC) type of diffuse large B-cell lymphoma (DLBCL) is clinically more aggressive than the germinal center (GCB) type. The gene expression profile of ABC DLBCL resembles that of mature B cells upon stimulation via their B-cell receptor (BCR). In app. 10% of ABC DLBCL cases, this signature can be explained by gain-of-function mutations in CARD11. Recent evidence suggests that autoantigen recognition by the BCR sustains the viability of ABC DLBCL in vitro (Young, PNAS 2015), although the functional relationship to CARD11 mutations remains unresolved. Antigen-independent, constitutively active BCR signaling is a fundamental oncogenic mechanism of CLL (Dühren-von Minden, Nature 2012). We therefore explored the hypothesis that autonomous BCR signaling akin to CLL could be operative in ABC DLBCL and would represent an alternative but functionally complementary oncogenic mechanism to activating CARD11 mutations in ABC DLBCL lymphomagenesis. Methods BCR transcripts were identified from archived fresh-frozen biopsies of 14 histologically confirmed, CD10-negative, IgM-expressing DLBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased isolation of full-length BCR transcripts facilitated by template switching (Koning, BJH 2016). Clonal full-length BCR were identified by single molecule sequencing on the PacBio platform. All biopsies were screened for mutations in CARD11 and CD79 by Sanger sequencing. To test for autonomous BCR signaling activity, murine triple KO (TKO) cells were transduced with functional DLBCL BCR (Dühren-von Minden, Nature 2012). TKO cells lack the rag2 and lambda5 genes and are therefore developmentally arrested at the pre-B-cell stage. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65 variant. When transduced with a functional BCR, autonomous or antigen-induced BCR signaling can be measured upon restoring functionality of the BCR signalling cascade with tamoxifen as calcium flux by flow cytometry and robust cellular proliferation. Results Re-examination by immunohistochemistry for CD10, PAX5, MUM1, and Bcl-6 was possible in 12 cases and identified three cases as GCB-type DLBCL. Nine cases were classified as non-GCB DLBCL, including one primary CNS DLBCL and three leg-type primary cutaneous DLBCL (LT DLBCL). One non-GCB DLBCL was found to carry a potentially activating CARD11 M212K mutation (Table). BCR of the three GCB DLBCL served as controls and lacked an autonomous BCR signal. In contrast, TKO cells transduced with five of the seven remaining DLBCL BCR (71%) showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. In accordance with our hypotheses, the CARD11-mutated non-GCB DLBCL case lacked autonomous BCR signaling. This case and the second, non-GCB DLBCL case lacking autonomous BR activity that carried wild-type CARD11 both expressed a BCR with the IGHV4-34 allele. Unexpectedly, the three LT DLBCL also did not exhibit autonomous BCR signaling. Conclusion Our data demonstrate autonomous BCR activity in a dominant fraction of non-GCB DLBCL. In striking similarity to CLL, these results point to an oncogenic role of a structurally normal BCR in non-GCB DLBCL that does not require binding of external autoantigens. Consistent with our hypotheses, antigen-independent signaling of a structurally normal BCR signaling cascade is a compelling candidate alternative mechanism to activating mutations in signal-transducing components of the BCR pathway. Both mechanisms may therefore induce the characteristic gene expression signature of ABC DLBCL. In addition, this finding readily explains the higher likelihood of ABC DLBCL with wild-type CD79 and CARD11 to respond to BCR signaling inhibition by BTK inhibitors (Wilson, Nat Med 2015). In contrast, LT DLBCL, which otherwise resembles ABC DLBCL, does not exhibit autonomous BCR signaling, suggesting yet another oncogenic mechanism that may explain its dismal prognosis. Finally, the non-GCB DLBCL lacking both autonomous BCR signaling and a potentially activating CARD11 mutation expressed the inherently autoreactive IGHV4-34 allele, opening the possibility that strong autoantigen stimulation might substitute for autonomous BCR signaling activity, thereby offering an alternative explanation for the BCR signaling pathway addiction in IGHV4-34-expressing cases. Table. Table. Disclosures Vermeer: Innate Pharma: Other: Investigator in a clinical trial.

Description: Objectives: Follicular lymphoma (FL) typically originates from premalignant mature B cells that carry the founder t(14;18) BCL2 translocation. Mutations in epigenetic modifiers and acquisition of N-glycosylation sites in CDR regions of the B-cell receptor (BCR) are recurrent secondary events in FL pathogenesis. Despite these oncogenic drivers, FL can remain indolent and clinically stable for years. The molecular events driving subclonal evolution into symptomatic progression and eventual transformation to aggressive lymphoma are insufficiently understood. FL cells are frozen in their B-cell development at the germinal center stage and undergo continuous somatic hypermutation mediated by expression of activation-induced deaminase (AID). We aim to identify crucial drivers of subclonal FL evolution by high-throughput mapping at single-cell resolution. Methods: Viable FL cells were isolated and cryopreserved from 23 histologically or immunocytologically confirmed FL samples from 13 patients with informed consent. Full-length VDJ/VJ transcripts were isolated by unbiased template-switching ARTISAN PCR and massive parallel NGS sequencing on the PacBio platform. The clonal primordial FL BCR (pBCR) was reconstructed from unmutated IGV/IGJ sequences with the CDR3 of the least mutated BCR. Since the IgTree program was unable to process the obtained numbers of BCR sequences, we developed the WILLOW algorithm for analysis of BCR evolution based on the principle of maximum parsimony and on distance from the pBCR. Intraclonal BCR variability was quantified by Shannon's diversity index. 5' single cell transcriptomics and VDJ/VJ sequencing was performed on 2 pools of highly purified FL cells from 5 lymph node biopsies on the 10x Genomics platform. Data were deconvoluted based on expressed variants by the Single Cell Sample Matcher (SCSM) algorithm. Clustering based on gene expression profiles was performed by shared nearest neighbour (SNN) modularity optimization within the R Seurat package. Genes whose expression differed significantly (adjusted p

Description: Introduction & Objectives: Primary Cutaneous Follicle Center Lymphoma (PCFCL) is a very indolent mature B-cell lymphoma that shares germinal center (GC) morphology with follicular lymphoma (FL) but lacks the characteristic t(14;18). Unlike FL, immunohistochemistry fails to detect expression of BCL2, CD10, and immunoglobulin in PCFCL. We investigated the B-cell receptor (BCR) to gain insight into the immunobiology of PCFCL. Materials & Methods: Whole Genome Sequencing (WGS) and RNAseq were performed on five PCFCL biopsies. Full-length heavy and light chain BCR transcripts were amplified by unbiased ARTISAN PCR (Koning et al., BJH 2016). More than 2000 sequences per BCR transcript were sequenced as full-length single molecules on the PacBio next-generation sequencing platform. Results: In addition to various minor translocations and deletions, WGSidentified a t(14;22) that resulted in juxtaposition of IGH and IGLL5 in one PCFCL case. No PCFCL case carried a t(14;18). Despite the absence of detectable surface Ig by immunohistochemistry, ARTISAN PCR and RNAseq-based de novo BCR assembly independently demonstrated expression of functional VDJ and VJ genes with heavily mutated V regions (VDJ: 7,1-16,0%; VJ: 4,6-11,1%) in all cases. Lack of intraclonal sequence variation indicated absence of ongoing somatic hypermutation (SHM). The t(14;22)-carrying PCFCL expressed an inconspicuous IgM. BCR of all remaining four PCFCL carried sequence motifs for N-linked glycosylation in antigen-binding regions that were apparently acquired by SHM. Three cases had undergone class switch recombination to IgG. The remaining case expressed IgM with extensive mutations. An L265P mutation in MYD88 was identified in one case, and two cases carried amplifications in chromosome 2 involving the proto-oncogene REL. Conclusions: GC morphology, extensive SHM, and class switch recombination indicate a shared origin of GC B cells for both PCFCL and FL. Clonal BCR sequences and previously identified copy number alterations prove that PCFCL represents a neoplastic clonal expansion. However, lack of ongoing SHM indicates that the immune follicles of PCFCL are not fully functional germinal centers. Since ongoing SHM is thought to contribute to lymphomagenesis by targeting non-BCR loci, absence of both ongoing SHM and the t(14;18) may explain the relatively benign clinical course of PCFCL compared to FL. As previously described for FL, continuous BCR stimulation through glycosylation-mediated binding of lectins on resident cells of the follicular microenvironment may cause clonal expansion of PCFCL cells and could play a decisive role in maintaining the follicular microarchitecture in both FL and PCFCL. In comparison to this BCR glycosylation, acquired somatic alterations in oncogenes that recurrently involved in other types of indolent B-cell lymphomas may constitute secondary driver events. Further comparisons to define the extent of malignant transformation between PCFCL, FL, and other B-lymphomas are warranted. Disclosures Vermeer: Innate Pharma: Other: Investigator in a clinical trial.