Predicting the effect of mutations in proteins is one of the most critical challenges in protein engineering; by knowing the effect a substitution of one (or several) residues in the protein's sequence has on its overall properties, could design a variant with a desirable function. New strategies and methodologies to create predictive models are continually being developed. However, those that claim to be general often do not reach adequate performance, and those that aim to a particular task improve their predictive performance at the cost of the method's generality. Moreover, these approaches typically require a particular decision to encode the amino acidic sequence, without an explicit methodological agreement in such endeavor. To address these issues, in this work, we applied clustering, embedding, and dimensionality reduction techniques to the AAIndex database to select meaningful combinations of physicochemical properties for the encoding stage. We then used the chosen set of properties to obtain several encodings of the same sequence, to subsequently apply the Fast Fourier Transform (FFT) on them. We perform an exploratory stage of Machine-Learning models in the frequency space, using different algorithms and hyperparameters. Finally, we select the best performing predictive models in each set of properties and create an assembled model. We extensively tested the proposed methodology on different datasets and demonstrated that the generated assembled model achieved notably better performance metrics than those models based on a single encoding and, in most cases, better than those previously reported. The proposed method is available as a Python library for non-commercial use under the GNU General Public License (GPLv3) license.
The purpose of genome sequencing is to determine the DNA sequence of a given organism. Current sequencing technologies can be classified by the type of output data. Whereas Nanopore technology generates long reads with high error rates, short read technologies - such as Illumina sequencing - generate shorter reads but with low error rate. Since de novo genome assembly of sequencing reads is defined as a NP-hard problem, it remains as one of the major challenges for defining reference genomes of different species. This paper aims to improve the quality of reads obtained through Oxford Nanopore Technologies (ONT). We developed an algorithm to associate the reads obtained from Illumina with the ones obtained with Nanopore. Low accuracy ONT reads were corrected with the high quality Illumina reads to achieve an improved sequencing data. The inclusion of this algorithm as a preprocessing step resulted in improved coverage, contig length, and mismatch rate when performing de novo genome assembly of a bacterial genome with well known tools.
Understanding the mechanisms of interaction between antigen-antibody systems is one of the most relevant problems in the field of Immunology, since, supported by computational tools and data mining techniques, it would be possible to implement new antibody design tools. As a first approach and taking advantage of microarray techniques, a biological pattern recognition pipeline is presented from multiple ProtoArray chips, based on data mining techniques and bioinformatic analysis. This new approach, proposes 3 key stages in data processing: cleaning, statistical-frequency clustering of antigens and characterization by bioinformatic methods. This methodology was tested in a set of 370,000 interactions (45 antibodies, 8,000 antigens), achieving an effective filtering and grouping, in addition, a group obtained is characterized, in order to test the proposed bioinformatic methods. It is expected that this new method allows the identification of patterns in a simple and efficient way from multidimensional sets generated by protein microarrays.
The immunoglobulin receptor represents a central molecule in acquired immunity. The complete set of immunoglobulins present in an individual is known as immunological repertoire. The identification of this repertoire is particularly relevant in immunology and cancer research and diagnostics. In a seminal work we provided a proof of concept of the novel ARTISAN-PCR amplification method, we adapted this technology for sequencing using Nanopore technology. This approach may represent a faster, more portable and cost-effective alternative to current methods. In this study we present the pipeline for the analysis of immunological repertoires obtained by this approach. This paper shows the performance of immune repertoires sequenced by Nanopore technology, using measures of error, coverage and gene usage identification.
In the bioinformatic methodology used in this study, first, Albacore Base calling software, was used to translate the electrical signal of Nanopore to DNA bases. Subsequently, the sequons, introduced during amplification, were aligned using bl2seq from Blast. Finally, selected reads were mapped using IMGT/HighV-QUEST and IgBlast.
Our results demonstrate the feasibility of immune repertoire sequencing by Nanopore technology, obtaining higher depth than PacBio sequencing and better coverage than pair-end based technologies. However, the high rate of systematic errors indicates the need of improvements in the analysis pipeline, sequencing chemistry and/or molecular amplification.
IgE-mediated allergic disease represents an increasing health problem. Although numerous studies have investigated IgE sequences in allergic patients, little information is available on the healthy IgE repertoire. IgM, IgG, IgA, and IgE transcripts from peripheral blood B cells of five healthy, non-atopic individuals were amplified by unbiased, template-switching, isotype-specific PCR. Complete VDJ regions were sequenced to near-exhaustion on the PacBio platform. Sequences were analyzed for clonal relationships, degree of somatic hypermutation, IGHV gene usage, evidence of antigenic selection, and N-linked glycosylation motifs. IgE repertoires appeared to be highly oligoclonal with preferential usage of certain IGHV genes compared to the other isotypes. IgE sequences carried more somatic mutations than IgM, yet fewer than IgG and IgA. Many IgE sequences contained N-linked glycosylation motifs. IgE sequences had no clonal relationship with the other isotypes. The IgE repertoire in healthy individuals is derived from relatively few clonal expansions without apparent relations to immune reactions that give rise to IgG or IgA. The mutational burden of normal IgE suggests an origin through direct class-switching from the IgM repertoire with little evidence of antigenic drive, and hence presumably low affinity for specific antigens. These findings are compatible with a primary function of the healthy IgE repertoire to occupy Fcε receptors for competitive protection against mast cell degranulation induced by allergen-specific, high-affinity IgE. This background knowledge may help to elucidate pathogenic mechanisms in allergic disease and to design improved desensitization strategies.
Lipoprotein lipase (LPL) mRNA expression in chronic lymphocytic leukaemia (CLL) is associated with an unmutated immunoglobulin profile and poor clinical outcome. We evaluated the subcellular localization of LPL protein in CLL cells that did or did not express LPL mRNA. Our results show that LPL protein is differently located in CLL cells depending on whether it is incorporated from the extracellular medium in mutated CLL or generated de novo by leukaemic cells of unmutated patients. The specific quantification of endogenous LPL protein correlates with mRNA expression levels and mutational IGHV status, suggesting LPL protein as a possible reliable prognostic marker in CLL.
Patients with leukemia who receive a T cell–depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD.
Follicular lymphoma (FL) is the second most common indolent B-cell lymphoma (1). FL is characterized by a heterogeneous clinical evolution and remains incurable despite recent progress. The accepted founding event of FL pathogenesis is the acquisition of the t(14;18)(q32;q21) during primary VDJ recombination in the bone marrow (2). This chromosomal translocation juxtaposes the antiapoptotic proto-oncogene BCL2 to the immunoglobulin heavy chain locus during VDJ recombination (3). The resulting dysregulation of BCL2 expression provides a survival advantage through activation of antiapoptotic programs. The consequences of this early genetic event become manifest much later during B-cell maturation, when FL cells clonally expand and adopt malignant growth in germinal centers (GC) (2). The precise mechanisms involved in the initiation of malignant growth of apoptosisresistant t(14:18)-positive B-cells remain unclear.
B cells recognize specific antigens by their membrane‐bound B‐cell receptor (BCR). Functional BCR genes are assembled in pre‐B cells by recombination of the variable (V), diversity (D) and joining (J) genes [V(D)J recombination]. When B cells participate in germinal centre reactions, non‐templated point mutations are introduced into BCR genes by somatic hypermutation (SHM) (Rajewsky, 1996). V(D)J recombination and SHM create virtually unlimited BCR repertoires.
Follicular lymphoma (FL) has a paradigmatic stepwise pathogenesis with the balanced translocation (14;18)(q32;q21) as the founding event (Kridel et al, 2012). Most healthy individuals harbour t(14;18)‐positive premalignant FL‐like cells (FLLC) (Roulland et al, 2011). FLLC have a memory phenotype and have often undergone immunoglobulin class switch recombination (CSR) on the translocated IGH allele. Frank FL develops only after repetitive entry of FLLC into germinal centres (GC) and multiple GC passages (Sungalee et al, 2014). Paradoxically, FLLC fail to switch the functional IGH allele, suggesting dependence on IgM signalling at this stage. In contrast, many diagnosed FL cases have undergone CSR of their B‐cell receptor (BCR) (Sachen et al, 2012; Scherer et al, 2015). Therefore, class‐switched FL cells have apparently lost the characteristic dependency of FLLC on IgM signalling for recruitment into the GC. In addition, expression of activation‐induced deaminase (AID) seems to be dissociated from somatic hypermutation (SHM) and aberrant SHM in class‐switched FL (Scherer et al, 2015).
In B-cells, activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes. AID introduces mutations in immunoglobulin variable regions (IGV) during B-cell receptor affinity maturation, but may also introduce aberrant mutations into non-immunoglobulin genes, most commonly BCL6. Follicular lymphoma (FL) B-cells constitutively express AID and undergo CSR, SHM and aberrant SHM. We have studied AID expression, the presence of SHM mutations, CSR, and aberrant SHM in BCL6 in a cohort of 75 FL patients. Whereas IgM-expressing (non-switched) FL were characterized by an expected positive correlation between AID and IGV and BCL6 mutations, isotype-switched FL showed dissociation between AID expression and aberrant SHM, and inverse correlation between SHM and AID expression. Our results unveil two manifest biological subgroups of FL and indicate that the specific dissociation between AID and SHM after isotype switch may correlate with the clinical outcome of this heterogeneous disease.
Antigen receptors of B cells (BCR) exhibit a virtually unlimited repertoire, created by V(D)J recombination through combinatorial and junctional diversity of genetic elements in B-precursor cells. B cells further increase the antigen affinity of their BCR through somatic hypermutation (SHM). VDJ recombination and SHM create unique and novel peptide sequences that are not encoded in germline (GL) and may, therefore, act as neoantigens for the adaptive immune system. Naturally occurring BCR-directed immunity could, therefore, influence expansion of individual B-cell clones. We have previously found evidence for HLA class I-dependent, BCR-directed immunosurveillance in follicular lymphoma (FL).1 We here report lack of evidence for this mechanism in other indolent B-cell lymphomas and an apparent dependence of possible immunosurveillance on the high SHM rate in FL.
Somatic mutations associated with distinctive types of cancer can be globally classified by the trinucleotide context of single nucleotide variants into mutational signatures. Distinct signatures can be attributed to underlying causative mechanisms such as UV irradiation or smoking. B-cell lymphomas have only been analyzed globally and carry a signature characterized by APOBEC activity (Alexandrov, 2014).
The two most common indolent B-cell neoplasms, chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), are characterized by distinctive pathogenic processes, but mutagenesis caused by the APOBEC enzyme activation-induced cytosine deaminase (AID) may be shared by both malignancies. AID deaminates deoxycytidine to deoxyuridine, which is processed by DNA repair enzymes into a larger spectrum of point mutations and also DNA double-strand breaks. AID activity and subsequent DNA repair create two different mutation signatures: the canonical signature characterized by C>T/G transitions at RCY motifs and the non-canonical defined by A>C transversions at WAN motifs. More recently a third AID signature characterized by C>T transitions at RCG motifs has been associated with AID-mediated CpG-methylation dependent mutagenesis.
We here investigate the hypothesis that AID mutational signatures contribute to the pathogenesis of FL, a lymphoma characterized by constitutional expression of AID, but also of CLL since high AID expression levels may account for a more aggressive disease, and a subset of CLL B-cells with AID expression display dissociation between class switch recombination and somatic hypermutation.
Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes,which are critically important for an effective immune response development. In addition, AID seems to contribute to B cell tolerance in mice and humans eliminating developing autoreactive B cells. As a trade-off of the beneficial roles of AID physiological activity, this enzyme can also contribute to cellular transformation and tumor progression through its off-target mutagenic activity. Mounting evidences indicate a role for AID in disease progression and poor prognosis of chronic lymphoid neoplasms. Since these leukemias are not immediately derived from germinal center B cells, normal AID regulation might not be fully functional. Specifically, in CLL we have shown that AID isanomalously expressed in the peripheral blood of patients with Unmutated (Um) VH genes, correlating with active CSR and poor clinical outcome (Oppezzo et al., Blood 2005). In addition, others and us have described that AID expression is mainly restricted to distinct proliferative subsets within the leukemic clone(Albesiano et al., Blood, 2003; Palacios et al., Blood, 2010 and Patten et al., Blood, 2012). Despite the fact that AID expression has been associated with adverse cytogenetics and worse outcome (Leuenberger, M., XVII INTERNATIONAL WORKSHOP ON CHRONIC LYMPHOCYTIC LEUKEMIA 2017 207 Downloaded by [University of Florida] at 09:55 17 December 2017 Mod Pathol., 2010) definitive experimental demonstration that AID contributes to CLL progression is missing. To obtain insight into this issue we analyzed mutation load and signatures in Um patients in which anomalous expression of AID in peripheral blood was sustainedthroughout disease evolution. We compared the mutational profile by Whole Exome Sequencing(WES) of purified CD19þ cells extracted from 5 progressivepatients expressing AID (Um/AIDpos) andfrom 5 progressive patients in which AID was notdetected (Um/AIDneg). Samples were obtained atdiagnosis and at second time point after the first treatment(AFT). Analysis was performed according toGATK-best practice guidelines, variant discovery wasperformed by Varscan algorithm. Unsupervised AIDmutational profile was assessed by the Bayesian nonnegativematrix factorization algorithm as previouslydescribed (Alexandrov et. al., Nature, 2013), with thisstrategy we identified non-canonical AID signatures(nc-AID), i.e. A>C mutations at WA motifs in sensestrand and T>G mutation at TW motifs in antisensestrand. For supervised analysis of canonical AID signatures(c-AID) we quantified C to T mutations at WRCYmotifs in the sense strand or T to C variants in YGRWmotifs in antisense strand.We found that the total number of somatic mutationsis significantly increased in Um/AIDpos compared withUm/AIDneg patients independently of the diseasetime of the analyzed sample. A higher frequency ofsomatic mutations were observed in Um/AIDpos samplesat debut of the disease compared with Um/AIDpos samples AFT. We next analyzed mutation signaturesin the different groups: Um/AIDpos at diagnosis;Um/AIDpos AFT; Um/AIDneg at diagnosis and Um/AIDneg AFT. Our results show no significant differencesobserved between the groups concerning c-AIDmutations. However, an increased and significant differencewas found in the group Um/AIDpos comparedwith Um/AIDneg. Interestingly, a higher frequency ofnc-AID mutations was observed in Um/AIDpos groupat diagnosis time compared with Um/AIDpos AFT,which might suggest selection for samples with lowerAID levels and/or mutation loads. In addition, theunsupervised analysis identified the presence otherpreviously described signatures (Alexandrov et. al.,Nature, 2013), such as Aging, and APOBEC in all samples.We also analyzed AID-catalyzed mutations thatwere found at the debut of the disease and maintainedduring disease evolution, as well as those thatappeared only in the AFT samples. Presently, mutationsin selected genes are being confirmed in a largercohort. We will discuss these results.AID expression could be specially problematic inchronic diseases, where even a small but continuouslevel of AID activity can lead to selectable geneticmutations over time, giving rise to more aggressivetumors and treatment resistance. Our results supportthis notion and describes for the first time the mutationalprofile of Um and progressive patients withanomalous AID expression through their evolution.Under the light of the hypothesis that AID expressionis a hallmark of an activated microenvironment resultingin disease progression our results will allow us toidentify AID mutated genes which could be implicatedin CLL tumor progression.This work was supported by ANII. ProyectoFMV_1_2014_1_104397 and CSIC-Udelar. Programa deIþD 2014.
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.
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.
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.
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 up to 30% of ABC DLBCL cases, this signature can be explained by gain-of-function mutations in CD79A, CD79B, or CARD11. Antigen-independent, constitutively active (BCR) signaling is a general oncogenic mechanism of CLL (Dühren-von Minden, Nature 2012). We hypothesized that autonomous BCR signaling akin to CLL may operate in ABC DLBCL and could explain the characteristic ABC gene expression signature.
BCR transcripts were identified from fresh-frozen biopsies from 12 histologically confirmed, IgM-expressing DLBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased amplification of BCR transcripts facilitated by template switching. Clonal full-length BCR sequences were identified by PacBio next-generation sequencing. Triple KO (TKO) cells were transduced with functional DLBCL BCR. TKO cells are arrested at the pre-B-cell stage due to lack of the rag2 and lambda5 genes. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65. When transduced with expression constructs encoding a functional BCR, autonomous or antigen-induced BCR signalling can be measured as calcium flux upon induction with tamoxifen.
TKO cells transduced with seven of the twelve DLBCL BCR (58%) showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. By histomorphology and immunohistochemistry for CD10, PAX5, MUM1, and Bcl-6, these cases were classified as non-GCB-type DLBCL, including one primary CNS DLBCL and two primary mediastinal (PM-)DLBCL. In accordance with our hypothesis, BCR from three DLBCL classified as GCB lacked an autonomous BCR signal. The remaining two negative cases were non-GCB-type and are currently being analyzed for activating CD79/CARD11 mutations.
To further explore the detection of autonomous BCR signaling in DLBCL, PM-DLBCL and ABC DLBCL cell lines were tested in vitro by comparing calcium flux with and without blockade of Syk signaling by the tyrosine kinase inhibitor R406. All four PM-DLBCL and two of five ABC DLBCL cell lines had higher calcium flux in the absence of R406, indicating autonomous signaling activity without antigenic stimulation or artificial BCR crosslinking.
In summary, we demonstrate autonomous BCR activity in a substantial fraction (72%) of non-GCB DLBCL (including PM-DLBCL). These results point to an oncogenic role of structurally normal BCR in non-GCB DLBCL akin to CLL. Autonomous BCR signaling is a candidate alternative mechanism to the previously described activating mutations of components of the BCR signaling cascade to induce the characteristic gene expression signature of ABC DLBCL. Formal delineation of autonomous BCR signalling to activating mutations in the BCR signalling cascade is ongoing.