Among B cell non-Hodgkin's lymphomas, MALT lymphomas express a unique antibody repertoire with frequent rheumatoid factor reactivity

We analyzed the structure of antigen receptors of a comprehensive panel of mature B non-Hodgkin's lymphomas (B-NHLs) by comparing, at the amino acid level, their immunoglobulin (Ig)VH-CDR3s with CDR3 sequences present in GenBank. Follicular lymphomas, diffuse large B cell lymphomas, Burkitt's lymphomas, and myelomas expressed a CDR3 repertoire comparable to that of normal B cells. Mantle cell lymphomas and B cell chronic lymphocytic leukemias (B-CLLs) expressed clearly restricted albeit different CDR3 repertoires. Lymphomas of mucosa-associated lymphoid tissues (MALTs) were unique as 8 out of 45 (18%) of gastric- and 13 out of 32 (41%) of salivary gland-MALT lymphomas expressed B cell antigen receptors with strong CDR3 homology to rheumatoid factors (RFs). Of note, the RF-CDR3 homology without exception included N-region–encoded residues in the hypermutated IgVH genes, indicating that they were stringently selected for reactivity with auto-IgG. By in vitro binding studies with 10 MALT lymphoma–derived antibodies, we showed that seven of these cases, of which four with RF-CDR3 homology, indeed possessed strong RF reactivity. Of one MALT lymphoma, functional proof for selection of subclones with high RF affinity was obtained. Interestingly, RF-CDR3 homology and t(11;18) appeared to be mutually exclusive features and RF-CDR3 homology was not encountered in any of the 19 pulmonary MALT lymphomas studied

Origin and pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma as revealed by global gene expression analysis

The pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL) and its relationship to other lymphomas are largely unknown. This is partly because of the technical challenge of analyzing its rare neoplastic lymphocytic and histiocytic (L&H) cells, which are dispersed in an abundant nonneoplastic cellular microenvironment. We performed a genome-wide expression study of microdissected L&H lymphoma cells in comparison to normal and other malignant B cells that indicated a relationship of L&H cells to and/or that they originate from germinal center B cells at the transition to memory B cells. L&H cells show a surprisingly high similarity to the tumor cells of T cell–rich B cell lymphoma and classical Hodgkin lymphoma, a partial loss of their B cell phenotype, and deregulation of many apoptosis regulators and putative oncogenes. Importantly, L&H cells are characterized by constitutive nuclear factor {kappa}B activity and aberrant extracellular signal-regulated kinase signaling. Thus, these findings shed new light on the nature of L&H cells, reveal several novel pathogenetic mechanisms in NLPHL, and may help in differential diagnosis and lead to novel therapeutic strategies

Salivary Gland Mucosa-Associated Lymphoid Tissue-Type Lymphoma From Sjogren's Syndrome Patients in the Majority Express Rheumatoid Factors Affinity-Selected for IgG

Objective: Patients with Sjӧgren's syndrome (SS) have an increased risk of developing malignant B cell lymphomas, particularly mucosa-associated lymphoid tissue (MALT)–type lymphomas. We have previously shown that a predominant proportion of patients with SS-associated salivary gland MALT lymphoma express somatically hypermutated IgM with strong amino acid sequence homology with stereotypic rheumatoid factors (RFs). The present study was undertaken in a larger cohort of patients with SS-associated MALT lymphoma to more firmly assess the frequency of RF reactivity and the significance of somatic IGV-region mutations for RF reactivity. Methods: B cell antigen receptors (BCRs) of 16 patients with SS-associated salivary gland MALT lymphoma were analyzed. Soluble recombinant IgM was produced of 12 MALT lymphoma samples, including 1 MALT lymphoma sample that expressed an IgM antibody fitting in a novel IGHV3-30–encoded stereotypic IGHV subset. For 4 of the 12 IgM antibodies from MALT lymphoma samples, the somatically mutated IGHV and IGKV gene sequences were reverted to germline configurations. Their RF activity and binding affinity were determined by enzyme-linked immunosorbent assay and surface plasmon resonance, respectively. Results: Nine (75%) of the 12 IgM antibodies identified in patients with SS-associated salivary gland MALT lymphoma displayed strong monoreactive RF activity. Reversion of the IGHV and IGKV mutations to germline configuration resulted in RF affinities for IgG that were significantly lower for 3 of the 4 somatically mutated IgM antibodies. In stereotypic IGHV3-7/IGKV3-15–encoded RFs, a recurrent replacement mutation in the IGKV3-15–third complementarity-determining region was found to play a pivotal role in the affinity for IgG-Fc. Conclusion: A majority of patients with SS-associated salivary gland MALT lymphoma express somatically mutated BCRs that are selected for monoreactive, high-affinity binding of IgG-Fc. These data underscore the notion that soluble IgG, most likely in immune complexes in inflamed tissues, is the principal autoantigen in the pathogenesis of a variety of B cell lymphomas, particularly SS-associated MALT lymphomas

Lymphoma-associated mutations in autoreactive memory B cells of patients with Sjögren's syndrome

We recently demonstrated that normal memory B lymphocytes carry a substantial number of de novo mutations in the genome. Here, we performed exome-wide somatic mutation analyses of bona fide autoreactive rheumatoid factor (RF)-expressing memory B cells retrieved from patients with Sjӧgren's syndrome (SS). The amount and repertoire of the de novo exome mutations of RF B cells were found to be essentially different from those detected in healthy donor memory B cells. In contrast to the mutation spectra of normal B cells, which appeared random and non-selected, the mutations of the RF B cells were greater in number and enriched for mutations in genes also found mutated in B-cell non-Hodgkin lymphomas. During the study, one of the SS patients developed a diffuse large B-cell lymphoma (DLBCL) out of an RF clone that was identified 2 years earlier in an inflamed salivary gland biopsy. The successive oncogenic events in the RF precursor clone and the DLBCL were assessed. In conclusion, our findings of enhanced and selected genomic damage in growth-regulating genes in RF memory B cells of SS patients together with the documented transformation of an RF-precursor clone into DLBCL provide unique novel insight into the earliest stages of B-cell derailment and lymphomagenesis.</p

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies

Biological and Clinical Implications of Gene-Expression Profiling in Diffuse Large B-Cell Lymphoma:A Proposal for a Targeted BLYM-777 Consortium Panel as Part of a Multilayered Analytical Approach

Gene-expression profiling (GEP) is used to study the molecular biology of lymphomas. Here, advancing insights from GEP studies in diffuse large B-cell lymphoma (DLBCL) lymphomagenesis are discussed. GEP studies elucidated subtypes based on cell-of-origin principles and profoundly changed the biological understanding of DLBCL with clinical relevance. Studies integrating GEP and next-generation DNA sequencing defined different molecular subtypes of DLBCL entities originating at specific anatomical localizations. With the emergence of high-throughput technologies, the tumor microenvironment (TME) has been recognized as a critical component in DLBCL pathogenesis. TME studies have characterized so-called “lymphoma microenvironments" and “ecotypes”. Despite gained insights, unexplained chemo-refractoriness in DLBCL remains. To further elucidate the complex biology of DLBCL, we propose a novel targeted GEP consortium panel, called BLYM-777. This knowledge-based biology-driven panel includes probes for 777 genes, covering many aspects regarding B-cell lymphomagenesis (f.e., MYC signature, TME, immune surveillance and resistance to CAR T-cell therapy). Regarding lymphomagenesis, upcoming DLBCL studies need to incorporate genomic and transcriptomic approaches with proteomic methods and correlate these multi-omics data with patient characteristics of well-defined and homogeneous cohorts. This multilayered methodology potentially enhances diagnostic classification of DLBCL subtypes, prognostication, and the development of novel targeted therapeutic strategies. Simple Summary: This review summarizes gene-expression profiling insights into the background and origination of diffuse large B-cell lymphomas (DLBCL). To further unravel the molecular biology of these lymphomas, a consortium panel called BLYM-777 was designed including genes important for subtype classifications, genetic pathways, tumor-microenvironment, immune response and resistance to targeted therapies. This review proposes to combine this transcriptomic method with genomics, proteomics, and patient characteristics to facilitate diagnostic classification, prognostication, and the development of new targeted therapeutic strategies in DLBCL

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC

Background: Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the β-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the β-catenin paradox. Methods: We analysed the expression patterns of SMAD4, p53 and β-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to s

Apparent Lack of BRAFV600E Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8+ T Cells in Langerhans Cell Histiocytosis

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAFV600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAFV600E can be a source of neoantigens capable of eliciting effective antitumor CD8+ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8+ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8+ T cell density in n = 101 LCH-lesions, with BRAFV600E mutated lesions displaying significantly lower CD8+ T cell:CD1a+ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8+ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAFV600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAFV600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAFV600E expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from a

Gene expression profile of AIDS-related Kaposi's sarcoma

BACKGROUND: Kaposi's Sarcoma (KS) is a proliferation of aberrant vascular structures lined by spindle cells, and is caused by a gammaherpes virus (HHV8/KSHV). Its course is aggravated by co-infection with HIV-1, where the timing of infection with HIV-1 and HHV8 is important for the clinical outcome. METHODS: In order to better understand the pathogenesis of KS, we have analysed tissue from two AIDS-KS lesions, and from normal skin by serial analysis of gene expression (SAGE). Semi-quantitative RT-PCR was then used to validate the results. RESULTS: The expression profile of AIDS-related KS (AIDS-KS) reflects an active process in the skin. Transcripts of HHV8 were found to be very low, and HIV-1 mRNA was not detected by SAGE, although it could be found using RT-PCR. Comparing the expression profile of AIDS-KS tissue with publicly available SAGE libraries suggested that AIDS-KS mRNA levels are most similar to those in an artificially mixed library of endothelial cells and leukocytes, in line with the description of KS lesions as containing spindle cells with endothelial characteristics, and an inflammatory infiltrate. At least 64 transcripts were found to be significantly elevated, and 28 were statistically downregulated in AIDS-KS compared to normal skin. Five of the upregulated mRNAs, including Tie 1 and sialoadhesin/CD169, were confirmed by semi-quantitative PCR to be elevated in additional AIDS-KS biopsies. Antibodies to sialoadhesin/CD169, a known marker of activated macrophages, were shown to specifically label tumour macrophages. CONCLUSION: The expression profile of AIDS-KS showed 64 genes to be significantly upregulated, and 28 genes downregulated, compared with normal skin. One of the genes with increased expression was sialoadhesin (CD169). Antibodies to sialoadhesin/CD169 specifically labelled tumour-associated macrophages, suggesting that macrophages present in AIDS-KS lesions belong to a subset of human CD169+ macrophages