DOI: 10.5061/dryad.612jm647m
title: Antigen-Independent, Autonomous B-cell Receptor Signalling in Diffuse Large B-cell Lymphoma

description: Diffuse large B-cell lymphoma (DLBCL) comprises two major cell-of-origin
 subtypes, germinal center B-cell (GCB) type and activated B-cell (ABC)
 type. ABC-DLBCL is characterized by chronic active B-cell receptor (BCR)
 signalling and NFκB activation, which is explained by activating mutations
 of the BCR signalling cascade in a minority of cases. We here demonstrate
 that autonomous BCR signalling, akin to its essential pathogenetic role in
 chronic lymphocytic leukemia (CLL), can explain chronic active BCR
 signalling in DLBCL. We show that 13 of 18 tested DLBCL-derived BCR
 induced spontaneous calcium flux in murine triple knock-out pre-B cells10
 in the absence of antigenic stimulation or external BCR crosslinking.
 Autonomous BCR signalling was associated with IgM isotype, dependent on
 somatic BCR mutations, and largely restricted to non-GCB DLBCL. Autonomous
 BCR signaling represents a novel immunological driver mechanism
 originating from individual BCR sequences and adds a new dimension to
 currently proposed genetics- and transcriptomics-based DLBCL
 classifications.
description: <strong>Cell lines and biopsies</strong> <br>
 Fresh-frozen biopsies of histologically confirmed DLBCL samples were
 identified in the pathology archive at Leiden University Medical Center
 (LUMC). The study was approved by the Scientific Review Committee of the
 LUMC Dept of Hematology under an applicable waiver of consent by the LUMC
 Ethical Committee (B16.048). <strong>Genetic
 analyses</strong> <br> Whole exome sequencing (WES) was
 performed on fragmented DNA with the SureSelect Human All Exon V7 kit
 (Agilent) capture on the HiSeq2000 (Illumina) platform to an average
 coverage of 50x. For the variant calling analysis, FASTQ files were
 processed using the Sarek workflow v2.7 and aligned to the human reference
 genome GRCh38 using the Burrows-Wheeler Algorithm (BWA) v0.7.17. 35,36
 Duplicated mapped reads were marked, local realignment of regions flanking
 indels, and recalibration of base quality scores were performed to obtain
 more accurate bases according to the Genome Analysis ToolKit (GATK) best
 practices version v4.1.7.0. 37 Single-nucleotide variants (SNV) and short
 insertions and deletions (INDELS) were called using Strelka2 v2.9.10. 38
 Only high confidence variants defined by quality scores (GQX) of at least
 15 for SNV and 30 for INDELS were kept. The resulting variant call files
 were annotated by Ensembl-VEP (v103) with four filtering steps. Variants
 were filtered for the NFkB signalling pathway of the Kyoto Encyclopedia of
 Genes and Genomes (www.kegg.jp/entry/map04064) and the most frequently
 mutated genes in DLBCL. Thereafter, variants were filtered by
 consequences, i.e. frameshift, in-frame deletion, missense, missense
 variant & splice region variant, splice region variant &
 synonymous variant, synonymous, in-frame insertion, stop gained, stop
 lost, frameshift variant & stop lost, missense variant &
 splice region variant, and coding sequence variant. Finally, variants were
 annotated for predicted effects by CADD phred, SIFT, and POLYPHEN scores,
 and according to clinical impact. Benign variants annotated in ClinVar
 202008 were discarded. Workflow quality control metrics were calculated
 and aggregated by MultiQC v1.8. 41 Recalibrated bam files from the variant
 calling workflow were used for the detection of copy number variations
 (CNV) by the somatic copy number variation workflow following the Broad’s
 recommended best practices using GATK4 CNV 37 with a panel of 24 normal
 tissue samples. The modelled segment files were filtered by genomic
 coordinates of the genes/regions of interest. Methods for processing the
 data: For whole exome sequencing (WES), DNA was amplified if necessary by
 isothermal alkaline genome amplification with Phi29 polymerase and random
 hexamer priming (REPLI-g kit; Qiagen). WES was performed on fragmented DNA
 with the SureSelect Human All Exon V7 kit (Agilent) capture on the
 HiSeq2000 (Illumina) platform to an average coverage of 50x.
description: # Antigen-independent, autonomous B cell receptor signaling drives
 activated B cell DLBCL
 [https://doi.org/10.5061/dryad.612jm647m](https://doi.org/10.5061/dryad.612jm647m) This dataset contains raw whole-exome sequencing (WES) data in paired-end FASTQ format for 15 patient samples diagnosed with diffuse large B-cell lymphoma (DLBCL). These data were generated to support downstream analyses, including variant calling, copy number variation (CNV) detection, structural variant identification, and molecular classification using the LymphGen algorithm. --- **Principal Investigator Contact Information** Name: Hendrik Veelken Institution: Leiden University Medical Center Email: [J.H.Veelken@lumc.nl](mailto:J.H.Veelken@lumc.nl) **Alternate Contact Information** Name: Cornelis van Bergen Institution: Leiden University Medical Center Email: [c.a.m.van_bergen@lumc.nl](mailto:c.a.m.van_bergen@lumc.nl) --- ## Description of the Data and File Structure ### File Format and Structure * **File type**: FASTQ (gzipped) * **Sequencing type**: Paired-end * **Platform**: Illumina HiSeq 2000 * **Capture Kit**: Agilent SureSelect Human All Exon V7 * **Reference genome**: GRCh38 * **Average coverage**: ~50× Each sample is represented by two FASTQ files: * `_1.fastq.gz`: Forward read * `_2.fastq.gz`: Reverse read Files follow the naming format: \[SampleID][SampleCase][ReadPair].fastq.gz * `SampleID`: Numeric identifier for the sample (e.g., 1, 3, 6...) * `SampleCase`: Case code used in the study * `ReadPair`: Either `1` (forward) or `2` (reverse) Example: `6_4760_1.fastq.gz` = Sample 6, Case 4760, forward read ### File List | Sample ID | Forward Read File | Reverse Read File | | --------- | ------------------------ | ------------------------ | | 5252 | 1\_5252\_1.fastq.gz | 1\_5252\_2.fastq.gz | | 3267 | 3\_3267\_1.fastq.gz | 3\_3267\_2.fastq.gz | | 3752 | 4\_3752\_1.fastq.gz | 4\_3752\_2.fastq.gz | | 4760 | 6\_4760\_1.fastq.gz | 6\_4760\_2.fastq.gz | | 3882 | 7\_3882\_1.fastq.gz | 7\_3882\_2.fastq.gz | | 4391 | 8\_4391\_1.fastq.gz | 8\_4391\_2.fastq.gz | | 2997 | 9\_2997\_1.fastq.gz | 9\_2997\_2.fastq.gz | | 3567 | 10\_3567\_1.fastq.gz | 10\_3567\_2.fastq.gz | | 5244 | 11\_5244\_1.fastq.gz | 11\_5244\_2.fastq.gz | | 3850 | 12\_3850\_1.fastq.gz | 12\_3850\_2.fastq.gz | | 3844 | 13\_3844\_1.fastq.gz | 13\_3844\_2.fastq.gz | | 3872 | 14\_3872\_1.fastq.gz | 14\_3872\_2.fastq.gz | | 208 | 15\_208\_1.fastq.gz | 15\_208\_2.fastq.gz | | 4328 | 16\_4328\_1.fastq.gz | 16\_4328\_2.fastq.gz | | 4609\_II | 17\_4609\_II\_1.fastq.gz | 17\_4609\_II\_2.fastq.gz | --- ## Experimental Context The data were generated and processed as follows: * **DNA Extraction**: Genomic DNA was extracted from frozen tissue. * **WES Library Preparation**: SureSelect Human All Exon V7 (Agilent) was used for capture. * **Sequencing Platform**: Illumina HiSeq 2000. * **Read Processing**: * Alignment with **BWA v0.7.17** to **GRCh38**. * Post-processing with **GATK v4.1.7.0** (duplicate marking, base recalibration). * **Variant calling** with **Strelka2 v2.9.10**. * **Annotation** using **Ensembl-VEP v103**, filtering by: * NF-κB pathway genes (KEGG map04064) * Frequent mutations in DLBCL (Chapuy et al., 2018) * Clinical impact scores (CADD, SIFT, PolyPhen) * Variants annotated as benign in ClinVar (version 202008) were excluded. * **Copy Number Variation**: * Detected using **GATK4 CNV**, with a panel of 24 normal samples. * **Structural Variants**: * Translocations involving **MYC**, **BCL2**, and **BCL6** were identified via **targeted locus capture-based sequencing**. --- ## Abbreviations | Term | Definition | | -------- | --------------------------------------- | | WES | Whole-Exome Sequencing | | FASTQ | Format for storing raw sequencing reads | | SNV | Single Nucleotide Variant | | INDEL | Insertion or Deletion | | CNV | Copy Number Variation | | GATK | Genome Analysis Toolkit | | VEP | Variant Effect Predictor | | DLBCL | Diffuse Large B-Cell Lymphoma | | CADD | Combined Annotation Dependent Depletion | | SIFT | Sorting Intolerant From Tolerant | | PolyPhen | Polymorphism Phenotyping | --- ## Code/Software The following tools and versions were used for data processing: * **Sarek Workflow v2.7** ([nf-core/sarek](https://nf-co.re/sarek)) * **BWA v0.7.17** – read alignment * **GATK v4.1.7.0** – duplicate marking, base recalibration, CNV detection * **Strelka2 v2.9.10** – variant calling * **Ensembl-VEP v103** – variant annotation * **MultiQC v1.8** – quality control reporting These tools were run in accordance with the best practice guidelines from the Broad Institute and the nf-core community. ---