Recent Advances in the Genetic of MALT Lymphomas

Mucosa-associated lymphoid tissue (MALT) lymphomas are a diverse group of lymphoid neoplasms with B-cell origin, occurring in adult patients and usually having an indolent clinical behavior. These lymphomas may arise in different anatomic locations, sharing many clinicopathological characteristics, but also having substantial variances in the aetiology and genetic alterations. Chromosomal translocations are recurrent in MALT lymphomas with different prevalence among different sites, being the 4 most common: t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), and t(3;14)(p14.1;q32). Several chromosomal numerical abnormalities have also been described, but probably represent secondary genetic events. The mutational landscape of MALT lymphomas is wide, and the most frequent mutations are: TNFAIP3, CREBBP, KMT2C, TET2, SPEN, KMT2D, LRP1B, PRDM1, EP300, TNFRSF14, NOTCH1/NOTCH2, and B2M, but many other genes may be involved. Similar to chromosomal translocations, certain mutations are enriched in specific lymphoma types. In the same line, variation in immunoglobulin gene usage is recognized among MALT lymphoma of different anatomic locations. In the last decade, several studies have analyzed the role of microRNA, transcriptomics and epigenetic alterations, further improving our knowledge about the pathogenic mechanisms in MALT lymphoma development. All these advances open the possibility of targeted directed treatment and push forward the concept of precision medicine in MALT lymphomas.

Intrinsic Strand-Incision Activity of Human UNG: Implications for Nick Generation in Immunoglobulin Gene Diversification

Uracil arises in cellular DNA by cytosine (C) deamination and erroneous replicative incorporation of deoxyuridine monophosphate opposite adenine. The former generates C → thymine transition mutations if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by C by the base excision repair (BER) pathway. The primary human UDG is hUNG. During immunoglobulin gene diversification in activated B cells, targeted cytosine deamination by activation-induced cytidine deaminase followed by uracil excision by hUNG is important for class switch recombination (CSR) and somatic hypermutation by providing the substrate for DNA double-strand breaks and mutagenesis, respectively. However, considerable uncertainty remains regarding the mechanisms leading to DNA incision following uracil excision: based on the general BER scheme, apurinic/apyrimidinic (AP) endonuclease (APE1 and/or APE2) is believed to generate the strand break by incising the AP site generated by hUNG. We report here that hUNG may incise the DNA backbone subsequent to uracil excision resulting in a 3´-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5´-phosphate. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2´ occurs via the formation of a C1´ enolate intermediate. UIP is removed from the 3´-end by hAPE1. This shows that the first two steps in uracil BER can be performed by hUNG, which might explain the significant residual CSR activity in cells deficient in APE1 and APE2.

Does MRD have a role in the management of iNHL?

Among indolent non-Hodgkin lymphomas (iNHLs), the analysis of measurable/minimal residual disease (MRD) has been extensively applied to follicular lymphoma (FL). Treatment combinations have deeply changed over the years, as well as the techniques to measure MRD, which is currently evaluated only in the setting of clinical trials. Here, we discuss the evidence on the role of molecular monitoring in the management of FL. Mature data support the quantification of molecular tumor burden at diagnosis as a tool to stratify patients in risk categories and of MRD evaluation at the end of treatment to predict progression-free survival and overall survival. Moreover, MRD deserves further studies as a tool to refine the clinical/metabolic response and to modulate treatment intensity/duration. Patients with a higher relapse probability can be identified, but the relevance of continuous molecular follow-up should be clarified by kinetic models of MRD analysis. Being the BCL2/heavy chain immunoglobulin gene hybrid rearrangement detectable in about 50% to 60% of advanced FL and in 30% of positron emission tomography/computed tomography–staged localized FL, technical advancements such as next-generation sequencing/target locus amplification may allow broadening the FL population carrying a molecular marker. Droplet digital polymerase chain reaction can better quantify MRD at low levels, and novel sources of DNA, such as cell-free DNA, may represent a noninvasive tool to monitor MRD. Finally, MRD in other iNHLs, such as lymphoplasmacytic lymphoma/Waldenström macroglobulinemia and marginal zone lymphoma, is beginning to be explored.

Integrated Epigenetic and Transcriptional Single Cell Analysis of t(11;14) Myeloma and Its BCL2 Dependency

Multiple myeloma is characterized by recurrent chromosomal translocations that involve the immunoglobulin gene enhancers and partners such as the cyclin D genes (CCND1, CCND2 or CCND3) or other genes like WHSC1 and MAF. t(11;14) results in upregulation of CCND1 with unique morphological, phenotypic markers, and drug sensitivity profiles with exquisite sensitivity to BCL2 inhibitors. It is evident now that this unique sensitivity profile is driven by the BH3-proapoptotic protein priming of BCL2 with high BCL2/MCL1 or BCL2/BCL2L1 ratios. However, the epigenetic mechanisms associated with t(11;14) and their impact on genes regulation and clinical response to venetoclax remains elusive. In the present study we compared the transcriptomics (scRNA-seq) and chromatin accessibility (scATAC-seq) of single plasma cells of MM patients with and without t(11;14) as well as pre- and post-venetoclax exposure in order to establish the epigenomic signature of t(11;14) and/or BCL2-sensitivity in myeloma. Serial BM aspirates (n=24) were collected from 15 relapsed or refractory myeloma patients (RRMM); harboring t(11;14) (n=6 pairs) and 9 without this translocation prior to initiation of salvage therapy and at time of relapse. All t(11;14) MM patients were treated with venetoclax. Unbiased chromatin accessibility and mRNA profiling of CD138 pos cells were performed using the chromium single cell ATAC and RNA-Seq 3' solution (10x Genomics), respectively. Cell Ranger, Seurat and ArchR were used for sample de-multiplexing, barcode processing, single-cell 3' gene, peaks counting, and data analysis. We first compared the scATAC-seq and scRNA-seq profiles found in CD138 pos MM cell isolated from patients harboring t(11;14) with the one obtained in patients without this translocation. As expected, t(11:14) patients had high chromatin accessibility at the CCND1 locus and high mRNA expression. Differentially accessible chromatin analysis identified 147518 peaks that were specific to t(11;14) patients. Of interest, motifs enrichment analysis of accessible peaks identified a "B cell-like" motifs signature with enriched TFs motifs such as TCF4 and PAX5 in t(11;14) patients compared to non t(11:14) enriched for IRF and STAT family of motifs. The integration of the scATAC-seq and scRNA-seq data confirmed the B cell signature of t(11;14) patients with upregulation of B cell markers such as MS4A1, VPREB3, CD79A, CD19, and down-regulation of plasma cell markers such as TDO2, EFEMP1, CD28, SLAMF7, and IL6R. Additionally, we found PAX1, PAX5, TCF3, TCF5, and SPI1 transcription factors to be highly expressed in t(11;14) while the non t(11:14) were enriched for IRF1-9 transcription factors. Of interest, the clustering analysis performed on scATAC-seq data identified 3 non t(11;14) patients with a chromatin accessibility profile similar to that of t(11;14) patients. They expressed B cell markers (PAX5, VPREB3 or FCRLA), overexpressed BCL2 and we are currently examining whether this B cell-like epigenetic signature determines sensitivity to venetoclax. In order to define the epigenetic contribution to the acquired resistance to venetoclax in t(11;14) myeloma, we compared the chromatin accessibility profiles of t(11;14) patients pre- vs. post-venetoclax treatment. Enriched motifs within accessible peaks differed significantly between pre- and post-venetoclax with RELA, REL, RELB and EGR1 motifs predominantly enrichmed in the pre-samples in contrast to JUN, JUNB, JUND and FOSL1/L2 motifs enrichment in the post-samples. Of note, integration analysis of scRNAseq (differentially expressed genes) and ATACseq data (differentially accessible peaks) identified MCL1 and ENSA (a gene 60 Kb centromeric to MCL1 on chr1q) as the top enriched genes and peaks in resistant samples suggesting that copy number gain at the MCL1 locus (which we confirmed by single cell CNV analysis) rather than epigenetic modifications is likely the main determinant of acquired resistant to venetoclax in t(11;14) MM. In the current study we have defined the epigenetic regulome and transcriptome associated with t(11;14) myeloma and its relatedness to B cell rather than plasma cell biology. Our studies also suggest that acquired resistance to venetoclax is largely driven by copy number gain at the MCL1 locus. Disclosures Bahlis: Karyopharm: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria. Neri: BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Detection of clonotypic DNA in the cerebrospinal fluid as a marker of central nervous system invasion in lymphoma

Diagnosis of parenchymal central nervous system (CNS) invasion and prediction of risk for future CNS recurrence are major challenges in the management of aggressive lymphomas, and accurate biomarkers are needed to supplement clinical risk predictors. For this purpose, we studied a next-generation sequencing (NGS)-based assay that detects tumor-derived DNA for clonotypic immunoglobulin gene rearrangements in the cerebrospinal fluid (CSF) of patients with lymphomas. Used as a diagnostic tool, the NGS-MRD assay detected clonotypic DNA in 100% of CSF samples from 13 patients with known CNS involvement. These included 7 patients with parenchymal brain disease only, whose CSF tested negative by standard cytology and flow cytometry, and 6 historical DNA aliquots collected from patients at median 39 months prior to accession, which had failed to show clonal rearrangements using standard polymerase chain reaction. For risk prognostication, we prospectively collected CSF from 22 patients with newly diagnosed B-cell lymphomas at high clinical risk of CNS recurrence, of whom 8 (36%) had detectable clonotypic DNA in the CSF. Despite intrathecal prophylaxis, a positive NGS-MRD assay in the CSF was associated with 29% cumulative risk of CNS recurrence within 12 months from diagnosis, in contrast with 0% risk among patients with a negative assay (P=0.045). These observations suggest that detection of clonotypic DNA can aid in diagnosis of suspected parenchymal brain recurrence in aggressive lymphoma. Furthermore, the NGS-MRD assay might enhance clinical risk assessment for CNS recurrence among newly diagnosed patients and help select those who might benefit most from novel approaches to CNS-directed prophylaxis.

CRIS: Complete Reconstruction of Immunoglobulin V-D-J Sequences from RNA-seq data

Motivation B-cells display remarkable diversity in producing B-cell receptors through recombination of immunoglobulin V-D-J genes. Somatic hypermutation of immunoglobulin heavy chain variable (IGHV) genes are used as a prognostic marker in B-cell malignancies. Clinically, IGHV mutation status is determined by targeted Sanger sequencing which is a resource intensive and low-throughput procedure. Here we describe a bioinformatic pipeline, CRIS (Complete Reconstruction of Immunoglobulin IGHV-D-J Sequences) that uses RNA sequencing (RNA-seq) datasets to reconstruct IGHV-D-J sequences and determine IGHV somatic hypermutation status. Results CRIS extracts RNA-seq reads aligned to immunoglobulin gene (Ig) loci, performs assembly of Ig-transcripts and aligns the resulting contigs to reference Ig sequences to enumerate and classify somatic hypermutations in the IGHV gene sequence. CRIS improves on existing tools that infer the B-cell receptor (BCR) repertoire from RNA-seq data using a portion IGHV gene segment by de novo assembly. We show that the somatic hypermutation status identified by CRIS using the entire IGHV gene segment is highly concordant with clinical classification in three independent chronic lymphocytic leukemia patient cohorts. Availability The CRIS pipeline is available under the MIT License from https://github.com/Rashedul/CRIS. Supplementary information Supplementary data are available at Bioinformatics Advances online.

Nanopore sequencing approach for immunoglobulin gene analysis in chronic lymphocytic leukemia

The evaluation of the somatic hypermutation of the clonotypic immunoglobulin heavy variable gene has become essential in the therapeutic management in chronic lymphocytic leukemia patients. European Research Initiative on Chronic Lymphocytic Leukemia promotes good practices and standardized approaches to this assay but often they are labor-intensive, technically complex, with limited in scalability. The use of next-generation sequencing in this analysis has been widely tested, showing comparable accuracy and distinct advantages. However, the adoption of the next generation sequencing requires a high sample number (run batching) to be economically convenient, which could lead to a longer turnaround time. Here we present data from nanopore sequencing for the somatic hypermutation evaluation compared to the standard method. Our results show that nanopore sequencing is suitable for immunoglobulin heavy variable gene mutational analysis in terms of sensitivity, accuracy, simplicity of analysis and is less time-consuming. Moreover, our work showed that the development of an appropriate data analysis pipeline could lower the nanopore sequencing error rate attitude.

Pandemic, epidemic, endemic: B cell repertoire analysis reveals unique anti-viral responses to SARS-CoV-2, Ebola and Respiratory Syncytial Virus

Immunoglobulin gene heterogeneity reflects the diversity and focus of the humoral immune response towards different infections, enabling inference of B cell development processes. Detailed compositional and lineage analysis of long read IGH repertoire sequencing, combining examples of pandemic, epidemic and endemic viral infections with control and vaccination samples, demonstrates general responses including increased use of IGHV4-39 in both EBOV and COVID-19 infection cohorts. We also show unique characteristics absent in RSV infection or yellow fever vaccine samples: EBOV survivors show unprecedented high levels of class switching events while COVID-19 repertoires from acute disease appear underdeveloped. Despite the high levels of clonal expansion in COVID-19 IgG1 repertoires there is a striking lack of evidence of germinal centre mutation and selection. Given the differences in COVID-19 morbidity and mortality with age, it is also pertinent that we find significant differences in repertoire characteristics between young and old patients. Our data supports the hypothesis that a primary viral challenge can result in a strong but immature humoral response where failures in selection of the repertoire risks off-target effects.