Somatic Hypermutation Targeting Research Articles

PIM1 Point Mutations Increase Migration of Diffuse Large B-Cell Lymphoma (DLBCL) Cells

PIM1 serine-threonine kinase is known proto-oncogene, highly expressed in lymphoid malignancies. It regulates crucial processes for the cancer cell, like proliferation, apoptosis, metabolism or migration. In DLBCLs, PIM1 gene is a target of somatic hypermutation and one of the most frequently mutated genes in DLBCL patients. PIM1 mutations are more frequent in ABC-type tumors, which have worse survival prognosis than GCB-type, and often co-occur with mutations in MYD88 and CD79B. Analyses of the available clinical data show that the most significant differences between the groups of DLBCL patients with or without PIM1 mutations are tumor mutational burden, age at diagnosis and extranodal (testicular) involvement. In line with this finding, PIM1, MYD88 and CD79B mutations have also been reported to be highly prevalent in extranodal B cell lymphomas, localized in central nervous system (PCNSL), testicles (PTL) or intravascular space (IVLBCL). Our goal was to assess the role of the most common PIM1 point mutations in the biology and pathogenesis of DLBCL cells. The HBL-1 cell line was chosen as a model of MYD88 L265P-positive ABC-DLBCL subtype. PIM1 knock-out was performed using CRISPR/Cas9 method. Sequences of wild type PIM1, kinase-dead PIM1K67M, serving as a an inactive kinase control, and 7 most frequent mutants (PIM1G28D, PIM1E70K, PIM1P81L, PIM1S97N, PIM1P125A, PIM1L164F, PIM1L184F) were reintroduced to the HBL-1 PIM1 KO cells using lentiviral vectors. PIM1 mutations did not affect the proliferation of DLBCL cells and did not increase resistance to doxorubicin, excluding the proliferation rate or treatment resistance as a cause of the poor prognosis associated with PIM1 mutations. We performed RNA-seq analysis of the HBL-1-PIM1-WT and HBL-1-PIM1-mutants. The most upregulated pathways in PIM1 mutants (including PIM1K67M) were those related to Interferon gamma, IL-15, STAT3, cMET and semaphorin-plexin signaling (involved in proliferation, actin dynamics and migration of cancer cells). We also found that PIM1 mutants universally overexpress cMET oncogene. Moreover, gene set and pathway enrichment analyses indicated that the PIM1-mutant cells exhibit features associated with altered actin polymerization, RHO GTPases and cell membrane dynamics, suggesting the increased migratory potential of the cells and their propensity to spread to extranodal sites. Higher surface level of cMET in PIM1 mutant cells was confirmed using FACS analysis. Since cMET and CXCR4 cooperate in signaling and trigger AKT/mTOR, which is required for CXCR4-mediated cell motility, we evaluated whether PIM1 mutant cells would exhibit increased phosphorylation of AKT. In line with this hypothesis, we noted higher pAKT and pERK1/2 in PIM1 mutant cells than in PIM1 WT cells. Finally, in transwell assays, PIM1 mutant cells exhibited markedly increased migratory potential. Taken together, these studies indicate that PIM1 mutants are not likely associated with differential drug responses, but rather facilitate tumor dissemination and are thus associated with inferior prognosis in DLBCL patients harboring PIM1 mutations.

RCHOP Plus High-Dose Methotrexate As First-Line Therapy in Large B-Cell Lymphoma with Testis Involvement

Primary testicular lymphoma (PTL) and diffuse large B-cell lymphoma with secondary testicular involvement (DLBCL-T) are rare subtypes of lymphomas associated with poor prognosis and a high risk of central nervous system (CNS) relapse. The optimal CNS prophylaxis strategy for these subtypes remains uncertain. High-dose methotrexate (HD-MTX) is debated for CNS prophylaxis, but its efficacy in this setting is unknown. This study aimed to retrospectively analyze the use of HD-MTX in combination with chemotherapy (R HD-MTX CHOP) as frontline treatment for PTL and DLBCL-T and to compare the biological characteristics of these two forms of testicular involvement. Data from patients who received R HD-MTX CHOP as first-line treatment for DLBCL-T or PTL between 2010 and 2020 were collected retrospectively. The study included 15 patients, 5 with PTL and 10 with DLBCL-T. The overall response rate was 86%, with 73% of patients achieving complete remission. Treatment-related adverse events were generally manageable, with febrile neutropenia being the most common. Two patients experienced CNS relapse, both from the DLBCL-T subgroup, with a median time of 10.7 months from diagnosis to CNS relapse. None of the PTL patients receiving HD-MTX experienced relapse during the median follow-up of 6.08 years (Figure 1A), compared to 8/10 DLBCL-T (p=0.007). All five PTL cases had a non-germinal center phenotype, while 7 out of 10 DLBCL-T cases were non-germinal center. One DLBCL-T case exhibited high-grade lymphoma. Myc expression was positive in 3 out of 10 patients assessed, of whom all were double expressors. Bcl-2 expression was positive in all 13 assessable patients, while Bcl-6 expression was positive in 6 patients. Myc, Bcl-2, and Bcl-6 rearrangements were assessed in 10 patients and were present in 1 patient who was found to have triple-hit lymphoma. No other patient had double-hit lymphoma or any significant rearrangement. Polysomy of Myc, Bcl-2, and/or Bcl-6 was found in 2 patients. PDL1 expression was observed in one out of six patients analyzed, while no PD1 expression was detected. In 8 out of 15 patients with available tissue material, next-generation sequencing using a panel targeting 43 lymphoid genes was performed. Among these 8 patients, 87% exhibited gain-of-function mutations in MYD88 (MYD88 L265P), and 62% had missense mutations in CD79B at position Y196. Multiple mutations in the PIM1 gene, a known target of aberrant somatic hypermutation, were also observed in 7 patients. Non-recurrent mutations were detected in various genes, including PRDM1, CD58, B2M, CREBBP, CIITA, CARD11, CDKN2A, EP300, CCDND3, FOXO1, IRF4, NFKBIE, and TCF3 (Figure 1B). Ig rearrangement was analyzed in 4/9 patients with available DNA. All 4 were clonal, with distinct but mutated VH segments (sequence identity between 81 and 89%). This study suggests that HD-MTX in combination with RCHOP chemotherapy is highly effective in preventing CNS relapse in PTL but less so in DLBCL-T. DLBCL-T had a higher risk of CNS relapse and poorer outcomes compared to PTL. Further studies are needed to determine the optimal timing and sequence of HD-MTX in DLBCL-T, as well as alternative treatment approaches. PTL and DLBCL-T have distinct clinical presentations and responses to frontline chemotherapy with HD-MTX prophylaxis. PTL may benefit from HD-MTX in preventing relapse, while alternative treatments should be explored for DLBCL-T. Future studies should consider PTL and DLBCL-T as separate entities to better understand their characteristics and optimize treatment strategies.

Large B-cell Lymphomas of Immune-Privileged Sites Relapse via Parallel Clonal Evolution from a Common Progenitor B Cell.

Genomic analyses reveal that primary and relapse LBCL-IP originate from a common progenitor cell with a small set of genetic alterations, followed by extensive parallel diversification, elucidating the clonal evolution of LBCL-IP.

Differences in the immunoglobulin gene repertoires of IgG versus IgA multiple myeloma allude to distinct immunopathogenetic trajectories.

The analysis of the immunogenetic background of multiple myeloma (MM) has proven key to understanding disease ontogeny. However, limited information is available regarding the immunoglobulin (IG) gene repertoire in MM cases carrying different heavy chain isotypes. Here, we studied the IG gene repertoire in a series of 523 MM patients, of whom 165 and 358 belonged to the IgA and IgG MM groups, respectively. IGHV3 subgroup genes predominated in both groups. However, at the individual gene level, significant (p<0.05) differences were identified regarding IGHV3-21 (frequent in IgG MM) and IGHV5-51 (frequent in IgA MM). Moreover, biased pairings were identified between certain IGHV genes and IGHD genes in IgA versus IgG MM. Turning to the imprints of somatic hypermutation (SHM), the bulk of rearrangements (IgA: 90.9%, IgG: 87.4%) were heavily mutated [exhibiting an IGHV germline identity (GI) <95%]. SHM topology analysis disclosed distinct patterns in IgA MM versus IgG MM cases expressing B cell receptor IG encoded by the same IGHV gene: the most pronounced examples concerned the IGHV3-23, IGHV3-30 and IGHV3-9 genes. Furthermore, differential SHM targeting was also identified between IgA MM versus IgG MM, particularly in cases utilizing certain IGHV genes, alluding to functional selection. Altogether, our detailed immunogenetic evaluation in the largest to-date series of IgA and IgG MM patients reveals certain distinct features in the IGH gene repertoires and SHM. These findings suggest distinct immune trajectories for IgA versus IgG MM, further underlining the role of external drive in the natural history of MM.

GEN-13 PAIRED MUTATIONAL ANALYSIS IN SECONDARY NERVOUS SYSTEM LYMPHOMA AND PCNSL SYSTEMIC RELAPSE REVEALS DRIVER MUTATION CANDIDATES IN THE CENTRAL NERVOUS SYSTEM

Abstract Primary central nervous system lymphoma (PCNSL) is an aggressive extranodal non-Hodgkin lymphoma confined to the brain, eyes, and the spinal cord. The mechanism of central nervous system (CNS) tropism in PCNSL has not been fully elucidated. Diffuse large B cell lymphomas (DLBCLs) occasionally present with distant recurrence, which can involve inside and outside the CNS. Secondary central nervous system lymphomas (SCNSLs) are CNS relapse of systemic lymphoma. PCNSLs also rarely present with systemic relapse. We have previously reported in our study of whole exome sequencing that PCNSLs harbor frequent mutations in genes of B cell receptor pathway members and aberrant somatic hypermutation (aSHM) target genes. Although several genetic alterations were identified as more frequent in PCNSLs compared with systemic lymphomas, specific genetic alterations which serve as the driver for CNS tropism in PCNSLs has not been identified. In order to search for mutations which might serve as driver mutations in the CNS, we have performed targeted sequencing in paired samples from patients with recurrent lymphomas, either SCNSLs or PCNSL systemic relapses, using Ion Torrent multiplex PCR. Mutational profiles were compared between the primary and recurrent tumor. Six cases (four SCNSL cases and two PCNSL systemic relapse cases) were analyzed. Of note, in the SCNSL cases, several de novo mutations were enriched only among the recurrent CNS tumors. Among these mutations, BTG2 mutations were observed in 3/4 (75%), and B2M and KLHL14 mutations were observed in 2/4 (50%) cases. In the two PCNSL systemic relapse cases, KMT2D mutations were enriched only in the recurrent systemic tumors. It is suggested that these de novo mutations in the recurrent CNS tumors might serve as driver mutations in the CNS. Further analysis in larger cohorts, and functional studies are required in order to validate these findings.

Towards a Unified Genetic Classification System for Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell neoplasm and the most common lymphoid malignancy worldwide. DLBCL, NOS is a genetically and biologically complex entity that remains uniformly treated at diagnosis despite the growing appreciation for subgroups with molecular distinctions. There are various approaches for assigning DLBCL into one or more molecular subgroups using either gene expression and/or mutation patterns. Several genetic classification systems have been proposed, each sharing commonalities. Distinctions between the definitions of individual classes and the ability to reproducibly assign individual patient samples to subgroups using each system remains a significant barrier to their implementation, further emphasized by up to 50% of patients that remain unclassified. The disparities among the genetic features considered by each classification scheme further limits the ability to consolidate, let alone adopt, these for research and clinical studies. We aimed to develop a classification system that unifies and extends the robustness of existing classification algorithms. Methods: We assembled whole genome sequencing (WGS) data from 900 tumors from DLBCL, Burkitt (BL), follicular (FL) and other mature B-cell neoplasms to comprehensively determine the set of significantly mutated genes (SMG) and common targets of aberrant somatic hypermutation (aSHM). These were intersected with the features used by the current DLBCL classification systems and a curated set of lymphoma genes to tabulate the mutation matrix. Common structural rearrangements involving oncogenes were determined from WGS data and the copy number state of focal high-level recurrent alterations was united with simple somatic mutations. For genes containing mutation hot-spots each was considered separately. Results: Clustering all cases using non-negative matrix factorization (NMF) analysis resulted in a solution with 7 distinct genetic subgroups (Figure 1). Many of these directly resemble clusters in the existing systems with either expanded coverage or refinement of a subgroup into two. First, the BNZ cluster was highly enriched for any aberration in BCL6, 3'UTR mutations in NFKBIZ, and truncating mutations in NOTCH2. Unlike the similar BN2/C1/NOTCH2 groups defined previously, the NFKBIZ mutations were weighted more highly than those of NOTCH2 in defining the BNZ subgroup. The cluster we named MCaP resembled the MCD/C5/MYD88 group. Tumors classified in this subgroup had evidence of aSHM with frequent non-coding mutations in PIM1,CD44, GRHPR, PIM2, and BCL11A among others. Another cluster, MP3, was enriched for coding mutations or aSHM in the MYC TSS (MYC_aSHM), which were generally correspondent with IG::MYC rearrangements. MP3 tumors were characterized by TP53 mutations, including hotspots, and significantly depleted for aberrations in BCL2, BCL6 and lower mutation rates at aSHM targets SGK1, PAX5, CIITA, RHOH and others. Notably, tumors in this subgroup were enriched for IRF4 translocations, suggesting potential overlap with large B-cell lymphoma with IRF4 rearrangement. Our approach further refined both the the ST2/C4 and EZB/C3/BCL2 classes into two smaller subgroups, aSEL/aSCI and EGB/ETB, respectively. The EGB tumors were enriched for GNAI2 mutations, and MYC translocations involving the BCL6 enhancer, and were more often associated with MEF2B hotspot mutations. Notably, ETB was significantly enriched for mutations in the lysine acetyltransferase (KAT) domain in CREBBP, while EGB was significantly enriched for non-KAT domain mutations. ETB tumors were further characterized by mutations in STAT6 and FOXO1 hotspot regions, MYC rearrangements with other non-IG partners, and TNFRSF14 mutations, largely resembling the mutational profile of follicular lymphomas. Conclusions: We present here a refined classification of DLBCL genetic subgroups that combines previously described classification algorithms and extends it to allow classification of more tumors into additional subgroups that preserve the major divisions of existing systems. This represents an important advancement that will facilitate further understanding of genomic complexity in this disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutational patterns along different evolution paths of follicular lymphoma.

Follicular lymphoma (FL) is an indolent disease, characterized by a median life expectancy of 18-20 years and by intermittent periods of relapse and remission. FL frequently transforms into the more aggressive diffuse large B cell lymphoma (t-FL). In previous studies, the analysis of immunoglobulin heavy chain variable region (IgHV) genes in sequential biopsies from the same patient revealed two different patterns of tumor clonal evolution: direct evolution, through acquisition of additional IgHV mutations over time, or divergent evolution, in which lymphoma clones from serial biopsies independently develop from a less-mutated common progenitor cell (CPC). Our goal in this study was to characterize the somatic hypermutation (SHM) patterns of IgHV genes in sequential FL samples from the same patients, and address the question of whether the mutation mechanisms (SHM targeting, DNA repair or both), or selection forces acting on the tumor clones, were different in FL samples compared to healthy control samples, or in late relapsed/transformed FL samples compared to earlier ones. Our analysis revealed differences in the distribution of mutations from each of the nucleotides when tumor and non-tumor clones were compared, while FL and transformed FL (t-FL) tumor clones displayed similar mutation distributions. Lineage tree measurements suggested that either initial clone affinity or selection thresholds were lower in FL samples compared to controls, but similar between FL and t-FL samples. Finally, we observed that both FL and t-FL tumor clones tend to accumulate larger numbers of potential N-glycosylation sites due to the introduction of new SHM. Taken together, these results suggest that transformation into t-FL, in contrast to initial FL development, is not associated with any major changes in DNA targeting or repair, or the selection threshold of the tumor clone.

Molecular subclusters of follicular lymphoma: a report from the United Kingdom’s Haematological Malignancy Research Network

AbstractFollicular lymphoma (FL) is morphologically and clinically diverse, with mutations in epigenetic regulators alongside t(14;18) identified as disease-initiating events. Identification of additional mutational entities confirms this cancer’s heterogeneity, but whether mutational data can be resolved into mechanistically distinct subsets remains an open question. Targeted sequencing was applied to an unselected population-based FL cohort (n = 548) with full clinical follow-up (n = 538), which included 96 diffuse large B-cell lymphoma (DLBCL) transformations. We investigated whether molecular subclusters of FL can be identified and whether mutational data provide predictive information relating to transformation. DNA extracted from FL samples was sequenced with a 293-gene panel representing genes frequently mutated in DLBCL and FL. Three clusters were resolved using mutational data alone, independent of translocation status: FL_aSHM, with high burden of aberrant somatic hypermutation (aSHM) targets; FL_STAT6, with high STAT6 & CREBBP mutation and low aSHM; and FL_Com, with the absence of features of other subtypes and enriched KMT2D mutation. Analysis of mutation signatures demonstrated differential enrichment of predicted mutation signatures between subgroups and a dominant preference in the FL_aSHM subgroup for G(C>T)T and G(C>T)C transitions consistent with previously defined aSHM-like patterns. Of transformed cases with paired samples, 17 of 26 had evidence of branching evolution. Poorer overall survival (OS) in the aSHM group (P = .04) was associated with older age; however, overall tumor genetics provided limited information to predict individual patient risk. Our approach identifies 3 molecular subclusters of FL linked to differences in underlying mechanistic pathways. These clusters, which may be further resolved by the inclusion of translocation status and wider mutation profiles, have implications for understanding pathogenesis as well as improving treatment strategies in the future.

Ig Enhancers Increase RNA Polymerase II Stalling at Somatic Hypermutation Target Sequences.

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

Identification of Recurrent Non-Coding Driver Mutations in Non-Hodgkin Lymphomas through Integrative Genomic Analysis of 777 Patients

IntroductionDiffuse large B-cell lymphoma (DLBCL) represents the most frequent NHL and comprises two molecular subgroups, namely germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subgroups, each having distinct gene expression and genetic features. Follicular lymphoma (FL) is an indolent cancer that shares genetic features with DLBCL, particularly the GCB subgroup. Despite a growing knowledge about molecular features of these cancers. There remains a limited understanding of the recurrence of noncoding mutations that may impact gene function or expression in DLBCL, owing, in part, to their genetic complexity and a paucity of whole genome sequence (WGS) data. We sought to uncover novel mutation patterns through global meta-analysis of a large cohort of DLBCLs comprising 353 discovery cases, an independent set of 300 validation cases and comparison to genomes from 124 FLs.MethodsDLBCL exomes with matched constitutional DNA (n=200) from multiple studies and genomes from prior publications (40), ICGC (41) and 72 additional patients (n=153) were analyzed using a unified somatic single nucleotide variant (SNV) pipeline. Significantly mutated genes (SMGs) were identified using OncodriveFML. Recurrent regions affected by noncoding mutations (“peaks”) were determined using a novel approach - an extension of the rainfall plot computation followed by wavelet analysis. Structural variants (SV) were detected using Manta and copy number variations (CNVs) were determined using read-depth strategies. A separate set of 124 FL genomes was analyzed similarly. RNA-seq data were available for 143 of the DLBCL genomes and were processed using Salmon and analyzed for differential expression using DeSeq2. Using a custom capture panel, 82 genes were sequenced in some of the discovery cases and an independent DLBCL cohort comprising 300 cases. This allowed us to confirm and determine the recurrence of mutations and SVs affecting coding exons and UTRs. RNA-seq and SNP6.0 arrays were also applied to all validation cases to confirm patterns of coordinated expression.ResultsWe detected 52 SMGs (Fig. 1A) and 4382 mutation peaks among the DLBCL meta-cohort. Up to 24 of these have not previously been ascribed to DLBCL (Fig. 1A, purple). Most mutation peaks proximal to genes affected the first intron or promoter, consistent with AID activity. Many of the affected genes are known targets of aberrant somatic hypermutation though some novel targets of this process were identified. Non-genic peaks tended to affect putative distal enhancers of BCL6, IRF4 and PAX5. A subset of the noncoding regions and novel SMGs exhibited specificity for mutation within either ABC or GCB DLBCLs, including some genes involved in NF-kB signalling. Less than 10% of peaks were mutated among the FLs and these were largely those with a higher mutation burden in GCB DLBCL. We identified numerous genes recurrently affected by SVs, SNVs and CNVs and determined the subset of those with expression altered in the presence of such candidate regulatory mutations. For example, MEF2B and TOX both showed patterns indicative of tumour suppressor function, including a large number of focal deletions (Fig. 1C,D). In contrast, we observed patterns consistent with a role of mutations promoting the expression of each of TCF4, ΙΚΒΚΕ, NFKBIZ (two regulators of NF-κB) and the Fc γ receptor locus (Fig. 1B). TCF4 harbored mainly SV breakpoints that may disrupt some of the many annotated isoforms and leave others intact whereas NFKBIZ was predominantly affected by mutations in the 3'UTR. The FC γ locus contained numerous examples of focal gains and rearrangements involving this region and these were also detected in some of the FLs.ConclusionThis integrative genomic analysis offers numerous insights into the biology of DLBCL and features that distinguish it from indolent cancers arising in the germinal center. The novel coding and noncoding mutations found herein and those enriched in either DLBCL subgroup will aid to extend our understanding of the molecular underpinnings of these and their relationship to FL. Additional genes involved in NF-κB have been found to be deregulated through unique genetic mechanisms that have evaded detection though exome-based analyses. Further, the expression and sequence of Fc γ receptors is thought to impact the efficacy of anti-CD20 therapies, though the prevalence of somatic alterations has not been thoroughly explored. [Display omitted] DisclosuresSehn:Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy. Connors:F Hoffmann-La Roche: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding; NanoString Technologies: Research Funding; Lilly: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Cephalon: Research Funding; Bristol-Myers Squibb: Research Funding; Bayer Healthcare: Research Funding; Seattle Genetics: Research Funding; Merck: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Scott:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; BCCA: Patents & Royalties: Patent describing molecular subtyping of DLBCL licensed to NanoString Technologies. Patent describing measurement of the proliferation signature in MCL.. Morin:Epizyme, Inc: Consultancy.

Comprehensive Genomic Characterization of Refractory Multiple Myeloma Reveals a Complex Mutational and Structural Landscape Associated with Drug Resistance

Comprehensive Genomic Characterization of Refractory Multiple Myeloma Reveals a Complex Mutational and Structural Landscape Associated with Drug Resistance

Genomic Landscape of Primary Cutaneous Diffuse Large B Cell Lymphoma, Leg Type

Primary cutaneous diffuse large B-cell lymphoma, leg type (DLBCL-LT) is an aggressive subtype of cutaneous B cell lymphoma (CBCL) with a poor prognosis. It is most often diagnosed on the legs of the elderly, frequently relapses, and has a tendency towards extracutaneous dissemination. Current treatment strategies are ineffective and revolve around surgical excision, multiagent chemotherapy (e.g. R-CHOP) and radiation therapy, with estimated 5-year survival rates of ~50%. Identifying genetic mutations and altered pathways specific for DLBCL-LT is crucial for the diagnosis and development of effective targeted therapies for this condition.We performed whole exome capture and massively parallel sequencing on 13 tumor samples of DLBCL-LT (4 matched and 9 unmatched) and 5 samples of cutaneous diffuse large B cell lymphoma, not otherwise specified (DLBCL-NOS) (1 matched and 4 unmatched). This is the largest whole exome cohort of cutaneous DLBCL to date. We applied algorithms routinely used by our lab to identify putative cancer promoting mutations and to compare the genetic landscape with DLBCL, both nodal and extranodal.Broadly, the landscape of mutations in DLBCL-LT is similar to that of activated B-cell like (ABC) DLBCL and most analogous to primary central nervous system lymphoma (PCNSL), a type of extranodal ABC DLBCL. Similar to PCNSL, we found frequent disease-defining oncogenic gene mutations in MYD88 (85%), CD79B (38%), and TBL1XR1 (38%), and loss of function mutations in tumor suppressor genes KMT2D (15%), CDKN1B (15%), and CD58 (8%). We observed frequent mutations in PIM1 (54%), OSBPL10 (31%), BTG2 (31%), and IGLL5 (15%), which are either known or proposed targets of activation-induced cytidine deaminase (AID) and aberrant somatic hypermutation (aSHM) also seen in PCNSL. Importantly and similarly to PCNSL, we found alterations at the PDL-1 and PDL-2 locus, but instead of copy number gains, we discovered frequent PD-L1 and PD-L2 translocations (40%) that may translate into overexpression of PD-L1 .This spectrum of mutations differed significantly compared to our DLBCL-NOS cohort, which did not have the above oncogenic mutations and lacked any PD-L1 or PD-L2 chromosomal alterations. Finally, we report novel oncogenic mutations in DLBCL-LT including MYD88 p.S243N, CD79B p.L199P, CCND3 p.T283P and p.P284S, BRAF p.V600E, and CARD11 p.R113Q and novel loss of function mutations in KMT2D, CDKN1B, CD58, MSH2, PIK3R1, MSH2, and BMF .Collectively, we identified recurrent mutations in multiple key cancer-related biological pathways, including epigenetic modulators (TBL1XR1, HIST1H1E, and KMT2D), NF-κB signaling pathway (MYD88, CD79B, and CARD11), MAPK signaling pathway (BRAF), PI3K signaling pathway (PIK3R1), JAK/STAT signaling pathway (PIM1), cell cycle regulators (CCND3, CDKN1B and BTG2), and immune escape (CD58). All of the DLBCL-LT samples in our cohort contained one or more gene mutations that can be targeted by agents that are currently FDA-approved or under clinical trials, such as BRAF inhibitors, JAK/STAT inhibitors, PIM inhibitors, PI3K inhibitors, NF-kB inhibitors, MYD88 inhibitors, and PD-1/PDL-1 inhibitors.Altogether, our data identified targetable putative driver gene mutations in DLBCL-LT. These mutational signatures bear broad similarities to PCNSL. Because of the relative rarity of this disease, we hope these efforts will allow for its inclusion, when relevant, in trials involving genotypically similar DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Position-Dependent Differential Targeting of Somatic Hypermutation.

Somatic hypermutation (SHM) generates much of the Ab diversity necessary for affinity maturation and effective humoral immunity. The activation-induced cytidine deaminase-induced DNA lesions and error-prone repair that underlie SHM are known to exhibit intrinsic biases when targeting the Ig sequences. Computational models for SHM targeting often model the targeting probability of a nucleotide in a motif-based fashion, assuming that the same DNA motif is equally likely to be targeted regardless of its position along the Ig sequence. The validity of this assumption, however, has not been rigorously studied in vivo. In this study, by analyzing a large collection of 956,157 human Ig sequences while controlling for the confounding influence of selection, we show that the likelihood of a DNA 5-mer motif being targeted by SHM is not the same at different positions in the same Ig sequence. We found position-dependent differential SHM targeting for about three quarters of the 38 and 269 unique motifs from more than half of the 292 and 1912 motif-allele pairs analyzed using productive and nonproductive Ig sequences, respectively. The direction of the differential SHM targeting was largely conserved across individuals with no allele-specific effect within an IgH variable gene family, but was not consistent with general decay of SHM targeting with increasing distance from the transcription start site. However, SHM targeting did correlate positively with the mutability of the wider sequence neighborhood surrounding the motif. These findings provide insights and future directions for computational efforts toward modeling SHM.

Transcription factor binding at Ig enhancers is linked to somatic hypermutation targeting.

Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)-three processes that are initiated by activation-induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer-like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E-box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eμ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eμ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eμ mutants. Our results provide novel insights into trans-acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.

Distinct subtypes of diffuse large B-cell lymphoma defined by hypermutated genes.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease whose personalized clinical management requires robust molecular stratification. Here, we show that somatic hypermutation (SHM) patterns constitute a marker for DLBCL molecular classification. The activity of SHM mutational processes delineated the cell of origin (COO) in DLBCL. Expression of the herein identified 36 SHM target genes stratified DLBCL into four novel SHM subtypes. In a meta-analysis of patients with DLBCL treated with immunochemotherapy, the SHM subtypes were significantly associated with overall survival (1642 patients) and progression-free survival (795 patients). Multivariate analysis of survival indicated that the prognostic impact of the SHM subtypes is independent from the COO classification and the International Prognostic Index. Furthermore, the SHM subtypes had a distinct clinical outcome within each of the COO subtypes, and strikingly, even within unclassified DLBCL. The genetic landscape of the four SHM subtypes indicated unique associations with driver alterations and oncogenic signaling in DLBCL, which suggests a possibility for therapeutic exploitation. These findings provide a biologically driven classification system in DLBCL with potential clinical applications.

AID preferentially targets the top strand in nucleosome sequences

AID initiates both somatic hypermutation (SHM) and class switch recombination (CSR) in Ig genes. AID-induced mutations are linked with transcription initiation and elongation. Transcription occurs in the context of chromatin and thus RNA PolII and AID need to deal with nucleosomes. Both nucleosome stability and positioning significantly influence the accessibility of AID to Ig genes and the SHM pattern. Interestingly, in the nucleosome, SHM process seems to have a preference for the top strand. To know whether the preferential targeting of SHM to the top strand is due to a post-AID event, we expressed an inhibitor of Uracil DNA glycosylase (UNG), Ugi, into DT40 cells containing the nucleosome positioning sequence (MP2) and compared the SHM pattern. We observed a similar preference to the top strand for the high-affinity nucleosome positioning sequence in UNG inhibited cells. Furthermore, to understand whether the primary sequence of nucleosome sequence is influencing preferential targeting, we introduced two copies of MP2 sequence in the reverse orientation (MP2R) into a variable Ig gene. We observed that the MP2R cells also demonstrated preferential targeting of the non-transcribed strand in nucleosome as compared to the transcribed strand, confirming that in nucleosome sequences AID has better access to Cs on the top strand. The preferential targeting of AID on the top strand suggests that RNA Pol-II stalls while it transcribes the stable nucleosomes, thus giving ample opportunity for the transcribed strand to form R-loops with the nascent RNA, thereby gives limited access to AID on the bottom strand.

Topologically Associated Domains Delineate Susceptibility to Somatic Hypermutation

Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes of activated B cells to support the process of antibody affinity maturation but also causes off-target mutations in other parts of the genome. We have used sensitive lentiviral SHM reporter vectors and a mutationally active human B cell line to identify dozens of regions of the genome that are intrinsically susceptible to SHM (hot regions) and many hundreds of regions that are resistant to SHM (cold regions). Hot and cold regions are frequently contained within topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while overall levels of transcription are equal, hot TADs are enriched for NIPBL (a component of the cohesin loader), super enhancers, markers of paused/stalled RNA polymerase 2, and multiple transcription factors implicated in B cell development and targeting of SHM. We demonstrate that at least some hot TADs contain enhancer elements that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs.

Beyond Hot Spots: Biases in Antibody Somatic Hypermutation and Implications for Vaccine Design.

The evolution of antibodies in an individual during an immune response by somatic hypermutation (SHM) is essential for the ability of the immune system to recognize and remove the diverse spectrum of antigens that may be encountered. These mutations are not produced at random; nucleotide motifs that result in increased or decreased rates of mutation were first reported in 1992. Newer models that estimate the propensity for mutation for every possible 5- or 7-nucleotide motif have emphasized the complexity of SHM targeting and suggested possible new hot spot motifs. Even with these fine-grained approaches, however, non-local context matters, and the mutations observed at a specific nucleotide motif varies between species and even by locus, gene segment, and position along the gene segment within a single species. An alternative method has been provided to further abstract away the molecular mechanisms underpinning SHM, prompted by evidence that certain stereotypical amino acid substitutions are favored at each position of a particular V gene. These “substitution profiles,” whether obtained from a single B cell lineage or an entire repertoire, offer a simplified approach to predict which substitutions will be well-tolerated and which will be disfavored, without the need to consider path-dependent effects from neighboring positions. However, this comes at the cost of merging the effects of two distinct biological processes, the generation of mutations, and the selection acting on those mutations. Since selection is contingent on the particular antigens an individual has been exposed to, this suggests that SHM may have evolved to prefer mutations that are most likely to be useful against pathogens that have co-evolved with us. Alternatively, the ability to select favorable mutations may be strongly limited by the biases of SHM targeting. In either scenario, the sequence space explored by SHM is significantly limited and this consequently has profound implications for the rational design of vaccine strategies.

A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data.

Analyses of somatic hypermutation (SHM) patterns in B cell Ig sequences have important basic science and clinical applications, but they are often confounded by the intrinsic biases of SHM targeting on specific DNA motifs (i.e., hot and cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized B1-8 H chain transgenic mice with nitrophenyl to stimulate nitrophenyl-specific λ+ germinal center B cells and sequenced the unexpressed κ L chains using next-generation methods. Most of these κ sequences had out-of-frame junctions and were presumably uninfluenced by selection. Despite being nonfunctionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We used 39,173 mutations to construct a quantitative SHM targeting model. The model showed targeting biases that were consistent with classic hot and cold spots, yet revealed additional highly mutable motifs. We observed comparable targeting for functional and nonfunctional sequences, suggesting similar biological processes operate at both loci. However, we observed species- and chain-specific targeting patterns, demonstrating the need for multiple SHM targeting models. Interestingly, the targeting of C/G bases and the frequency of transition mutations at C/G bases was higher in mice compared with humans, suggesting lower levels of DNA repair activity in mice. Our models of SHM targeting provide insights into the SHM process and support future analyses of mutation patterns.

Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells.

The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.