Activation-induced Cytidine Deaminase Research Articles

Abstract PO1-13-05: Genomic characteristics and its therapeutic implications in breast cancer patients with detectable molecular residual disease

Abstract Background: Breast cancer constitutes approximately 30% of cancers in women, with a mortality-to-incidence ratio of 15%. While early and middle-stage breast cancer patients can undergo radical surgical resection, postoperative recurrence remains a significant challenge for clinicians. Molecular residual disease (MRD) is the main cause of postoperative recurrence of breast cancer. However, the baseline tumor genomic characteristics and therapeutic implications of breast cancer patients with detectable MRD are still unknown. Methods: In this study, we enrolled 80 patients with breast cancer who underwent next-generation sequencing (NGS)-based genetic testing of 1,021 cancer-related genes performed on baseline tumor and postoperative plasma from Army Specialty Medical Center between June 2017 and January 2023. The criteria for the patient’s enrollment were: (1) pathological diagnosis as breast cancer, (2) no distal metastasis, (3) radical surgery and R0 resection, (4) 1021 cancer-related gene testing of the tumor, (5) plasma MRD testing performed within 3 months after surgery and before adjuvant therapy. Results: A total of 18 patients in our cohort had detectable MRD. Baseline clinical characteristics found that patients with higher clinical stages were more likely to have detectable MRD. Analysis of single nucleotide variations and small insertions/deletions in baseline tumors showed that somatic mutations in MAP3K1, ATM, FLT1, GNAS, POLD1, SPEN, and WWP2 were significantly enriched in patients with postoperative MRD. Oncogenic signaling pathway analysis revealed that alteration of the Cell cycle pathway was more likely to occur in patients with detectable MRD (p=0.0125). Mutational signature analysis showed that defective DNA mismatch repair and activation-induced cytidine deaminase (AID) mediated somatic hypermutation (SHM) were associated with detectable MRD. Somatic copy number variation analysis found that amplification of CDKN2A, HOXB13, PPM1D, MPL, and VHL was significantly enriched in patients with detectable MRD. According to the OncoKB database, 77.8% (14/18) of patients with detectable MRD had U.S. Food and Drug Administration-approved mutational biomarkers and targeted therapy. Conclusion: For the first time, our study reports genomic characteristics of breast cancer patients with detectable MRD. The tumor biology is probably the main driver of detectable MRD. We also found the vast majority of patients with detectable MRD have the opportunity to access targeted therapy. Keywords: Breast cancer, MRD, Genomic character, Cell cycle pathway, Defective DNA mismatch repair, Activation-induced cytidine deaminase Figure 1. Somatic mutational landscape and clinical actionability of breast cancer patients with detectable MRD A. Oncoplot of top 20 genes altered in patients with detectable MRD. B. Forest plot of different mutant genes between patients with detectable and undetectable MRD. OR means odds ratio. Inf means infinity. ＊ means P-value < 0.05. C. The ten cancer-related signaling pathways were affected by somatic mutations both in patients with detectable and undetectable MRD. ＊ means P-value < 0.05. D&E. The fraction and etiology of each signature in patients with detectable (D) or undetectable (E) MRD. F. The number of patients with detectable MRD in different actionable alterations. gBRCA1 means germline BRCA1 mutation. sBRCA1 means somatic BRCA1 mutation. sBRCA2 means somatic BRCA2 mutation. TMB-H means TMB-High. Citation Format: Xu Yan, Shu Zhang, Lu zhou, Yan Jiang, Jing Xu, Gang Zhang, Lu Shen. Genomic characteristics and its therapeutic implications in breast cancer patients with detectable molecular residual disease [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-05.

Genomic Characteristics and Its Therapeutic Implications in Breast Cancer Patients with Detectable Molecular Residual Disease.

Molecular residual disease (MRD) is the main cause of postoperative recurrence of breast cancer. However, the baseline tumor genomic characteristics and therapeutic implications of breast cancer patients with detectable MRD after surgery are still unknown. In this study, we enrolled 80 patients with breast cancer who underwent next-generation sequencing-based genetic testing of 1,021 cancer-related genes performed on baseline tumor and postoperative plasma, among which 18 patients had detectable MRD after surgery. Baseline clinical characteristics found that patients with higher clinical stages were more likely to have detectable MRD. Analysis of single nucleotide variations and small insertions/deletions in baseline tumors showed that somatic mutations in MAP3K1, ATM, FLT1, GNAS, POLD1, SPEN, and WWP2 were significantly enriched in patients with detectable MRD. Oncogenic signaling pathway analysis revealed that alteration of the Cell cycle pathway was more likely to occur in patients with detectable MRD (p=0.012). Mutational signature analysis showed that defective DNA mismatch repair and activation-induced cytidine deaminase (AID) mediated somatic hypermutation (SHM) were associated with detectable MRD. According to the OncoKB database, 77.8% (14/18) of patients with detectable MRD had U.S. Food and Drug Administration-approved mutational biomarkers and targeted therapy. Our study reports genomic characteristics of breast cancer patients with detectable MRD. The cell cycle pathway, defective DNA mismatch repair, and AID-mediated SHM were found to be the possible causes of detectable MRD. We also found the vast majority of patients with detectable MRD have the opportunity to access targeted therapy.

Abstract 403: APOBEC3 enzymes induce chromosomal translocations in solid cancer

Abstract Introduction: Chromosomal translocations are common oncogenic drivers in human cancers, and an increasing number of translocations are being considered as crucial diagnostic and prognostic markers in clinics. In B-cell lymphoma, a number of chromosomal translocations are directed by the off-target activity of the activation-induced cytidine deaminase (AID; a B-cell specific apolipoprotein B mRNA-editing enzyme, catalytic polypeptode-like (APOBEC) family enzyme). However, in solid tumors, no such enzymes capable of directing chromosomal translocations have been identified. Given recent findings on APOBEC-mediated mutagenesis in solid cancers, APOBECs may be involved in the formation of chromosomal translocations in solid tumors, like AID does in B cells. In human, the APOBEC3 (A3) family consists of seven members (A3A-A3H) and is associated with C <t or="" c<g="" substitutions.="" recent="" evidence="" shows="" that="" a3a="" might="" be="" involved="" in="" chromosomal="" instability="" leading="" to="" drug="" resistance="" lung="" cancer.="" however,="" it="" has="" not="" been="" explored="" whether="" a3s="" generate="" dna="" dsbs,="" eventually="" translocations.="" this="" study,="" we="" investigated="" the="" role="" of="" and="" their="" mechanisms="" translocations="" solid="" cancer.Methods: We applied high-throughput genome-wide translocation sequencing (HTGTS) approach to map genome-wide chromosomal translocations induced by human A3s in mouse fibroblast and human epithelial cells. We correlated patterns of translocations induced by A3s with several other cellular features including transcription, DNA accessibility, DNA replication timing, and mutational signatures. We validated the contribution of A3 to the generation of oncogenic translocations in lung cancer. Results: Ectopic expression of A3s significantly increased chromosomal translocations in mouse fibroblasts and human epithelial cells. A3s and AID preferentially induced translocations in different parts of the genome, but some of the translocation hotspots were shared among them. Hierarchical clustering of hotspots showed that A3-mediated translocations were distinguished from AID-mediated translocations. Notably, the junction distribution of breakpoint clusters showed three distinct translocation patterns based on the locations of breakpoint clusters: (1) AID-preferred; (2) A3-preferred; and (3) AID/A3-mixed. AID-mediated translocations were heavily enriched near the transcription start site, whereas A3-mediated translocations were dispersed across the gene body and transcription end site. Furthermore, DNA replication timing and genomic architectures were linked to the formation of A3-mediated chromosomal translocations in solid cancer. Finally, A3 contributed to the formation of oncogenic rearrangements in lung cancer. Conclusions: A3 enzymes induced chromosomal translocations with distinct patterns compared to AID and facilitated chromosomal translocation formation in solid cancers. Citation Format: Taek-Chin Cheong, Qi Wang, Ahram Jang, Elif Karaca-Atabay, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 403.

Abstract A052: ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell

Abstract Objective: Ovulation is a major risk factor of epithelial ovarian cancer (EOC), presumably acts from the cancer initiation stage. High-grade serous carcinoma (HGSC) is the most prevalent and fatal EOC, mainly arising from fallopian tube fimbrial epithelium (FTE). TP53 missense mutations are considered the earliest event of carcinogenesis since they form “p53 signatures” in FTE and were found in virtually all HGSC. Previously, we discovered that a subset of ovulatory follicular fluids (FF) harbored high levels of ROS, which were both mutagenic and oncogenic, causing DNA double-strand breaks (DSB) and tumorigenesis. However, DSB is unlikely the cause of TP53 mutations which are mostly CG: TA base substitutions involving deamination. We hypothesized that during ovulation FF exposes the fallopian tube fimbria and causes TP53 mutations by activating the activation-induced cytidine deaminase (AID). Materials and Methods: By conducting an HPRT gene mutation assay, we determined the mutagenic activity of FF on FTE cells with or without AID-knockdown. AID gene activation and protein nuclear translocation were analyzed in human FTE tissues and cells with different degrees of transformation, as well as in the oviducts of mice under controlled ovulation. The binding of AID to the TP53 gene and the subsequent deamination were determined by ChIP-PCR and uracil-qPCR, respectively. Results: After FF exposure, the mutation frequency of HPRT in FTE cells increased by 4.1 folds, at levels 40% higher than the ethyl methane sulfonate control and could be diminished by antioxidants or knockdown of the AID gene. AID activation, including mRNA and protein inductions and nuclear translocation, was evident in FTE cells and tissue after FF exposure and in mouse oviducts after ovulation. Two waves of AID activations were noted, peaking at 45 minutes (early activity) and 8.5 hours (late activity) after ovulation or FF exposure. TNF-α and ROS were found to be responsible for the activities in FF. Mechanistically, the early activity worked through the MAPK, while the late activity worked through the NF-κB pathway. The induced AID in both activities physically binds to and biochemically deaminates the TP53 gene irrelative to the known mutational hotspots of HGSC. However, it did not act on PTEN, the other prevalent tumor suppressor gene in HGSC. Meanwhile, ovulatory FF also activates the putative tumor-initiating and stemness gene LGR6 in FTE at locations where AID was also activated. Conclusion: The serial studies characterized the mutagenic activities of ovulatory FF, which acts on the LGR6-expressing cells in the fallopian tube fimbria and targets the TP53 gene. The study also identified ROS as the mutagen and pointed to strategies of ovulation inhibition and antioxidation for the prevention of EOC. Citation Format: Tang-Yuan Chu, Sanchana Subramani, Hsuan-Shun Huang. ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A052.

5-Chloro-2'-deoxycytidine Induces a Distinctive High-Resolution Mutational Spectrum of Transition Mutations In Vivo.

The biomarker 5-chlorocytosine (5ClC) appears in the DNA of inflamed tissues. Replication of a site-specific 5ClC in a viral DNA genome results in C → T mutations, which is consistent with 5ClC acting as a thymine mimic in vivo. Direct damage of nucleic acids by immune-cell-derived hypochlorous acid is one mechanism by which 5ClC could appear in the genome. A second, nonmutually exclusive mechanism involves damage of cytosine nucleosides or nucleotides in the DNA precursor pool, with subsequent utilization of the 5ClC deoxynucleotide triphosphate as a precursor for DNA synthesis. The present work characterized the mutagenic properties of 5ClC in the nucleotide pool by exposing cells to the nucleoside 5-chloro-2'-deoxycytidine (5CldC). In both Escherichia coli and mouse embryonic fibroblasts (MEFs), 5CldC in the growth media was potently mutagenic, indicating that 5CldC enters cells and likely is erroneously incorporated into the genome from the nucleotide pool. High-resolution sequencing of DNA from MEFs derived from the gptΔ C57BL/6J mouse allowed qualitative and quantitative characterization of 5CldC-induced mutations; CG → TA transitions in 5'-GC(Y)-3' contexts (Y = a pyrimidine) were dominant, while TA → CG transitions appeared at a much lower frequency. The high-resolution mutational spectrum of 5CldC revealed a notable similarity to the Catalogue of Somatic Mutations in Cancer mutational signatures SBS84 and SBS42, which appear in human lymphoid tumors and in occupationally induced cholangiocarcinomas, respectively. SBS84 is associated with the expression of activation-induced cytidine deaminase (AID), a cytosine deaminase associated with inflammation, as well as immunoglobulin gene diversification during antibody maturation. The similarity between the spectra of AID activation and 5CldC could be coincidental; however, the administration of 5CldC did induce some AID expression in MEFs, which have no inherent expression of its gene. In summary, this work shows that 5CldC induces a distinct pattern of mutations in cells. Moreover, that pattern resembles human mutational signatures induced by inflammatory processes, such as those triggered in certain malignancies.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients’ B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

The prognostic value of mutational signatures in pancreatic cancer.

689 Background: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor overall survival (OS), however there are significant outliers. Understanding the biological factors driving PDAC cancer may reveal new biomarkers and treatment strategies. Mutational signatures, imprinted in the cancer genome, encode the life history of the cancer and may be used to stratify tumors into clinically-relevant subgroups. Methods: In this study we performed mutational signature analysis (MutationalPatterns, COSMIC v3.3) on whole genomes from 434 PDAC patients, comprising 167 patients with resected primary tumors, and 267 advanced PDACs from patients enrolled in the COMPASS trial. We evaluated whether the mutational signatures detected associate with OS using the log-rank test in the overall cohort and stratified by disease stage, adjusting for multiple hypotheses with 5% false discovery rate (FDR). We further characterized these associations in exploratory analyses. Results: Among the 55 COSMIC signatures detected in the overall cohort, the presence of three signatures significantly associated with OS after FDR correction. SBS85, a signature associated with indirect effects of activation-induced cytidine deaminase, was present in 18/434 (4.1%) samples, and was significantly associated with improved OS in the overall study population (log-rank p=6.5e-4, FDR-adjusted-p=0.026). SBS93, a signature of unknown etiology previously seen in other gastrointestinal cancers, was present in 4/434 (0.9%) samples, and was significantly associated with worse OS in the overall study population (log-rank, p=7e-6; FDR-adjusted-p=1.1e-3). SBS33, also of unknown etiology and previously seen in other gastrointestinal cancers, was present in 73/434 (16.8%) samples. It was seen more frequently in advanced disease (Fisher’s-exact p=1.9e-4), but was significantly associated with worse OS only in the resected cohort (log-rank, p=2.3e-4, FDR-adjusted-p=0.012). Conclusions: We identified three mutational signatures—SBS85, SBS93, and SBS33—that have not previously been described in PDAC and associate with OS. Further characterization of these novel signatures in PDAC and their underlying biology may uncover new treatment avenues.

MCT1-governed pyruvate metabolism is essential for antibody class-switch recombination through H3K27 acetylation

Monocarboxylate transporter 1 (MCT1) exhibits essential roles in cellular metabolism and energy supply. Although MCT1 is highly expressed in activated B cells, it is not clear how MCT1-governed monocarboxylates transportation is functionally coupled to antibody production during the glucose metabolism. Here, we report that B cell-lineage deficiency of MCT1 significantly influences the class-switch recombination (CSR), rendering impaired IgG antibody responses in Mct1f/fMb1Cre mice after immunization. Metabolic flux reveals that glucose metabolism is significantly reprogrammed from glycolysis to oxidative phosphorylation in Mct1-deficient B cells upon activation. Consistently, activation-induced cytidine deaminase (AID), is severely suppressed in Mct1-deficient B cells due to the decreased level of pyruvate metabolite. Mechanistically, MCT1 is required to maintain the optimal concentration of pyruvate to secure the sufficient acetylation of H3K27 for the elevated transcription of AID in activated B cells. Clinically, we found that MCT1 expression levels are significantly upregulated in systemic lupus erythematosus patients, and Mct1 deficiency can alleviate the symptoms of bm12-induced murine lupus model. Collectively, these results demonstrate that MCT1-mediated pyruvate metabolism is required for IgG antibody CSR through an epigenetic dependent AID transcription, revealing MCT1 as a potential target for vaccine development and SLE disease treatment.

CD30 plays a role in T-dependent immune response and Tcell proliferation.

CD30 is a member of the tumor necrosis factor receptor (TNFR) superfamily and expressed in both normal and malignant lymphoid cells. However, the role of CD30 in lymphopoiesis is not known. In this study, we showed CD30 was expressed both in T and B cells, but its deficiency in mice had no effect on T- and B-cell development. In fact, CD30 deficiency attenuated B-cell response to T-cell-dependent antigens. The impaired B cell response in CD30-deficient mice is caused by the reduction of activation-induced cytidine deaminase (AID) expression. Moreover, CD30-deficient mice exhibited decreased TCR-mediated T cell proliferation and slightly impaired TCR signaling. High-throughput RNA sequencing analysis revealed that CD30 deficiency led to a decrease of FOXO-autophagy axis in T cells upon TCR stimulation. Thus, CD30 positively regulates T-cell-dependent immune response and T cell proliferation.

Enhanced C-To-T and A-To-G Base Editing in Mitochondrial DNA with Engineered DdCBE and TALED.

Mitochondrial base editing with DddA-derived cytosine base editor (DdCBE) is limited in the accessible target sequences and modest activity. Here, the optimized DdCBE tools is presented with improved editing activity and expanded C-to-T targeting scope by fusing DddA11 variant with different cytosine deaminases with single-strand DNA activity. Compared to previous DdCBE based on DddA11 variant alone, fusion of the activation-induced cytidine deaminase (AID) from Xenopus laevis not only permits cytosine editing of 5'-GC-3' sequence, but also elevates editing efficiency at 5'-TC-3', 5'-CC-3', and 5'-GC-3' targets by up to 25-, 10-, and 6-fold, respectively. Furthermore, the A-to-G editing efficiency is significantly improved by fusing the evolved DddA6 variant with TALE-linked deoxyadenosine deaminase (TALED). Notably, the authors introduce the reported high-fidelity mutations in DddA and add nuclear export signal (NES) sequences in DdCBE and TALED to reduce off-target editing in the nuclear and mitochondrial genome while improving on-target editing efficiency in mitochondrial DNA (mtDNA). Finally, these engineered mitochondrial base editors are shown to be efficient in installing mtDNA mutations in human cells or mouse embryos for disease modeling. Collectively, the study shows broad implications for the basic study and therapeutic applications of optimized DdCBE and TALED.

Activation-induced cytidine deaminase an antibody diversification enzyme interacts with chromatin modifier UBN1 in B-cells

Activation-induced cytidine deaminase (AID) is the key mediator of antibody diversification in activated B-cells by the process of somatic hypermutation (SHM) and class switch recombination (CSR). Targeting AID to the Ig genes requires transcription (initiation and elongation), enhancers, and its interaction with numerous factors. Furthermore, the HIRA chaperon complex, a regulator of chromatin architecture, is indispensable for SHM. The HIRA chaperon complex consists of UBN1, ASF1a, HIRA, and CABIN1 that deposit H3.3 onto the DNA, the SHM hallmark. We explored whether UBN1 interacts with AID using computational and in-vitro experiments. Interestingly, our in-silico studies, such as molecular docking and molecular dynamics simulation results, predict that AID interacts with UBN1. Subsequently, co-immunoprecipitation and pull-down experiments established interactions between UBN1 and AID inside B-cells. Additionally, a double immunofluorescence assay confirmed that AID and UBN1 were co-localized in the human and chicken B-cell lines. Moreover, proximity ligation assay studies validated that AID interacts with UBN1. Ours is the first report on the interaction of genome mutator enzyme AID with UBN1. Nevertheless, the fate of interaction between UBN1 and AID is yet to be explored in the context of SHM or CSR.

Cold-blooded vertebrates evolved organized germinal center-like structures.

Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)-specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non-M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA-associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igμ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.

The off-target effects of AID in carcinogenesis.

Activation-induced cytidine deaminase (AID) plays a crucial role in promoting B cell diversification through somatic hypermutation (SHM) and class switch recombination (CSR). While AID is primarily associated with the physiological function of humoral immune response, it has also been linked to the initiation and progression of lymphomas. Abnormalities in AID have been shown to disrupt gene networks and signaling pathways in both B-cell and T-cell lineage lymphoblastic leukemia, although the full extent of its role in carcinogenesis remains unclear. This review proposes an alternative role for AID and explores its off-target effects in regulating tumorigenesis. In this review, we first provide an overview of the physiological function of AID and its regulation. AID plays a crucial role in promoting B cell diversification through SHM and CSR. We then discuss the off-target effects of AID, which includes inducing mutations of non-Igs, epigenetic modification, and the alternative role as a cofactor. We also explore the networks that keep AID in line. Furthermore, we summarize the off-target effects of AID in autoimmune diseases and hematological neoplasms. Finally, we assess the off-target effects of AID in solid tumors. The primary focus of this review is to understand how and when AID targets specific gene loci and how this affects carcinogenesis. Overall, this review aims to provide a comprehensive understanding of the physiological and off-target effects of AID, which will contribute to the development of novel therapeutic strategies for autoimmune diseases, hematological neoplasms, and solid tumors.

IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sμ) and the 3' regulatory region (3'RR) (Sμ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sμ-3'RRrec counts per sample: Sμ-3'RRrecHigh cases (mostly unmutated CLL) and Sμ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sμ-3'RRrec appeared to be ongoing in Sμ-3'RRrecHigh CLL cells and comparison of Sμ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sμ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sμ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sμ-3'RRrec in Sμ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sμ-3'RRrec, even in the absence of AID for the latter.

The APOBEC3B c.783delG Truncating Mutation Is Not Associated with an Increased Risk of Breast Cancer in the Polish Population.

The APOBEC3B gene belongs to a cluster of DNA-editing enzymes on chromosome 22 and encodes an activation-induced cytidine deaminase. A large deletion of APOBEC3B was associated with increased breast cancer risk, but the evidence is inconclusive. To investigate whether or not APOBEC3B is a breast cancer susceptibility gene, we sequenced this gene in 617 Polish patients with hereditary breast cancer. We detected a single recurrent truncating mutation (c.783delG, p.Val262Phefs) in four of the 617 (0.65%) hereditary cases by sequencing. We then genotyped an additional 12,484 women with unselected breast cancer and 3740 cancer-free women for the c.783delG mutation. The APOBEC3B c.783delG allele was detected in 60 (0.48%) unselected cases and 19 (0.51%) controls (OR = 0.95, 95% CI 0.56-1.59, p = 0.94). The allele was present in 8 of 1968 (0.41%) familial breast cancer patients from unselected cases (OR = 0.80, 95% CI 0.35-1.83, p = 0.74). Clinical characteristics of breast tumors in carriers of the APOBEC3B mutation and non-carriers were similar. No cancer type was more frequent in the relatives of mutation carriers than in those of non-carriers. We conclude the APOBEC3B deleterious mutation p.Val262Phefs does not confer breast cancer risk. These data do not support the hypothesis that APOBEC3B is a breast cancer susceptibility gene.

Unraveling Mechanisms of Autoimmune Skin Blistering: Applying Single-Cell Transcriptomics to Pemphigus B Cells

Clinical Implications•Single-cell RNA sequencing of antigen-specific B cells results in novel, potentially druggable targets for therapy.•CD137L/CD137 signaling and BATF are critically involved in patients with active pemphigus.•Integration of published omics datasets on patients with pemphigus may hold great promise. •Single-cell RNA sequencing of antigen-specific B cells results in novel, potentially druggable targets for therapy.•CD137L/CD137 signaling and BATF are critically involved in patients with active pemphigus.•Integration of published omics datasets on patients with pemphigus may hold great promise. Autoimmunity against the skin is typically attributed to autoantibodies (with their corresponding B cells producing them) or T cells that react against autoantigens, resulting in disease that can be clinically and immunologically differentiated into distinct patterns in most patients (Gudjonsson et al., 2020Gudjonsson J.E. Kabashima K. Eyerich K. Mechanisms of skin autoimmunity: cellular and soluble immune components of the skin.J Allergy Clin Immunol. 2020; 146: 8-16Google Scholar). Among those, autoimmune skin conditions with potentially life-threatening blistering encompass entities from the pemphigus and pemphigoid groups, with the respective autoantigens being well-defined at the molecular level, facilitating research and diagnosis (Hammers and Stanley, 2016Hammers C.M. Stanley J.R. Mechanisms of disease: pemphigus and bullous pemphigoid.Annu Rev Pathol. 2016; 11: 175-197Google Scholar). In patients with pemphigus, autoantibodies against desmosomal transmembrane adhesion proteins desmoglein (DSG) 3 and DSG1 are formed and result in pathologies on the skin and/or mucous membranes that are explained by the presence or absence of those autoantibody reactivities: patients with mucosal-dominant pemphigus vulgaris (mPV) feature anti-DSG3 antibodies, whereas patients with mucocutaneous pemphigus vulgaris (mcPV) display anti-DSG3 and anti-DSG1 antibodies. In pemphigus foliaceus (PF), only the skin is affected, and patients show only anti-DSG1 antibodies in circulation. Intracellular signaling processes are involved in targeted keratinocytes, as evidenced by various cell biologic studies, complementing the direct steric effects mediated by these pathogenic autoantibodies (Hammers and Stanley, 2016Hammers C.M. Stanley J.R. Mechanisms of disease: pemphigus and bullous pemphigoid.Annu Rev Pathol. 2016; 11: 175-197Google Scholar). Although the role and pathogenicity of autoantibodies in these patients are increasingly understood, the characteristics of the B cells that produce them are not fully understood or elucidated. In their article published in the Journal of Investigative Dermatology, Egami et al., 2023Egami S. Watanabe T. Fukushima-Nomura A. Nomura H. Takahashi H. Yamagami J. et al.Desmoglein-specific B-cell-targeted single-cell analysis revealing unique gene regulation in patients with pemphigus [epub ahead of print].J Invest Dermatol. 2023; (accessed XXX)https://doi.org/10.1016/j.jid.2023.03.1661Google Scholar performed state-of-the-art single-cell transcriptomic analysis on autoimmune antigen-specific memory B cells from patients with pemphigus to unravel new pathways of pathophysiologic relevance. Over the past decade, much progress has been made in decoding autoimmune mechanisms at all stages of this paradigmatic model disease, with important input stemming from omics-based high-throughput technologies: antibody and/or B-cell receptor repertoires of patients with pemphigus were studied in great detail and partly over time by antibody phage display (Payne et al., 2005Payne A.S. Ishii K. Kacir S. Lin C. Li H. Hanakawa Y. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Google Scholar), Epstein−Barr virus−transformed hybridoma cells (Di Zenzo et al., 2012Di Zenzo G. Di Lullo G. Corti D. Calabresi V. Sinistro A. Vanzetta F. et al.Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface.J Clin Invest. 2012; 122: 3781-3790Google Scholar), and single-cell cloning after sorting (Cho et al., 2019Cho A. Caldara A.L. Ran N.A. Menne Z. Kauffman R.C. Affer M. et al.Single-cell analysis suggests that ongoing affinity maturation drives the emergence of pemphigus vulgaris autoimmune disease.Cell Rep. 2019; 28: 909-922.e6Google Scholar), yielding information on clonal diversity, processes of affinity maturation, antigen-binding properties, and synergistic effects of mAbs in mediating tissue pathology. By including next-generation sequencing approaches into these repertoire analyses, subclass-specific autoantibody repertoires (i.e., IgG4/IgG1, IgA1/IgA2) could be studied, providing new insights into their origins and formation in human disease (Ellebrecht et al., 2018Ellebrecht C.T. Mukherjee E.M. Zheng Q. Choi E.J. Reddy S.G. Mao X. et al.Autoreactive IgG and IgA B cells evolve through distinct subclass switch pathways in the autoimmune disease pemphigus vulgaris.Cell Rep. 2018; 24: 2370-2380Google Scholar). Proteomics-based deconvolution of pemphigus autoantibody repertoires finally allowed for unprecedented tracking of single-serum autoantibodies over time in patients, suggesting a much more clonally diverse and diverse landscape of autoantibodies in pemphigus (Chen et al., 2017Chen J. Zheng Q. Hammers C.M. Ellebrecht C.T. Mukherjee E.M. Tang H.Y. et al.Proteomic analysis of pemphigus autoantibodies indicates a larger, more diverse, and more dynamic repertoire than determined by B cell genetics.Cell Rep. 2017; 18: 237-247Google Scholar). Early genomic works applied microarrays to identify signatures of pemphigus vulgaris (PV)−associated differentially expressed genes (DEGs), comparing PBMCs from patients of differing clinical activity with those from controls (Dey-Rao et al., 2013Dey-Rao R. Seiffert-Sinha K. Sinha A.A. Genome-wide expression analysis suggests unique disease-promoting and disease-preventing signatures in pemphigus vulgaris.Genes Immun. 2013; 14: 487-499Google Scholar). Similar techniques were then used on single antigen-specific B cells to identify molecular effects of treatment, revealing pattern differences in IL1β and CD27 gene expression in the corticosteroid and rituximab treatment groups (Hébert et al., 2019Hébert V. Petit M. Maho-Vaillant M. Golinski M.L. Riou G. Derambure C. et al.Modifications of the transcriptomic profile of autoreactive B cells from pemphigus patients after treatment with rituximab or a standard corticosteroid regimen.Front Immunol. 2019; 10: 1794Google Scholar) or increases in the surface expression of the KCNN4 potassium channel on B cells after rituximab administration (Caillot et al., 2018Caillot F. Derambure C. Berkani N. Riou G. Maho-Vaillant M. Calbo S. et al.Long-term increase of Kcnn4 potassium channel surface expression on B cells in pemphigus patients after rituximab treatment.J Invest Dermatol. 2018; 138: 2666-2668Google Scholar). Total RNA sequencing of peripheral blood CD3−CD19+ lymphocytes from patients with pemphigus beautifully confirmed murine experimental data on reduced FcγRIIB signaling as an important driver for pathogenic class switching of autoantibodies also in human disease development (Nomura et al., 2022Nomura H. Wada N. Takahashi H. Kase Y. Yamagami J. Egami S. et al.IgM to IgG class switching is a necessary step for pemphigus phenotype induction in desmoglein 3-specific B cell receptor knock-in mouse.J Immunol. 2022; 208: 582-593Google Scholar). A trove of new transcriptomics data has been generated recently by studying lesional pemphigus skin samples that, in ectopic lymphoid-like structures, contain autoreactive B and T cells. Total RNA sequencing−based and microarray-based analyses not only led to the identification of an IL-17+ T-cell−governed autoimmune response (extending the concept of pemphigus just being a classical T helper 2−driven disease [Holstein et al., 2021Holstein J. Solimani F. Baum C. Meier K. Pollmann R. Didona D. et al.Immunophenotyping in pemphigus reveals a TH17/TFH17 cell-dominated immune response promoting desmoglein1/3-specific autoantibody production.J Allergy Clin Immunol. 2021; 147: 2358-2369Google Scholar]) but also to new potential epigenetic regulators (Huang et al., 2022Huang Z.X. Qu P. Wang K.K. Zheng J. Pan M. Zhu H.Q. Transcriptomic profiling of pemphigus lesion infiltrating mononuclear cells reveals a distinct local immune microenvironment and novel lncRNA regulators.J Transl Med. 2022; 20: 182Google Scholar) and new insights into the in vivo biology of autoimmune B cells (Zhou et al., 2020Zhou S. Liu Z. Yuan H. Zhao X. Zou Y. Zheng J. et al.Autoreactive B cell differentiation in diffuse ectopic lymphoid-like structures of inflamed pemphigus lesions.J Invest Dermatol. 2020; 140: 309-318.e8Google Scholar). Single-cell−sequencing technologies allow for the detection of pathologic dysregulations even in small subpopulations of cells, with regard to changes on the genetic, transcriptional, epigenetic, or proteomic levels, depending on the methodology applied. This technique can either be applied to complex samples (with preserved cellular heterogeneity) or to samples enriched for specific cell types, such as B cells. This latter approach was applied by Egami et al., 2023Egami S. Watanabe T. Fukushima-Nomura A. Nomura H. Takahashi H. Yamagami J. et al.Desmoglein-specific B-cell-targeted single-cell analysis revealing unique gene regulation in patients with pemphigus [epub ahead of print].J Invest Dermatol. 2023; (accessed XXX)https://doi.org/10.1016/j.jid.2023.03.1661Google Scholar on peripheral blood samples from three patients with pemphigus (one with PF, one with mcPV, and one with mPV), resulting in 103 DSG-specific sorted B cells and 128 non−DSG-specific sorted B cells as input material for single-cell RNA sequencing. For the one patient with PF, additional DSG1-specific B cells (31) were single-cell sorted after treatment, for comparison with 37 cells before treatment from that patient. The authors show that with their sorting and sequencing approach, B-cell receptor repertoire data can be obtained, also serving as an internal technical control for their experimentation. The authors then go on to identify, among upregulated DEGs, signatures of CD137L-mediated T-cell costimulation (with CD137L expression on antigen-specific B cells confirmed by flow cytometry), B-/T-cell differentiation (e.g., BATF), and inflammation (e.g., S100A8/A9, CCL3). In light of earlier published works and their translational potential, these findings are particularly intriguing. CD137L/CD137 signaling is involved in follicular T helper cell activation, a cell type recently implicated in pemphigus pathophysiology (Holstein et al., 2021Holstein J. Solimani F. Baum C. Meier K. Pollmann R. Didona D. et al.Immunophenotyping in pemphigus reveals a TH17/TFH17 cell-dominated immune response promoting desmoglein1/3-specific autoantibody production.J Allergy Clin Immunol. 2021; 147: 2358-2369Google Scholar). Moreover, the transcription factor BATF is known to be expressed in B cells, directly controlling activation-induced cytidine deaminase and thus acting as a regulator of class-switch recombination and somatic hypermutation (Ise et al., 2011Ise W. Kohyama M. Schraml B.U. Zhang T. Schwer B. Basu U. et al.The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells.Nat Immunol. 2011; 12: 536-543Google Scholar). Comparing DEGs before and after immunosuppressive treatment in a patient with PF, the authors identified, as expected, downregulation of genes and signaling implied in B-cell−mediated antibody production (i.e., CD27, peroxisome proliferator–activated receptor, APRIL), consistent with improvements in clinical activity and decreased serum antibody titers from treatment with corticosteroids and azathioprine. Studying cellular mechanisms of autoimmunity is challenging, especially so when rare diseases such as PV or PF are concerned; yet, at the same time, these conditions are very well-characterized with regard to their autoantigens, positioning them as model diseases for (theoretically) unbiased identification of new pathways and, potentially, treatment targets. As shown, Egami et al., 2023Egami S. Watanabe T. Fukushima-Nomura A. Nomura H. Takahashi H. Yamagami J. et al.Desmoglein-specific B-cell-targeted single-cell analysis revealing unique gene regulation in patients with pemphigus [epub ahead of print].J Invest Dermatol. 2023; (accessed XXX)https://doi.org/10.1016/j.jid.2023.03.1661Google Scholar succeeded in establishing an appropriate methodology for the isolation of antigen-specific B cells and subsequent single-cell RNA sequencing, holding great potential for translation into personalized medicine and studying more patients with PV, PF, and other dermatologic autoimmune diseases as well. From this and other works, it also becomes clear that the resulting datasets are becoming increasingly complex (and less intuitively accessible), requiring sophisticated bioinformatic analyses and the close collaboration of clinician scientists, basic researchers, informaticians, and statisticians. Finally, another future challenge (and chance) lies in the integration of various single-cell and non-single-cell omic datasets concerning one particular disease or group of diseases from laboratories worldwide, and to conclude and appropriately test promising treatment targets resulting from these strides. Christoph M. Hammers: http://orcid.org/0000-0001-5631-2415 The author states no conflict of interest. Desmoglein-Specific B-Cell−Targeted Single-Cell Analysis Revealing Unique Gene Regulation in Patients with PemphigusJournal of Investigative DermatologyPreviewAutoreactive B cells are assumed to play a critical role in pemphigus; however, the characteristics of these cells are not yet fully understood. In this study, 23 pemphigus vulgaris or pemphigus foliaceus samples were used to isolate circulating desmoglein (DSG)-specific B cells. Transcriptome analysis of the samples was performed at the single-cell level to detect genes involved in disease activity. DSG1- or DSG3-specific B cells from three patients’ differentially expressed genes related to T cell costimulation (CD137L) as well as B-cell differentiation (CD9, BATF, TIMP1) and inflammation (S100A8, S100A9, CCR3), compared with nonspecific B cells from the same patients. Full-Text PDF Open Access

Clinical utility of circulating tumor DNA qualification and quantification in classical Hodgkin lymphoma

Background: Classical Hodgkin lymphoma (cHL), the most common lymphoma in young adults, is a disease of poorly understood heterogeneity. The need for enriching tumor cells before assaying has limited the granularity of its molecular sub-classification. Although the cure rates of patients with cHL have increased over time, new biomarkers are still needed for more precise treatment decision-making in this complex patient population. Patients with cHL frequently have significant amounts of circulating tumor DNA (ctDNA), suggesting an alternative approach for studying this enigmatic tumor. Methods: IOSI-EMA003 (NCT03280394) is a prospective, observational, multi-center study of adult patients with newly diagnosed cHL aiming at: (i) identifying subgroups of patients with phenotype- and outcome-associated molecular signatures; (ii) testing and validating baseline ctDNA load as a prognostic biomarker; (iii) testing if ctDNA can be used as biomarker for the early identification of chemoresistance. Blood samples were collected at staging and disease response assessment. PET scans were centrally and blindly reviewed. ctDNA was analyzed by using deep targeted and low pass whole genome sequencing to measure ctDNA load, capture mutations and profile ctDNA fragmentation patterns. Single-cell RNA sequencing has been used to deconvolute the composition of the tumor microenvironment. Results: A total of 215 patients were enrolled. Based on ctDNA fragmentation patterns reflecting chromatin accessibility in the regulatory region of germinal center (GC) B-cell genes, we segregated cHL into two previously unknown subgroups that we named SNCD (for sub-nucleosomal cfDNA) and NCD (for nucleosomal cfDNA). We hypothesized that SNCD cHL stems from a cell that is closer to the GC B-cell differentiation stage than NCD cHL. SNCD cHL and NCD cHL differed in many aspects, including activation induced cytidine deaminase (AID)-hypermutation profile, neoantigen load, immune editing mechanism and response to chemotherapy and checkpoint inhibitors. Clinically, SNCD cHL displayed less sensitivity to both chemotherapy and anti-PD1 antibodies. Immune editing of SNCD cHL points loss of MHC-I, recruitment of Treg and upregulation of LAG3 as prominent immune checkpoint. High load of pre-treatment ctDNA nominated high-risk patients more accurately than clinico-radiological features. In addition, persistence of residual 18FDG avid lesions along with measurable ctDNA after two chemotherapy courses was a better proof of chemoresistance than the sole persistence of residual 18FDG avid lesions. Also, persistence of measurable ctDNA after the end of therapy invariably predicted progression. Conclusions: Collectively, our results provide a roadmap for cHL subtyping and for the clinical use of ctDNA as a biomarker to aid risk stratification and guide treatment decisions in a more personalized approach. Encore Abstract - previously submitted to ASCO 2023 The research was funded by: Research Advisory Board of the Ente Ospedaliero Cantonale, ABREOC 2019-22514, Bellinzona, Switzerland; European Research Council (ERC) Consolidator Grant CLLCLONE, ID: 772051; Swiss Cancer Research Foundation, ID 3746, 4395, 4660, 5257, Bern, Switzerland; Swiss National Science Foundation, ID 320030_169670/1, 310030_192439, 320036_179318, Berne, Switzerland; The Leukemia & Lymphoma Society, Translational Research Program, ID 6594-20, New York; AFRI, Ente Ospedaliero Cantonale, Bellinzona, Switzerland; ASH Global Research Award, Washington DC, USA; Worldwide Cancer Research (ID: 10583); ISREC Foundation; Helmut Horten Foundation, Switzerland; Fondazione Fidinam, Lugano, Switzerland; Nelia & Amadeo Barletta Foundation, Lausanne, Switzerland; Fond’Action, Lausanne, Switzerland; Fondazione Jacob, Lugano, Switzerland; Fondazione Dr. Ettore Balli, Lugano, Switzerland; Fondazione Ticinese per la Ricerca sul Cancro, Bellinzona, Switzerland; Jacques & Gloria Gossweiler Foundation, Berne, Switzerland. Keywords: Diagnostic and Prognostic Biomarkers, Hodgkin lymphoma, Liquid biopsy Conflicts of interests pertinent to the abstract. M. C. Pirosa Educational grants: BeiGene A. Condoluci Honoraria: AbbVie, AstraZeneca and Janssen Research funding: Gilead and Pfizer A. Moccia Honoraria: Janssen and Takeda A. Stathis Honoraria: Eli Lilly, Bayer, Roche, Novartis, Janssen Oncology, AstraZeneca Research funding: ADC Therapeutics, Pfizer, Roche, Novartis, Bayer, Eli Lilly, MEI Pharma, Cellestia, Debiopharm Group, Merck/MSD, Abbvie, Amgen, AstraZeneca, Incyte, Loxo, and Philogen C. Carlo-Stella Consultant or advisory role: Celgene/Bristol-Myers Squibb, ADC Therapeutics, Karyopharm 767 Therapeutics, Roche, and Sanofi Honoraria: Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, AstraZeneca, Incyte, Novartis, Takeda, ADC Therapeutics Research funding: ADC Therapeutics, Sanofi, Roche G. Gaidano Honoraria: Abbvie, Astra Zeneca, BeiGene, Incyte, Janssen E. Zucca Honoraria: BeiGene, BMS, Celltion Healthcare, Curis, Eli/Lilly, Incyte, Ipsen, Janssen, Kyte, Merck, Miltenyi, Roche Research funding: AstraZeneca, BMS, Incyte, Janssen, Merck, Roche Educational grants: Abbvie, BeiGene, Janssen and Roche D. Rossi Honoraria: AbbVie, AstraZeneca, BeiGene, BMS, Janssen Research funding: AbbVie, AstraZeneca, Janssen Educational grants: AstraZeneca and Janssen.

Circulating tumor DNA of classical Hodgkin lymphoma.

7516 Background: Classical Hodgkin lymphoma (cHL) is the most common lymphoma in young adults. The morphologic peculiarity that makes cHL unique are the few tumor cells surrounded by a polytypic inflammatory microenvironment. It is also the cancer with the highest sensitivity to immune checkpoint inhibitors. But why is it so immunogenic and how it escapes the immune control at the same time have only been partially elucidated. Although cure rates of cHL have constantly increased over the years there is still a high need for identification of biomarkers for more precise treatment decisions. The need for enriching tumor cells before assaying has limited cHL biological studies. Patients with cHL have significant amounts of circulating tumor DNA (ctDNA), suggesting an alternative approach for studying this enigmatic tumor. Methods: IOSI-EMA003 (NCT03280394) is a prospective, observational, multi-center study of adult patients with previously untreated cHL aiming at: i) identifying subgroups of patients with phenotype- and outcome-associated molecular signatures; ii) testing and validating baseline ctDNA load as a prognostic biomarker; iii) testing if ctDNA can be used for the early identification of chemoresistance. Blood samples were collected at staging and disease response assessment. PET scans were centrally and blindly reviewed. ctDNA was analyzed by using deep targeted and shallow whole genome sequencing to measure ctDNA load, capture mutations and profile ctDNA fragmentation patterns. The composition of the tumor microenvironment of cHL was probed by single cell RNA-seq. Results: A total of 215 patients were enrolled. Based on ctDNA fragmentation patterns reflecting chromatin accessibility in the regulatory region of germinal center (GC) B-cell genes, we segregated cHL into two subgroups that we named SNCD (for sub-nucleosomal cfDNA) and NCD (for nucleosomal cfDNA). We hypothesized that SNCD cHL stems from a cell that is closer to the GC B-cell differentiation stage than NCD cHL. SNCD cHL and NCD cHL differed in many aspects, including activation induced cytidine deaminase (AID)-hypermutation profile, neoantigen load, immune editing mechanism and response to chemotherapy and checkpoint inhibitors. Clinically, SNCD cHL displayed less sensitivity to both chemotherapy and anti-PD1 antibodies. Immune editing of SNCD cHL points loss of MHC-I, recruitment of Treg and upregulation of LAG3 as prominent immune checkpoint. High load of pre-treatment ctDNA nominated high-risk patients more accurately than clinico-radiological features. In addition, persistence of residual 18FDG avid lesions and measurable ctDNA was a better proof of chemoresistance than the sole persistence of residual 18FDG avid lesions. Conclusions: Our results provide a roadmap for cHL subtyping and molecular classification. We also propose ctDNA as a quantifiable and radiation-free biomarker to be used in clinical trials testing a more personalized treatment approach. Clinical trial information: NCT03280394 .

Genetic and transcriptomic analyses of diffuse large B‐cell lymphoma patients with poor outcomes within two years of diagnosis

Background: R-CHOP immunochemotherapy is the first-line treatment for DLBCL, but approximately 30%–40% of patients still experience refractory or relapsed (R/R) disease. We aimed to characterize the molecular features of DLBCL derived from patients with early R/R disease and develop a prognosis model to identify those high-risk patients. Methods: We performed a comprehensive genomic and transcriptomic characterization on 161 R-CHOP-treated DLBCL samples. These patients were grouped by follow-up outcome status after R-CHOP treatment. The first group had R/R disease within two years of diagnosis (poor outcome; n = 50), and the second group remained in remission at two years following R-CHOP treatment (good outcome; n = 111). In an addition, external cohorts were included in the validation analysis (GSE117556, n = 374; GSE181063, n = 810). Results: Patients with poor outcomes more often had an advanced stage of the disease, a high international prognostic index (IPI) and a non-GCB subtype of the tumor. We identified a set of frequent somatically mutated targets (Figure 1A), including PIM1, TP53, MPEG1 and ROBO1, as well as a specific mutational signature (activation-induced cytidine deaminase related) in DLBCLs with poor outcomes. Transcriptomic analyses further showed a distinct gene expression pattern and a less inflamed tumor microenvironment in these patients (Figure 1B). Finally, we developed an 11-gene signature as an independent prognostic marker for DLBCL patients treated with R-CHOP (Figure 1C). The 11-gene signature could further stratify DLBCL patients based on their IPI scores (Figure 1D). Additionally, the use of 11-gene signature to identify high-risk patients was also validated in two independent cohorts, suggesting the robustness of the risk model. Furthermore, our model effectively identified high-risk patients, including those with double-hit and MCD genetic subtypes that are associated with poor survival outcomes. FIGURE 1 (A) The mutation pattern between poor outcome and good outcome DLBCLs. (B) Tumor-infiltrating immune cells in DLBCL patients with poor and good outcomes. (C-D) Kaplan–Meier survival analysis of PFS between high-risk and low-risk groups (C) and the association of 11-gene risk scores and IPI risk groups (D). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Diagnostic and Prognostic Biomarkers, Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

B cell-specific knockout of AID protects against atherosclerosis

Antigen-naive IgM-producing B cells are atheroprotective, whereas mature B cells producing class-switched antibodies promote atherosclerosis. Activation-induced cytidine deaminase (AID), which mediates class switch recombination (CSR), would thus be expected to foster atherosclerosis. Yet, AID also plays a major role in the establishment of B cell tolerance. We sought to define whether AID affects atherosclerotic plaque formation. We generated Ldlr-/- chimeras transplanted with bone marrow from Aicda-/- or wild-type (WT) mice, fed a HFD for 14 weeks. Decreased B cell maturation in Ldlr-/-Aicda-/- mice was demonstrated by 50% reduction in splenic and aortic BAFFR expression, a key signaling component of B2 cell maturation. This was associated with increased plasma IgM in Ldlr–/-Aicda-/- compared with Ldlr-/-WT animals. Importantly, Ldlr-/-Aicda-/- mice had reduced atherosclerotic lesion area (0.20 ± 0.03mm2) compared with Ldlr-/-WT (0.30 ± 0.04mm2, P < 0.05), although no differences in plaque composition were noted between groups. In addition, immunofluorescence analysis revealed increased splenic B and T cell areas independent of cell number. AID depletion directly inhibits atherosclerotic plaque formation.