BCL6 Research Articles

Abstract P13: Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity

Abstract IG translocations occur in various B-cell neoplasms and result in the deregulation of (onco)genes due to their juxtaposition with regulatory elements in the immunoglobulin loci, predominantly the immunoglobulin heavy chain (IGH) locus (14q32). Some of these IGH translocations are pathognomonic for distinct disease entities, like those involving MYC, BCL2 or CCND1 in Burkitt, Follicular or Mantle Cell Lymphomas, respectively. In Chronic Lymphocytic Leukemia (CLL), the BCL3 gene (19q13), a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family, is a known partner gene in these translocations. Two subsets of IG::BCL3 translocation-positive B-cell neoplasms have been described that exhibit differences in chromosomal aberrations, IGHV mutation status, and histopathology [Martín-Subero et al. 2007]. In CLL, IG::BCL3 translocation is linked to a younger age at diagnosis, a more aggressive clinical course, and an atypical tumor cell phenotype [Michaux et al. 1997; Au et al. 2002]. A total of 89 B-cell neoplasms, primarily diagnosed as CLL, displaying IG::BCL3 translocation detected by fluorescence in situ hybridization (FISH), were examined. Analysis of DNA methylation and copy number aberrations (CNA) was conducted for 86 IGH::BCL3-translocated B-cell neoplasms using 850K EPIC BeadChip array data. Evaluation of chromosomal aberrations typical for CLL was moreover performed by FISH and the IGHV mutation status was determined. IGH::BCL3 breakpoint junctions were sequenced in 23 samples using targeted capture and whole-genome sequencing approaches. RNA expression of BCL3 was assessed using the HTG mRNA pan B-cell panel. In UMAP analysis of DNA methylation, CLLs with IG::BCL3 translocation exhibited segregation from other CLLs, irrespective of IGH translocations. Supervised analysis revealed global hypomethylation of B-cell neoplasms with IG::BCL3 translocation compared to other CLL. A total of 66/69 CLLs with IG::BCL3 translocation showed an unmutated IGHV status (96%). Copy number determination using BeadChip data (n=86) and FISH analysis (n=85) showed a significantly higher incidence of trisomy 12 in IG::BCL3-translocated cases compared to the general CLL population (p=0.011). Breakpoint sequencing indicated IG::BCL3 junctions to be recurrently associated with aberrant class-switch recombination at the IGH locus, involving IGHA (n=11/23), IGHG (n=5/23) and IGHM segments (n=5/23). Breakpoints at the BCL3 locus displayed two groups. 80 cases (all CLL) were located upstream of BCL3 and two other B-cell neoplasms (NHL, DLBCL) were located downstream of BCL3. BCL3 expression analysis in 15 IG::BCL3-translocated CLLs confirmed its transcriptional upregulation compared to the general CLL population. Our genetic and epigenetic analyses support the view that CLL with IG::BCL3 translocation may constitute a genetically and epigenetically distinct subtype of B-cell neoplasms, diverging from the typical CLL. Citation Format: Cosima Drewes, Cristina López, Nnamdi Okeke, Sina Hillebrecht, Petra Schütz, Anja Fischer, Anja Mottok, Susanne Bens, Christof Schneider, Stephan Stilgenbauer, Eugen Tausch, Reiner Siebert. Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P13.

Exploring the pathogenesis and key genes associated of acute myocardial infarction complicated with Alzheimer’s disease

Despite mounting evidence linking Acute Myocardial Infarction (AMI) to Alzheimer’s disease (AD), the shared mechanism of these two conditions’ occurrence remains unclear. This research aims to delve deeper into the molecular process of the occurrence of the two diseases. We retrieved the gene expression profiles of AD (GSE5281) and AMI (GSE66360) from the Gene Expression Omnibus database. Then, a total of 22 common differentially expressed genes (DEGs) including one downregulated gene and 21 upregulated genes were chosen for further analysis. Following the discovery of the common DEGs between AMI and AD, we performed protein–protein interaction analysis and hub gene identification analysis. Next, ten important hub genes were identified. Additionally, the key genes were identified by the least absolute shrinkage and selection operator and support vector machine‐recursive feature elimination and multivariable logistic regression analysis. The BCL6 was identified to be the most connected with AMI and AD. Finally, the BCL6 gene was validated in the GSE40680 (AMI) and GSE122063 (AD) datasets. Our research indicates that AMI and AD share a comparable pathophysiology. The Hub genes, especially BCL6, were essential in developing AMI and AD. In addition, these hub genes and shared pathways can offer fresh perspectives for additional mechanism investigation.

Investigation of iso-propylchaetominine anticancer activity on apoptosis, cell cycle and Wnt signaling pathway in different cancer models

Dysregulation of the Wnt signaling pathway contributes to the development of many cancer types. Natural compounds produced with biotechnological systems have been the focus of research for being a new drug candidate both with unlimited resources and cost-effective production.In this study, it was aimed to reveal the effects of isopropylchaetominine on cytotoxic, cytostatic, apoptotic and Wnt signaling pathways in brain, pancreatic and prostate cancer.The IC50 values of isopropylchaetominine in U-87 MG, PANC1, PC3 and LNCaP cells were calculated as 91.94 μM, 41.68 μM, 54.54 μM and 7.86 μM in 72nd h, respectively. The metabolite arrests the cell cycle in G0/G1 phase in each cancer cells. Iso-propylchaetominine induced a 4.3-fold and 1.9-fold increase in apoptosis in PC3 and PANC1 cells, respectively. The toxicity of isopropylchaetominine in healthy fibroblast cells was assessed using the annexin V method, and no significant apoptotic activity was observed between the groups treated with the active substance and untreated. In U-87 MG, PANC1, PC3, and LNCaP cells under treatment with isopropylchaetominin, the expression levels of DKK3, TLE1, AES, DKK1, FRZB, DAB2, AXIN1/2, PPARD, SFRP4, APC and SOX17 tumor suppressor genes increased significantly. Decreases in expression of Wnt1, Wnt2, Wnt3, Wnt4, Wnt5, Wnt6, Wnt10, Wnt11, FRZ2, FRZ3, FRZ7, TCF7L1, BCL9, PYGO, CCND2, c-MYC, WISP1 and CTNNB1 oncogenic genes were detected.All these result shows that isopropylchaetominine can present promising new treatment strategy in different cancer types.

Efficacy and Safety of Zanubrutinib-Containing Regimens in Patients with Secondary CNS Relapse in Diffuse Large B-Cell Lymphoma

Background: In the era of immune-targeted therapy represented by rituximab, despite improved remission and survival rates in patients with diffuse large B-cell lymphoma (DLBCL), central nervous system (CNS) relapse, occurring in some DLBCL patients after achieving remission, remains a clinical challenge. Zanubrutinib is one of a new generation of BTK inhibitors that have higher blood-brain barrier permeability compared to other BTK inhibitors. Research Design and Methods: We performed a multicenter retrospective study to evaluate the efficacy and safety of Zanubrutinib-containing regimens for salvage treatment in 28 relapsed DLBCL patients with CNS involvement treated in 6 centers from January 2020 to December 2021. Results: The IPI score and CNS-IPI score did not have good enough predictive significance for lack of consistency of the median time from the initial diagnosis to CNS recurrence. Of the 28 patients, 16 had targeted next-generation sequencing (NGS) data. NGS test was performed on their tissue samples that were obtained at initial diagnosis and/or at the timepoint of CNS relapse in DLBCL. 8 at the time of initial diagnosis and 10 at the time of recurrence, with 2 of them having undergone NGS sequencing at both initial diagnosis and recurrence. Median study follow-up from CNS relapse was 8.55 months, all patients were evaluated for response with 20/28 (71.4 %) showing a response: 11 CR, 9 PR, and 4 SD, 4 PD. The median PFS is 6.6 months, and the median OS has not yet been reached. Notably, MYD88, CD79B, or TP53 mutations tested at diagnosis in these patients were associated with significantly more favorable treatment response, while patients with a dual rearrangement of MYC and BCL2 and/or BCL6 genes did not get a treatment response to Zanubrutinib-containing regimens. It has well-tolerated toxicity with few adverse events requiring treatment discontinuation or dose reduction. Conclusion: Zanubrutinib-containing regimens extended PFS and OS in relapsed DLBCL patients with CNS involvement.

DA-EPOCH-R for High Grade B-Cell Lymphoma Patients with MYC and BCL2 and/or BCL6 Rearrangements: Clinical Results of the Induction Phase of the HOVON-152 Trial

Introduction Patients (pts) with high grade B-cell lymphoma (HGBL) with MYC and BCL2 and/or BCL6 gene rearrangements (double hit and triple hit (DH/TH)) have poor outcomes to standard R-CHOP. Retrospective studies reported improved disease-free survival (DFS) through intensification with dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R). This regimen was studied in a small prospective trial including 24 DH/TH and 19 single MYC rearranged (SH) lymphomas (Dunleavy, Lancet Haematol 2019). No prospective studies evaluating DA-EPOCH-R exclusively for pts with DH/TH HGBL have been reported. The HOVON-152 trial aimed to improve outcomes in untreated DH/TH HGBL pts by investigating the efficacy of the immune checkpoint inhibitor nivolumab as consolidation treatment in pts achieving complete metabolic response (CMR) after DA-EPOCH-R induction. Here, we present the efficacy and safety profile of the induction phase with DA-EPOCH-R. Methods HOVON-152 is a prospective, multi-center, single arm phase II trial. Inclusion criteria were pts with newly diagnosed HGBL-DH/TH (according to the WHO 2016 classification), age ≥ 18 years, WHO performance status (PS) 0-3 and Ann Arbor stage II-IV. During the screening period for rearrangement status, pts could receive 1 cycle of R-CHOP or DA-EPOCH-R, followed by 5 cycles of DA-EPOCH-R. All pts received intrathecal CNS prophylaxis. All diagnostic lymphoma samples were centrally reviewed. PET-CT scans were performed at diagnosis, after 3 cycles and at end-of-induction (local review, central review will be reported at the conference). Pts in CMR after induction treatment (Deauville 1-3 or a negative lymphoma biopsy in case of Deauville 4) proceeded to nivolumab consolidation (480 mg every 4 weeks for one year). The HOVON-152 aimed to improve 12 months DFS of pts in CMR after induction from an expected 70% to 85% with nivolumab consolidation. Secondary objectives included evaluation of response rates, overall survival (OS) and safety. With a power of 0.90 a sample size of 97 pts was calculated. Here, we report efficacy (CMR rate) and safety of DA-EPOCH-R induction treatment. Logistic univariate analysis is used to analyze baseline characteristics associated with response. Adverse events (AEs) were defined according to the common terminology criteria for adverse events (CTC AE 5.0), counted by the highest grade per system organ class per patient. Results From August 2018 - March 2022, 97 pts have been enrolled (study inclusion completed). One patient was excluded due to CNS localization. The median age was 62 years (range 35-79); 90 (83%) pts had stage III-IV disease and 52 (54%) had (intermediate-)high international prognostic index (IPI) (Table 1). Central pathology review confirmed DH/TH in all pts. 65 pts (67%) had a BCL2 DH, 11 (12%) a BCL6 DH and 16 (17%) a TH. Dose adjustments were performed conform protocol. The maximum dose-level (DL) achieved was DL1 in 41 (43%), DL2 in 22 (23%), DL3 in 25 (25%), DL4 in 7 (7%) and DL5 in 1 (1%) of the pts. Vincristine dose was reduced in 28/81 (35%) pts. After DA-EPOCH-R induction, 63/96 (66%, 95% CI 55-75%) pts achieved CMR. WHO PS 2-3 (odds ratio (OR) 0.15, 95% CI 0.03-0.85, p=0.03), elevated LDH (OR 0.24, 95%CI 0.10-0.59, p=0.002) and bulky disease defined as ≥10 cm mass (OR 0.23, 95%CI 0.09-0.56, p=0.001), were significantly associated with a lower chance of achieving CMR. During treatment, 7 (7%) pts experienced a grade 5 AE (ileus, intestinal perforation, multi-organ failure/sepsis). Thereof, 4 patients died during DA-EPOCH-R and 3 patients died after DA-EPOCH-R. 31 (32%) experienced a grade 4 AE (e.g. sepsis, perforation, hemorrhage, thrombocytopenia and neutropenia), 21 (22%) a grade 3 AE (e.g. anemia, mucositis, infections and electrolyte disturbances) and 16 (17%) pts a grade 2 AE. Conclusion We report the largest prospective series of DH/TH HGBL patients treated with DA-EPOCH-R. DA-EPOCH-R induction was feasible, with toxicity and dose adjustments as previously described. The observed CMR rate of 66% was lower than previously reported in a prospective cohort of mixed DH/TH and SH patients (74%). For patients achieving CMR, the nivolumab consolidation phase is ongoing. Translational side studies investigating predictive factors to identify patients not achieving CMR are ongoing. For these patients, novel strategies to improve first-line treatment are warranted.

Oral follicular lymphoma: a clinicopathologic and molecular study.

Follicular lymphoma is a hematolymphoid neoplasm that originates from germinal center B cells. It is made up of a combination of small cleaved centrocytes and a varying quantity of larger non-cleaved centroblasts to describe the clinical, microscopic, immunohistochemical, and molecular features of oral follicular lymphomas. Follicular lymphomas affecting the oral cavity were retrieved from pathology files. Immunohistochemistry was performed to confirm the diagnosis, and fluorescence in situ hybridization (FISH) was employed to detect rearrangements in BCL2, BCL6, and MYC genes. Clinical and follow-up data were obtained from the patient's medical and pathology files. Twenty cases were obtained. There was an equal sex distribution (10 males: 10 females) and a mean age of 60.9 years (range: 10-83 years-old). Lesions presented as asymptomatic swellings, usually in the palate (10 cases) and the buccal mucosa (7 cases). Five patients presented with concomitant nodal involvement. Microscopic evaluation depicted the follicular growth pattern with diffuse areas in six cases. Grades 1 and 2 follicular lymphomas represented 12 cases, while grade 3A neoplasms accounted for other 8 cases. Two cases showed rearrangements in MYC, BCL2, and BCL6 genes, while single BCL2 translocation was found in eight cases. Two cases had no translocation. Three patients deceased and the 2-year overall survival achieved 88%. Follicular lymphoma affecting the oral cavity is uncommon, usually affects the palate as a non-ulcerated swelling and the presence of a systemic disease most always be ruled out.

STAT5-Feedback Controls Distinct Metabolic States for Dynamic Transitions between Cellular Activation and Quiescence in Acute Lymphoblastic Leukemia

Background and significance: B-cell acute lymphoblastic leukemia (B-ALL) is characterized by dynamic shifts between activation and quiescence. These cycles are predicated by fluctuations in oncogenic signaling, which mimic pre-BCR and cytokine receptor signaling, e.g. IL7 receptor signaling and phosphorylation of STAT5. These fluctuations in cytokine receptor and STAT5 signaling are mitigated by powerful feedback regulators, including CISH and SOCS (suppressors of cytokine signaling) family members. While B-ALL cells depend on oncogenic STAT5 signaling, CISH and SOCS2 negative feedback regulators of STAT5 are highly expressed in aggressive B-ALL and high expression levels are strongly linked to poor clinical outcome, positive MRD and increased likelihood of bone marrow relapse in children (COG P9906) and adults (ECOG E2993). To determine whether high levels of CISH and SOCS2 are biomarkers of high oncogenic STAT5 signaling or mechanistically contribute to clonal fitness and adaptability of B-ALL, we performed genetic experiments in mouse models for B-ALL. Here we examined the mechanisms and consequences of active and inactive states of STAT5 by studying constitutively active (CA) and non-phosphorylatable Y694F (YF) mutations of STAT5. Results: Consistent with a central role of STAT5-feedback control in B-ALL survival, inducible ablation of Cishfl/fl resulted in rapid depletion of B-ALL cells. Socs2-/- B-ALL cells rapidly underwent cellular senescence, lacked colony forming and leukemia-initiating capacity in serial transplantation experiments. Likewise direct interference with STAT5-function, by mutations resulting in constitutive activation (STAT5-CA) or an inactive state (STAT5-YF). Consistent with a requirement of adaptive STAT5-feedback control in B-ALL, both Stat5-CA and Stat5-YF resulted in rapid cell death. Induction of Stat5-CA caused accumulation of biomass indicated by their increased cell size, whereas Stat5-YF caused rapid cell shrinkage ( Figure A). To determine whether the mTOR cell growth pathway was responsible for cell death induced by Stat5-CA, we tested whether the genetic deletion of Mtor or pharmacological inhibition of protein synthesis were sufficient to rescue B-ALL cell death. As expected, B-ALL cells retaining intact Mtor rapidly underwent energy crisis and cell death, while genetic deletion of Mtor reversed the toxic effects of Stat5-CA, suggesting that Stat5-CA is associated with excessive protein synthesis and defective ER turnover. Indeed, Stat5-CA increased protein synthesis rate and ER content compared to EV control, and these effects were opposed by Stat5-YF. To compare the metabolic outcomes of Stat5-CA and Stat5-YF, we performed mass spectrometry-based metabolomic analyses ( Figure B). Consistent with the role of mTOR in promoting glycolysis, Stat5-CA increased the content of glycolytic intermediates in B-ALL cells. In contrast, Stat5-YF increased phospholipid intermediates and phosphatidylethanolamine (PtdEtn). Opposed by Myc, Stat5-YF increased the expression levels of Bcl6 and autophagic genes, suggesting that Bcl6-dependent programs promote PtdEtn-LC3B conjugation, thereby autophagosome formation. As expected, microscopic validation with LC3B-GFP reporter revealed that Stat5-YF increased the number of LC3B-GFP puncta, upon pharmacological inhibition of autophagosome-lysosome fusion ( Figure A). Consistent with STAT5-CA promoting Myc- as opposed to STAT5-YF driving Bcl6-dependent programs, a flow cytometry analysis of Myc-eGFP/Bcl6-mCherry dual reporter expression in murine B-ALL cells demonstrated that changes between glycolytic Myc+ and autophagic Bcl6+ states were the direct consequence of Stat5-CA and Stat5-YF induction, respectively. Conclusions: Our results demonstrate feedback regulators of STAT5, including SOCS2 and CISH are not only biomarkers of increased oncogenic STAT5 activity and a more aggressive course of disease linked to poor overall outcomes. Importantly, negative STAT5 feedback control mechanisms are essential to enable nimble adaptations of B-ALL cells in response to fluctuations of oncogenic signaling strength and availability of nutrients during distinct metabolic states of cellular activation (MYC) and quiescence (BCL6).

Mutational Landscape of Bone Marrow CD19 and CD138 Cells in Waldenström Macroglobulinemia (WM) and IgM Monoclonal Gammopathy of Undetermined Significance (IgM MGUS)

Background Waldenström's Macroglobulinemia (WM) is an incurable B-cell neoplasm characterized by serum monoclonal immunoglobulin M (IgM) and clonal lymphoplasmacytic cells infiltrating the bone marrow (BM). Smoldering WM (sWM) is the asymptomatic/indolent form with a high risk of progressing to symptomatic WM requiring treatment whereas IgM monoclonal gammopathy of undetermined significance (IgM MGUS) is an early precursor stage of WM. Despite recurrent and activating mutations, including MYD88, CXCR4, ARID1A, KMT2D, and CD79B have been identified, the genetic basis for WM and the risk of progression of IgM MGUS to WM remain to be fully elucidated. Methods Based on the mutational profiling of WM and our previous wide transcriptome analyses on BM CD19+ and CD138+ cells of patients with WM and IgM MGUS, we decided to investigate the mutation status of these patients as follows: WM (n=10), sWM (n=7), and IgM MGUS (n=5), by performing high throughput targeted AmpliSeq next generation sequencing on 117 target genes potentially involved in B-cell lymphomagenesis and in the risk of progression of IgM MGUS to WM (Trojani A. et al. Cancers, 2021). Results We identified genetic aberrations in multiple genes, including previously described classic mutations in MYD88 (L260P alias L265P) found in 82.3% (14/17) of WM/sWM patients and in 60% (3/5) of IgM MGUS subjects. The CXCR4 mutation (S338Ter) was detected in 23.5% of CD19+ cells of WM patients, whereas it was absent in IgM MGUS subjects. Interestingly, we also identified new mutated genes, including WNK2 somatic mutations affecting 47% of WM patients, for which a recurrent allelic variant (V1635Ter) was observed, and BCL9 with a recurrent frameshift variant (P516Lfs) identified in 29.4% of WM patients exclusively in the CD19 cell fraction. The BCL9 (P516Lfs) variant was also detected in the B cells of one IgM MGUS patient. Moreover, sequencing evaluation revealed recurrently frameshift or missense mutations involving NFKB2 (L473Afs), PTPN13 (P1546Tfs), CARD11 (S622del), KMT2C (I823T), and ATM in WM and IgM MGUS patients (Figure 1). Of interest, KMT2C (also known as MLL3) exhibited a somatic hypermutated phenotype in both CD19+ and CD138+ cells of WM and IgM MGUS samples. The KMT2C mutations were not mutually exclusive with those involving KMT2D in our cohort of patients. Conclusion The present genomic analysis revealed new insights into the mutational landscape of Waldenström Macroglobulinemia, providing evidence of the recurrence of some variants in subjects with IgM MGUS as well. Notably, we uncovered new genetic mutations with somatic variants recurrently observed (n≥3) in KMT2C, WNK2, BCL9, PTPN13, CARD11 and NFKB2 genes. These recurring somatic events provide a genomic basis for future research and a better understanding of the WM pathogenesis as well as the risk of transformation of IgM MGUS to WM.

Patients with Chronic Lymphocytic Leukemia Carrying t(14;19) Display a Distinctive Transcriptomic Profile and Adverse Outcome, Which Might be Overcome Continuous Therapy with BTK Inhibitors. an Italian Campus CLL and Eric Study

Introduction Rearrangements of the BCL3 gene due to the translocation t(14;19)(q32;q13) can be identified in up to 1% of chronic lymphocytic leukemias (CLL) with stimulated karyotype and is associated with an adverse outcome. In this study, we aimed at unraveling the clinico-molecular features of CLL harboring t(14;19), including the response to novel therapies. Methods CLL with t(14;19) were collected within databases from the Italian Campus CLL , European Research Initiative on CLL, German CLL study group and Ohio State University. Treatment-free survival (TFS), time to next treatment (TTNT), overall survival (OS) curves were compared with the Log-rank tests. For RNA sequencing (RNA-seq), RNA was extracted from 10 6 purified B-cells, processed by TruSeq Stranded Total RNA Ribo-Zero Gold, sequenced by Illumina at an average read depth of 120x10 6 per sample. Linear and circular RNAs (circRNA) were identified and quantified with CirComPara2 software. Differential expression analysis was conducted imposing a 0.01 Benjamini-Hochberg adjusted p value threshold by using the DESeq2 and limma-voom R/Bioconductor packages for gene and circRNA expression, respectively. Pathway enrichment was conducted using the gene set enrichment analysis (GSEA) method from the clusterProfiler R. Results We gathered data from 88 patients with t(14;19) CLL from 16 centers, 52% were males, the median age at diagnosis was 58±13years, 93% (68/73) had an unmutated IGHV status [U-IGHV, including 25% stereotyped subset #8], 60% carried trisomy 12 (+12), 52% a complex karyotype (CK, 52% ≥3 aberration, 19% ≥5), 39% atypical phenotype (22/57, Matutes score <3) and 15% TP53 abnormality (abn, deletion and/or mutation). To dissect the role of t(14;19) in CLL pathobiology, we analyzed the transcriptomes of 25 t(14;19) samples (comparable in terms of TFS and OS to the whole cohort), 22 control CLL without t(14;19) (11 with +12 and 11 with both normal FISH and karyotype, 10 being U-IGHV) and B cells from 9 healthy donors. Among the differentially expressed genes between t(14;19) cases and control CLL, 708 were upregulated and 1230 downregulated. In particular, among the top upregulated genes we found BCL3, CD79b (confirming their atypical phenotype), CDKN2A, U2, TP63 and LAG3 whereas among the downregulated CD274 and TIGIT. Of note, the t(14;19) cases displayed a significant enrichment (p-adjusted<0.01) of distinct Gene Ontology pathways, primarily downregulation of leukocyte chemotaxis, immune effector process and cytokine-mediated signaling. Further, we detected over 23000 circRNAs expressed from 7024 genes. Most circRNAs were backspliced from known exons (97%), whereas 1.8% and 1.2% involved intronic and intergenic regions, respectively. We found 60 circRNA upregulated and 197 downregulated circRNAs in t(14;19) cases compared to control CLL, including 28 also differentially expressed against normal B cells, such as circCDK14, not annotated and generated by antisense gene. After a median follow-up of 7.6 years, the median TFS and OS were 2 and 12.6 years, respectively. In univariate analysis, no clinic-biological variables correlated with TFS and OS. The poor outcome of t(14;19) patients was similar to CLL cases without t(14;19) but harboring TP53 abn or CK5, and even shorter than CK3 or +12 patients (historic data from the Italian Campus CLL). By analyzing the first-line therapy, 28 patients received FCR/BR, 6 venetoclax-based (VEN) therapy (5 within the CLL13 trial), 12 BTK inhibitor (BTKi, 10 ibrutinib and 2 acalabrutinib), 34 other therapies. TTNT was longer with BTKi (p<0.001, Figure 1). At 3-year 59%, 67%, 100% and 35% for patients treated with FCR/BR, VEN, BTKi and others did not need a further line of therapy. In the relapse setting, 11 patients received VEN and 29 BTKi; a trend for a longer TTNT was observed with BTKi (3-year, 16% vs 64%, p=0.09) Discussion In this international retrospective study we report that t(14;19) i) is recurrent in young CLL with sterotyped #8; ii) portends a negative prognostic impact superimposable to CK5 or TP53 cases; iii) display by a distinct gene expression profile characterized by the deregulation of immune checkpoints, offering a hint for innovative therapeutic approaches, and of circRNAs, whose pathogenetic role in CLL is still unknown and deserves further investigation. Finally, for this aggressive subset of CLL, a continuous therapy with a BTKi resulted in improved treatment outcomes

BCL6 Protein Expression Identifies Cases without BCL6 Gene Rearrangement in Non-Germinal Centre Diffuse Large B- Cell Lymphoma Cases

BCL6 gene rearrangements are the most frequent cytogenetic abnormality in Diffuse Large B-Cell Lymphoma (DLBCL), occurring in up to 64% of cases, suggesting a poorer prognosis and response to therapy. The aim of this study was to calculate the accuracy and predictive power of BCL6 IHC in identifying BCL6 gene rearrangements. A search for DLBCL cases was performed on the laboratory information system. We retrospectively analysed 46 cases of DLBCL, and correlated BCL6 protein expression with gene rearrangement status. Of the 46 cases, 39 (84.78%) showed positive BCL6 protein expression by IHC. In comparison, only 10 (21.74 %) samples presented with gene rearrangements. There were 8 cases positive for BCL6 IHC with gene translocations and 5 cases were negative for both protein expression and gene rearrangements. BCL6 IHC had a sensitivity of 80% and a specificity of 14%. Furthermore, the Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were 21% and 71%, respectively. BCL6 rearrangement was more common in cases with a Non-Germinal Centre (GC) Cell of Origin (COO) (45.5%) as compared to the GC group (14.3%). When stratifying the analysis according to the COO, the sensitivity and NPV of BCL6 IHC in the non-GC group were both 100%, while the same parameters were considerably poorer in the GC cases. BCL6 protein expression did not correlate with the presence of BCL6 gene rearrangements in HIV related DLBCL cases. However, lack of BCL6 protein expression may be used to identify cases without a BCL6 gene rearrangement, particularly in non-GC COO.

The devolution of a mature plasma cell dyscrasia into a fatal plasmablastic lymphoma

Introduction: Plasmablastic lymphoma is a rare, aggressive, non-Hodgkin’s lymphoma with an untreated prognosis as poor as three months. There exists scant literature describing transformation of plasmablastic lymphoma from a more benign dyscrasia, the mature plasmacytoma. This case report describes the transformation of plasmablastic lymphoma from a mature plasma cell neoplasm/plasma cell myeloma in an atypical combination of patient characteristics. Case Report: A 66-year-old man presented with acute onset right lower extremity pain and rapidly progressive mobility loss. He was found to have a lytic lesion in the lateral right iliac wing. Biopsy revealed the lesion to be plasmablastic lymphoma with Epstein–Barr virus (EBV) positivity by in situ hybridization with a Ki-67 proliferation index >99%, and strongly staining CD138 and MUM-1. CD20 and PAX-5 were negative. A bone marrow biopsy from the right iliac crest showed mature plasma cells without evidence of plasmablastic lymphoma cytology found in the initial specimen. These specimens showed CD138 positivity with 15–20% plasma cells with Kappa positive clonality by in situ hybridization, and diffusely Epstein–Barr virus negative by in situ hybridization. Further plasma cell fluorescence in situ hybridization study showed a clone with a TP53 deletion and an immunoglobulin heavy chain gene rearrangement that did not translocate to one of the common plasma cell dyscrasia translocation partners (FGFR3, CCND1, MAF, or MAFB). Additionally, a near-tetraploid subclone was observed in approximately 60% of nuclei. Also, there was gain of BCL2 gene or chromosome 18/18q, gain of BCL6 gene or chromosome 3/3q and MYC amplification. There was no MYC and BCL2 and/or BCL6 rearrangements. Our patient was neither HIV-positive nor immunocompromised, rather Epstein–Barr virus positive with a quantitative polymerase chain reaction level greater than 67,000. He was started on Daratumumab combined with etoposide, vincristine, doxorubicin, cyclophosphamide, and prednisone. Conclusion: This case exhibits a unique presentation of plasmablastic lymphoma in terms of disease presentation, unique risk factors, including HIV-negativity and male-assigned sex, and the creativity of treatment utilized.

Foxp3 and Bcl6 deficiency synergistically induces spontaneous development of atopic dermatitis-like skin disease.

Atopic dermatitis (AD) is a common chronic skin disease caused by immune dysfunction, specifically the hyperactivation of Th2 immunity. AD is a complex disease with multiple factors contributing to its development; however, the interaction between these factors is not fully understood. In this study, we demonstrated that the conditional deletion of both the forkhead box p3 (Foxp3) and B-cell lymphoma 6 (Bcl6) genes induced the spontaneous development of AD-like skin inflammation with hyperactivation of type 2 immunity, skin barrier dysfunction, and pruritus, which were not induced by the single deletion of each gene. Furthermore, the development of AD-like skin inflammation was largely dependent on IL-4/13 signaling but not on immunoglobulin E (IgE). Interestingly, we found that the loss of Bcl6 alone increased the expression of thymic stromal lymphopoietin (TSLP) and interleukin (IL)-33 in the skin, suggesting that Bcl6 controls Th2 responses by suppressing TSLP and IL-33 expression in epithelial cells. Our results suggest that Foxp3 and Bcl6 cooperatively suppress the pathogenesis of AD. Furthermore, these results revealed an unexpected role of Bcl6 in suppressing Th2 responses in the skin.

Characterization of the genetic and epigenetic landscape of B‐cell neoplasms with IG::BCL3‐translocation

Introduction: The BCL3 gene (19q13) encodes a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family. It is recurrently deregulated in B-cell neoplasms by a juxtaposition to regulatory elements of one of the immunoglobulin loci, mostly the immunoglobulin heavy chain (IGH) locus (14q32). Two distinct subsets of IG::BCL3 translocation-positive B-cell neoplasms, differing in the number of chromosomal aberrations, IGHV mutation status and histopathology, have been described [Martín-Subero et al., 2007]. In B-cell chronic lymphocytic leukaemia (CLL), the IG::BCL3 translocation has been associated with younger age at diagnosis, a more aggressive clinical course and an atypical tumour cell phenotype [Michaux et al., 1997; Au et al., 2002]. Methods: A total of 89 B-cell neoplasms with IG::BCL3 translocation detected by fluorescence in situ hybridisation (FISH), including predominately cases diagnosed as CLL, were studied. Breakpoints at the BCL3 and IGH loci were sequenced in a subset of samples using targeted capture and breakpoint-spanning PCR approaches. Chromosomal aberrations detected by FISH as well as the IGHV mutation status were analysed. In addition, DNA methylation and copy number aberration (CNA) analyses were performed for 48 samples with CLL using the 850K EPIC BeadChip array. Results: Targeted capture-based sequencing data showed that in the majority of cases BCL3 translocations are associated with aberrant class-switch recombination at the IGH locus, often involving IGHA segments (n = 4/10). From the 69 IG::BCL3-translocated cases with available IGHV mutation status, a total of 51 (74%) were classified as unmutated and 18 (26%) as mutated. This indicates an overrepresentation of cases with unmutated IGHV status among the IG::BCL3-translocated cases compared to the general CLL population. Based on EPIC BeadChip data (total n = 48) 25 (52%) IG::BCL3-translocated cases carried a trisomy 12, 11 cases (23%) a deletion 13q, 7 cases (15%) a deletion 17p and 3 cases (6%) a deletion 11q. FISH analysis of a partially overlapping set of neoplasms (n = 54) was in line with the EPIC BeadChip data and revealed 17 IG::BCL3-translocated cases (32%) with trisomy 12, 7 cases (13%) with deletion 17p and deletion 13q each, and 4 cases (7%) with deletion 11q. Thus, IG::BCL3-translocated cases showed a striking skewing towards the presence of trisomy 12 compared to the general CLL population (p = 0.011). In the DNA methylation analyses, the IG::BCL3-translocated CLLs segregated separately from other CLLs with and without IGH translocations. Conclusions: Our large multi-OMICs study of IG::BCL3-translocated B-cell neoplasms corroborates previous reports on the pathogenetic heterogeneity of these lymphomas and moreover provides evidence that IG::BCL3-translocated CLL might form a genetically and epigenetically distinct subtype of B-cell neoplasms distinct from common CLL. Keywords: Chronic Lymphocytic Leukemia (CLL), Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

Clinical characteristics of 11 patients with chronic lymphocytic leukemia with t (14;19) (q32;q13)

Objective: To analyze the clinicopathological characteristics of 11 cases of chronic lymphocytic leukemia (CLL) with t (14;19) (q32;q13) . Methods: The case data of 11 patients with CLL with t (14;19) (q32;q13) in the chromosome karyotype analysis results of the Blood Diseases Hospital, Chinese Academy of Medical Sciences from January 1, 2018, to July 30, 2022, were retrospectively analyzed. Results: In all 11 patients, t (14;19) (q32;q13) involved IGH::BCL3 gene rearrangement, and most of them were accompanied by +12 or complex karyotype. An immunophenotypic score of 4-5 was found in 7 patients and 3 in 4 cases. We demonstrated that CLLs with t (14;19) (q32;q13) had a mutational pattern with recurrent mutations in NOTCH1 (3/7), FBXW7 (3/7), and KMT2D (2/7). The very-high-risk, high-risk, intermediate-risk, and low-risk groups consisted of 1, 1, 6, and 3 cases, respectively. Two patients died, 8 survived, and 2 were lost in follow-up. Four patients had disease progression or relapse during treatment. The median time to the first therapy was 1 month. Conclusion: t (14;19) (q32;q13), involving IGH::BCL3 gene rearrangement, is a rare recurrent cytogenetic abnormality in CLL, which is associated with a poor prognosis.

Neutrophil-Membrane-Coated Biomineralized Metal-Organic Framework Nanoparticles for Atherosclerosis Treatment by Targeting Gene Silencing.

Antisense oligonucleotides (ASOs) are promising tools for gene silencing and have been exploited as therapeutics for human disease. However, delivery of therapeutic ASOs to diseased tissues or cells and subsequent escape from the endosomes and release of ASO in the cytosol remain a challenge. Here, we reported a neutrophil-membrane-coated zeolitic imidazolate framework-8 (ZIF-8) nanodelivery platform (AM@ZIF@NM) for the targeted transportation of ASOs against microRNA-155 (anti-miRNA-155) to the endothelial cells in atherosclerotic lesions. Neutrophil membrane could improve plaque endothelial cells targeting through the interaction between neutrophil membrane protein CD18 and endothelial cell membrane protein intercellular adhesion molecule-1 (ICAM-1). The ZIF-8 "core" provided high loading capacity and efficient endolysosomal escaping ability. Delivery of anti-miR-155 effectively downregulated miR-155 expression and also saved the expression of its target gene BCL6. Moreover, RELA expression and the expression of its downstream target genes CCL2 and ICAM-1 were correspondingly reduced. Consequently, this anti-miR-155 nanotherapy can inhibit the inflammation of atherosclerotic lesions and alleviate atherosclerosis. Our study shows that the designed biomimetic nanodelivery system has great application prospects in the treatment of other chronic diseases.

Clinicopathological, cytogenetic, and molecular profiles of primary cutaneous diffuse large B-cell lymphomas

We analyzed the clinicopathological, cytogenetic, and molecular features of 18 primary cutaneous diffuse large B-cell lymphomas (PCDLBCLs)and 15 DLBCLs secondarily localized to the skin (SCDLBCLs), highlighting biological similarities and differences between the 2 groups. PCDLBCLs were subclassified after histopathological review as PCDLBCL-leg type (PCDLBCL-LT, 10 cases) and the PCDLBCL-not otherwise specified (PCDLBCL-NOS, 8 cases). Immunohistochemistry for Hans' algorithm markers, BCL2, and MYC was performed. The molecular study included the determination of the cell of origin (COO) by Lymph2Cx assay on NanoString platform, FISH analysis of IgH, BCL2, BCL6, and MYC genes, as well as the mutation analysis of MYD88 gene. In immunohistochemistry analysis, BCL2 and MYC hyperexpression was more frequent in LT than in NOS cases and, according to Hans' algorithm, PCDLBCL-LTs were mostly of the non-GC type (8/10), whereas in PCDLBCL-NOS, the GC type prevailed (6/8). The determination of COO using Lymph2Cx supported and further confirmed these results. In FISH analysis, all but one LT cases versus 5 of 8 PCDLBCL-NOS showed at least one gene rearrangement among IgH, BCL2, MYC, or BCL6. In addition, MYD88 mutations were more frequently present in LT than in NOS subtypes. Interestingly, MYD88-mutated patients were older, with a non-GC phenotype and had worse OS, compared to MYD88 WT cases. Overall, SCDLBCL did not show, at the genetic and expression level, different profiles than PCDLBCL, even if they bear a significantly worse prognosis. At survival analysis, the most important prognostic factors in patients with PCDLBCL were age and MYD88 mutation, whereas relapse and high Ki-67 expression were relevant in patients with SCDLBCL. Our study comprehensively analyzed the clinicopathological and molecular features of PCDLBCL-LT, PCDLBCL-NOS, and SCDLBCL, underlining the differences among them and the importance of properly identifying these entities at the time of diagnosis.

Signet ring cell-like diffuse large B-cell lymphoma involving the breast: a case report

BackgroundDiffuse large B-cell lymphoma (DLBCL) with signet ring cell components is extremely rare. Here, we present a case of DLBCL with signet ring cell components involving the breast, which can be easily confused with invasive lobular carcinoma of the breast or metastatic signet ring cell carcinoma of gastrointestinal origin.Case presentationA 66-year-old woman presented with a painless mass in her left breast. Enhanced magnetic resonance imaging (MRI) of the breast revealed a 42 × 29 × 28 mm mass in the left breast. Histological examination revealed a diffuse or scattered arrangement of round cells mixed with signet ring-like cells. Immunohistochemically, the neoplastic cells were positive for PAX-5, CD79a, CD20, Bcl-6, and MUM-1 but and negative for cytokeratin, ER, PR, E-cadherin, and P120. The Ki-67 proliferation index was approximately 70%. Fluorescence in situ hybridisation (FISH) demonstrated non-rearrangement of Bcl-2, Bcl-6, and c-MYC genes. Immunohistochemistry and FISH examination confirmed the diagnosis of DLBCL. Subsequently, immunofluorescence showed both IgM and IgG deposits in the signet ring-like lymphocytes. After confirming the diagnosis, the patient received four courses of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy in a specialist hospital and achieved partial remission; however, she unfortunately died of secondary pneumocystis pneumonia infection 3 months later.ConclusionMalignant lymphoma with signet ring cell morphology is quite uncommon, and this variant can be a diagnostic pitfall. We emphasise that pathologists should consider lymphoma in the differential diagnosis of malignant breast tumours.

Prognostic and survival impact of BCL9 and RPS6KB1 copy number variation detected from circulating free DNA in hepatocellular carcinoma

ABSTRACT Background Circulating cell-free DNA (cfDNA) is a noninvasive substitute to liver biopsy for hepatocellular carcinoma (HCC) molecular profiling. This study aimed to use cfDNA to investigate copy number variation (CNV) in the BCL9 and RPS6KB1 genes and its impact on prognosis in HCC. Methods Real-Time Polymerase Chain Reaction was used to determine the CNV and cfDNA integrity index in 100 HCC patients. Results CNV gain in BCL9 and RPS6KB1 genes was detected in 14% and 24% of patients, respectively. Gain in CNV of BCL9 associated with risk of HCC in alcohol drinkers and hepatitis C seropositivity. In patients with RPS6KB1 gain, HCC risk increased with a high body mass index, smoking, schistosomiasis, and Barcelona clinical liver cancer stage (BCLC) A. Gain in both genes showed a high risk of HCC with elevated liver enzymes, Schistosomiasis, BCLC C, and PS > 1. The integrity of cfDNA was higher in patients with CNV gain in RPS6KB1 than those harboring CNV gain in BCL9. Lastly, BCL9 gain and BCL9 + RPS6KB1 gain led to higher mortality rates and reduced survival times. Conclusion cfDNA was used to detect BCL9 and RPS6KB1 CNVs, which influence prognosis and can be used as independent predictors of HCC patient survival.

Effect of 50-Hz magnetic fields on the expression of activation-induced deaminase, B-cell lymphoma 6 and serum levels of interleukin-6, interleukin-21

Background Investigations showed different effects of magnetic fields (MFs) on the immune system. During humoral immune responses, genes of activation-induced deaminase (AID) and B-cell lymphoma-6 (Bcl-6) are expressed and interleukin (IL)-6 and IL-21 are produced. These factors play significant roles in class switching, affinity maturation of antibodies and activations of B cells germinal centers (GCs). Therefore, this study investigated the effect of 50-Hz MFs exposure with different densities on these factors. Materials and methods Eighty rats were divided into four exposures and control groups. The treatment groups were exposed to magnetic flux densities of 1, 100, 500, and 2000 μT (50 Hz, 2 h/d for 60 d). To activation of the immune system, all the animals were immunized with human serum albumin on days 31, 44, and 58 of exposure. Reverse transcription-quantitative polymerase chain reaction was used to assay the expression levels of AID and Bcl-6 genes in the spleen. The serum levels of IL-6 and IL-21 were also detected by enzyme-linked immunosorbent assay at the pre-and post-immunization phases. Results AID expression was significantly declined at 1μT magnetic flux density, while no change was observed in the expression of Bcl-6. Serum IL-6 was increased only in the 500 μT group at the post-immunization phase. Conclusions It seems exposure to 50-Hz MFs at 1 μT density, suppresses AID and may cause a decline in class switching and affinity maturation of Abs. On the other hand, exposure to 500 μT, may activate them. These findings demonstrate the various potential effects of MFs on the humoral immune system.

Detection of Selection Signatures in Anqing Six-End-White Pigs Based on Resequencing Data

As a distinguished Chinese indigenous pig breed that exhibits disease resistance and high meat quality, the Anqing six-end-white (AQ) pig represents a valuable germplasm resource for improving the quality of the pig breeding industry. In this study, 24 AQ pigs that were distantly blood-related and 6 Asian Wild Boar (AWB) were selected for 10× deep-genome resequencing. The signatures of the selection were analyzed to explore the genetic basis of their germplasm characteristics and to identify excellent germplasm-related functional genes based on NGS data. A total of 49,289,052 SNPs and 6,186,123 indels were detected across the genome in 30 pigs. Most of the genetic variations were synonym mutations and existed in the intergenic region. We identified 275 selected regions (top 1%) harboring 85 genes by applying a crossover approach based on genetic differentiation (FST) and polymorphism levels (π ratio). Some genes were found to be positively selected in AQ pigs' breeding. The SMPD4 and DDX18 genes were involved in the immune response to pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV). The BCL6 and P2RX6 genes were involved in biological regulation of immune T cells and phagocytes. The SLC7A4 and SPACA4 genes were related to reproductive performance. The MSTN and HIF1A genes were related to fat deposition and muscle development. Moreover, 138 overlapping regions were detected in selected regions and ROH islands of AQ pigs. Additionally, we found that the QTLs with the most overlapping regions were related to back fat thickness, meat color, pH value, fatty acid content, immune cells, parasitic immunity, and bacterial immunity. Based on functional enrichment analysis and QTLs mapping, we conducted further research on the molecular genetic basis of germplasm traits (disease resistance and excellent meat quality). These results are a reliable resource for conserving germplasm resources and exploiting molecular markers of AQ pigs.