Mutations In TNFAIP3 Research Articles

Large B-cell lymphomas with CCND1 rearrangement have different immunoglobulin gene breakpoints and genomic profile than mantle cell lymphoma

Mantle cell lymphoma (MCL) is genetically characterized by the IG::CCND1 translocation mediated by an aberrant V(D)J rearrangement. CCND1 translocations and overexpression have been identified in occasional aggressive B-cell lymphomas with unusual features for MCL. The mechanism generating CCND1 rearrangements in these tumors and their genomic profile are not known. We have reconstructed the IG::CCND1 translocations and the genomic profile of 13 SOX11-negative aggressive B-cell lymphomas using whole genome/exome and target sequencing. The mechanism behind the translocation was an aberrant V(D)J rearrangement in three tumors and by an anomalous IGH class-switch recombination (CSR) or somatic hypermutation (SHM) mechanism in ten. The tumors with a V(D)J-mediated translocation were two blastoid MCL and one high-grade B-cell lymphoma. None of them had a mutational profile suggestive of DLBCL. The ten tumors with CSR/SHM-mediated IGH::CCND1 were mainly large B-cell lymphomas, with mutated genes commonly seen in DLBCL and BCL6 rearrangements in 6. Two cases, which transformed from marginal zone lymphomas, carried mutations in KLF2, TNFAIP3 and KMT2D. These findings expand the spectrum of tumors carrying CCND1 rearrangement that may occur as a secondary event in DLBCL mediated by aberrant CSR/SHM and associated with a mutational profile different from that of MCL.

The genomic, transcriptomic, and immunological profile of patients with recurrent/refractory NSCLC.

8039 Background: Lung cancer is the leading cause of cancer-related deaths worldwide, and often presents at advanced stages, with > 50% of patients presenting as stage III or IV. Patients with recurrent stage III NSCLC (R/R) previously treated with definitive chemoradiation and immunotherapy consolidation may have a difference in overall survival compared to de novo stage IV (DN). In this study, we aim to compare the molecular and immune landscapes of these two patient populations to identify potential genomic patterns and biomarkers of resistance. Methods: 3728 NSCLC specimens underwent sequencing of DNA (592-gene panel or whole exome) or RNA (whole transcriptome) or immunohistochemistry at Caris Life Sciences (Phoenix, AZ). Patients were classified as R/R (n = 26) if they received Durvalumab and chemoradiation within 12 months (m) before tissue collection and treated with Pembrolizumab within 6 m after tissue collection. Patients treated with Pembrolizumab within 6 months after tissue collection and had no prior (or post) treatment with Durvalumab and chemoradiation at any point in time were classified as DN (n = 3702). Tumor microenvironment (TME) cell fractions were estimated from bulk RNA sequencing using QuanTIseq. Significance was determined using chi-square, Mann Whitney U and adjusted for multiple comparisons where applicable (q < 0.05). Results: TP53 (91 vs 68%) and KEAP1 (30 vs. 14%, both p < 0.05, q > 0.05) mutation prevalence was higher in R/R, whereas a decreased prevalence of KRAS mutations was noted in this group (13 vs. 33%) as compared to the DN group. Compared to DN patients, R/R patients were associated with an increased prevalence of mutations in BCL2 (4.3 vs. 0%, q < 0.05), BCL9 (4.3 vs. 0.2%, q < 0.05), HNF1A (4.3 vs. 0.2%), TNFAIP3 (4.3 vs. 0.2%), AKT1 (4.3 vs. 0.4%), MAP3K1 (4.3 vs. 0.5%), and SPEN (4.3 vs. 0.6%; all p < 0.05, q > 0.05). Further, copy number amplification of NUTM1 (4.5 vs. 0%), SMO (4.3 vs. 0.5%), CHIC2 (4.5 vs. 0.2%; all q < 0.05), PIK3CA (9 vs. 1.2%) and FLCN (4.5 vs. 0.3%; both p < 0.05, q > 0.05) was more common in R/R compared to DN. R/R TME was associated with decreased B cell (2.9 vs 4.4%, q < 0.05) and regulatory T cell fractions (1.3 vs. 2.6%, p < 0.05, q > 0.05). Conclusions: R/R patient tumors had distinct molecular alterations and immune landscapes, including an increased prevalence of biomarkers associated with immunotherapy resistance ( TP53 and KEAP1 mutations) and lower tumor B- and T reg- cell fractions, which may suggest decreased likelihood of response to immunotherapy compared to patients with DN NSCLC. The RR population had a lower-than-expected percentage of KRASmutations. These distinct molecular differences highlight the need for NGS testing in the recurrent setting, despite initial NGS testing at diagnosis. Molecularly targeted interventions may be options in the future for patients with R/R Stage III NSCLC.

A20 Haploinsufficiency: A Systematic Review of 177 Cases

A20 haploinsufficiency is an autoinflammatory disease caused by defective inactivation of the NF-κB pathway. We conducted a systematic literature review of articles reporting patients with TNFAIP3 sequence variants from 2016 to August 2023 following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Data from 177 patients from 65 articles were retrieved (108 women). The principal features were mucosal ulcers (n= 129); fever (n= 93) followed by gastrointestinal (n= 81); skin features (n= 76); autoimmunity (n= 61), including thyroiditis (n= 25) and lupus (n= 16); and joint involvements (n= 54). Five patients had died at the time of publication. In 54 of 63 patients, CRP was significantly elevated during flares, with a median of 51 mg/l. The most commonly used treatment included corticosteroids and nonsteroidal anti-inflammatory drugs (n= 32), TNF blockers (n= 29), colchicine (n= 28), and methotrexate (n= 14). TNFAIP3 variants impacted the ovarian tumor domain in 92 cases and a Zinc finger domain in 68 cases. Geographic origin, reported sex, and variant type significantly impacted phenotype. A better understanding of the wide A20 haploinsufficiency phenotype could facilitate the diagnosis process. Much remains to be elucidated about pathogenesis and treatment to improve outcome in patients with A20 haploinsufficiency.

Recurrent Mutations in Refractory/Relapsed Diffuse Large B-Cell Lymphoma by Targeted Gene Sequencing

Introduction: Whole-genome sequencing of diffuse large B-cell lymphoma (DLBCL) has identified recurrent mutations involved in pathogenesis and potentially affecting response to therapy. In this pilot study, a targeted gene panel was created to identify mutations associated with relapse/refractoriness. Material and Methods: A 14-gene targeted panel was designed to sequence thirteen patients who were in remission and 8 cases that had relapsed/refractory to treatment. A paired diagnostic biopsy and a relapse biopsy were sequenced to find genes repeatedly altered in relapse. Results: A total of 751 nonsynonymous and truncating mutations were identified. Truncated mutations in NOTCH1, TNFAIP3, and CD58 were associated with poor treatment outcomes. In cases that did not respond to treatment, a high number of mutations were found in the EZH2 gene, followed by the DNA-binding domain of TP53 and MYD88. Termination mutations in the intracellular domain of NOTCH were found in 75% of non-responsive cases. Co-occurrence of loss of function mutations of TNFAIP3 and missense mutations in MYD88 was associated with a non-responsive cohort. Discussion: The study highlights mutations associated with chemotherapeutic response in DLBCL with implications for initial diagnostic biopsy response prediction.

Joint Analyses of Serum Proteins and Circulating Tumor DNA Improves Risk Assessment and Subtype Stratification in Large B-Cell Lymphomas

Introduction Inadequate dismantling of clinical and biological heterogeneity is an obstacle for improving outcome in large B-cell lymphoma (LBCL). Circulating tumor DNA (ctDNA)-based stratification holds great potential in resolving these challenges. However, the assessment is limited to genetic content, and excludes host response from the estimates. Previously, we have shown that an inflammatory signature identified from the pretreatment serum proteins translates to poor survival, and multiple serum proteins correlate with LBCL genetic subtypes. To further improve risk stratification and molecular characterization of LBCL from the liquid biopsy, we co-analyzed serum protein and ctDNA levels in LBCL patients treated in two Nordic Lymphoma Group (NLG) trials. Materials and methods Our discovery cohort consisted of 122 patients less than 65 years of age with clinically high-risk (age adjusted IPI ≥ 2) LBCL, who were treated in NLG-LBC-06 trial with immunochemotherapy. Plasma and serum samples were collected prior to treatment. Cell free DNA was extracted from plasma and sequenced with in-house adapted duplex sequencing platform using custom targeted panel of 347 lymphoma driver genes and immunoglobulin loci. An inflammation score was composed as log transformed mean of the most pertinent inflammatory serum proteins detected in the inflammatory signature (IL10, IL2RA, CXCL9, IL18, GZMB, PD-L1 and IFNG), and their levels were measured using antibody-based Luminex assay (pg/ml, R&D Systems). In addition, CCL17 and TNFRSF13B levels were analyzed to uncover their associations with genetic subtypes. An independent cohort of 91 patients treated in our previous NLG-LBC-05 trial was used for validation. Results Combined analysis of serum proteins and ctDNA identified a group of patients (n=12, 10%) with high serum inflammation scores and ctDNA burden (≥ 3.75 hGE/ml), which translated to poor survival compared to the patients with low concentrations of either inflammatory proteins or ctDNA, or both (progression free survival, PFS, hazard ratio 7.33, 95%-CI 2.59-20.79, P < 0.001, Figure 1A). This finding was confirmed in the independent validation cohort. In both cohorts, the combined analysis showed higher specificity in survival prediction as compared to the ctDNA-based assessment alone, indicating that inflammatory serum markers alongside with ctDNA can identify those patients who, despite of having high ctDNA burden, have excellent survival. Beyond inflammation, the levels of serum TNFRSF13B and CCL17 correlated with mutations associating with non-germinal center diffuse large B-cell lymphoma (non-GCB DLBCL)/MCD subtype and primary mediastinal B-cell lymphoma/grey-zone lymphoma (PMBCL/GZL), respectively. For example, serum TNFRSF13B levels correlated positively with mutations in MCD genes, such as CD79B, SETD1B, PIM2 and PRDM1, and negatively with mutations in EZB genes EZH2 and CREBBP in both cohorts (P < 0.05, Figure 1B). Supporting our earlier findings, high CCL17 levels revealed a group of patients diagnosed with DLBCL not otherwise specified or high-grade lymphoma but with a mutational landscape resembling PMBCL/GZL, including mutations in TNFAIP3, ITPKB, B2M, BTG1 and STAT6 (P < 0.05). Conclusion Our findings demonstrate that co-analysis of inflammatory serum markers along with the ctDNA improves ctDNA-based survival prediction. Moreover, analysis of serum proteins refines molecular classification. Altogether, joint analysis of serum proteins and ctDNA provides new tools to dismantle LBCL heterogeneity.

Understanding endoscopic and clinicopathological features of patients with very early onset inflammatory bowel disease: Results from a decade of study

BackgroundVery early onset inflammatory bowel disease (VEOIBD) is associated with a unique disease course and distinct endoscopic features. AimsThis study aims to provide a comprehensive description of the endoscopic and histologic features observed in a large cohort of patients with VEOIBD from a tertiary medical center. MethodsA retrospective review of medical records from 2011 to 2021 was conducted to analyze clinical data, including disease phenotypes, endoscopic and histologic findings. Next generation sequencing was performed. ResultsA total of 225 VEOIBD subjects were included in this study. Monogenic defects were identified in 161 patients. Monogenic IBD patients more commonly had CD-like disease. Colonic involvement was more prevalent among those with monogenic IBD (P<0.001). Pseudo-polyps were significantly more common in the monogenic IBD group (P<0.001), while ileal edema and ulcers were significantly more prevalent in non-monogenic IBD cases. IL10RA deficiency were characterized by colonic ulcers and pseudo-polyps without upper gastrointestinal tract lesions, while patients with TNFAIP3 mutations demonstrated both upper and lower gastrointestinal tract involvement. The non-monogenic IBD patients showed a higher incidence of chronic architectural changes of crypt, increased apoptosis and eosinophils infiltration. ConclusionsEndoscopic and histologic analysis of children with VEOIBD plays a crucial role in facilitating accurate diagnosis. Various forms of monogenic IBD exhibit distinct endoscopic and pathologic changes.

Multi-Dimensional Molecular and Tumor-Microenvironment Analysis of Classic Hodgkin Lymphoma

Introduction Classic Hodgkin Lymphoma (CHL) is currently classified into histological subtypes based on morphology and antigen marker expression. However, additional biological features might help guide treatment strategies and inform prognosis. Here, we aimed to uncover disease heterogeneity and biologic subtypes of CHL based on multi-dimensional molecular profiling ( Fig A) capturing somatic gene mutations, malignant cell expression phenotypes and altered tumor microenvironment (TME) architecture. METHODS We performed exome/targeted sequencing on flow-sorted or micro-dissected HRS cells from fresh-frozen CHL biopsies (n=116) of patients treated at BC Cancer. In addition, we constructed tissue microarrays (TMA) from this cohort on which we performed GeoMx® Whole Transcriptome Assay (WTA) to obtain gene expression profiles from CD30+ HRS cells and multiplexed imaging analyses using multi-colour immunofluorescence (mIF) to delineate the spatial TME ecosystem. Cox regression analysis was performed to identify outcome differences according to molecular features in CHL patients treated with ABVD-like chemotherapy (n=104). Results Mutational and copy number analyses identified known recurrent driver events including mutations and copy number changes in SOCS1, STAT6, TNFAIP3, B2M, REL and the PDL1/PDL2 locus. These mutational findings provided a statistically powered framework to investigate the association between genomic alterations with pathological and clinical disease parameters including outcomes. Mutations affecting BCL7A, TNFAIP3, and 2p15 (REL) amplifications were strongly enriched in EBV- CHL compared to EBV+ CHL. In line with “thymic mutations” reported in grey zone lymphoma, STAT6, GNA13, ITPKB, and TBL1XR1 mutations were significantly enriched in CHL with involvement of the anterior mediastinum. ZNF217, SPEN and CD58 mutations were significantly associated with progression-free survival (PFS) (all P &amp;lt; .01). When investigating outcome correlation according to age group, genomic alteration in STAT6 including both single nucleotide variants and STAT6 amplification, was the most significant feature associated with unfavorable PFS in patients &amp;lt;45 years of age ( P = .013). Alterations in the antigen recognition system, including loss of major histocompatibility complex (MHC) is one of the most studied mechanisms of immune escape in CHL. Mutations in CSF2RB, GNA13, TNFAIP3, and STAT6 were significantly associated with MHC-I loss. Strikingly, all cases harboring CSF2RB mutations (n=23) also showed loss of MHC-I. GeoMx analysis revealed that IFN-a and IFN-g pathways were significantly upregulated in MHC-I+ HRS cells. Mutations in HIST1H1E and BCL7A were significantly enriched in cases with loss of both MHC-I and MHC-II expression. To define mutational subtypes of CHL, we next applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events. Cluster1 (C1) is characterized by mutations in TNFAIP3, CSF2RB and GNA13, younger age, loss of MHC-I, and up-regulation of GCB and fatty acid metabolism signatures. C2 was associated with old age at initial diagnosis, EBV positivity and significant upregulation of the IFN-g pathway. C3 is characterized by REL and STAT6 amplification andexpression of a DNA repair signature. C4 includes samples from mostly younger patients with STAT6 and B2M mutations and high expression of CCL17 by HRS cells. MIF analyses further identified correlations between each mutational NMF cluster and TME composition ( Fig B), and in particular specific CD4+ T cell subsets. The TME of tumors was significantly enriched by C1: FOXP3+Tregs, C2: LAG3+CD4+ cells, and C3: PD1+CD4+ cells. C4 tumors showed no positive correlation with any of these immune cell compartments. C2 tumors were also significantly associated with the abundance of CD68+ macrophages. Conclusion Our multi-dimensional profiling approach enabled us to delineate molecular profiles of HRS cells that are linked to distinct TME patterns. These linkages have implications for current pathogenesis models, molecular subtyping of CHL, and identification of cellular vulnerabilities that might be therapeutically exploitable via targeting of HRS cell phenotypes and/or immune escape mechanisms.

Spectrum and Clinical Features of Gene Mutations in Chinese Follicular Lymphoma

Background: Follicular lymphoma (FL) is the most common indolent lymphoma with variable biologic presentation and heterogeneous clinical outcomes, and some of patients experiences early progression and have inferior survival outcomes. Next-generation sequencing (NGS) identified recurrently mutated genes in FL. While few studies on gene mutations in Chinese FL patients have been identified in the real world, this present study aimed to characterize the genomic landscape and provide insights into the biomarkers of high-risk FL populations. Methods: 590 FL patients were reviewed, and 100 patients with sufficient samples were enrolled in our cohort. We performed targeted sequencing of 521 lymphoma-related genes based on NGS in this study. Results: A total of 207 genetic alterations were detected in 99/100 patients. T NFRSF14, CARD11, and NOTCH3 mutations were common in FL1-2 patients compared to FL3a patients (p = 0.025, 0.0036, and 0.0047, respectively). SOCS1 mutations were more common in patients with Ki-67 index &amp;gt; 30% (p =0.324), and PRDM1 mutations were more common in patients who experienced progression of disease within 24 months (POD24) (33.3% vs. 0%, p =0.008). We observed a trend that patients with a high mutation load (with ≥ 6 mutated genes) showed decreased PFS compared with patients with &amp;lt; 6 mutated genes (p=0.068). For patients who received R-CHOP-like regimens, the multivariate COX proportional hazard modeling identified TP53, TNFAIP3, and SOCS1 mutations as independent risk factors of PFS (HR 6.76, 95% CI 1.81 to 25.18, p =0.004; HR 3.68, 95% CI 1.06 to 12.82, p =0.041; HR 5.07, 95% CI 1.81 to 21.73, p =0.029). TP53 and TNFAIP3 were significant independent predictors of PFS when testing with either FLIPI (p=0.029) or FLIPI2 (p=0.023) in multivariable analysis. Conclusion: Our study depicted genomic characterization of real-world Chinses FL patients and demonstrated TP53, TNFAIP3 and SOCS1 mutations can help identify high-risk patients.

Clonal Relationship and Mutation Analysis in Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia Associated With Diffuse Large B-cell Lymphoma

Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B-cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88 L265P (&gt;95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high-throughput sequencing of immunoglobulin gene rearrangements showed evidence of &gt;1 clonal B-cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B-cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88 L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88 WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1. Together, we demonstrate variable clonal B-cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one-quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients.

Clinicopathologic and mutational profiles of primary breast diffuse large B cell lymphoma in a male patient: case report and literature review

IntroductionPrimary breast lymphoma (PBL) is rare, and most cases occur in female patients, with few reported cases in male patients. The clinical presentation is similar to that of breast cancer, but the condition needs to be well understood, as treatment options and clinical course vary. Hence, we provide a relatively rare case of primary breast diffuse large B cell lymphoma (PB-DLBCL) in a male, including its complete clinicopathological features, radiological findings, genomic mutational profiles, and clinical course.Case presentationA 45-year-old male presented with a lump in his right breast for 1 week and was pathologically diagnosed with breast malignancy after a breast puncture biopsy at the local hospital. He came to our hospital for further treatment and underwent breast ultrasound and systemic positron emission tomography/computed tomography (PET/CT) imaging, followed by right mastectomy and sentinel lymph node biopsy. Histomorphology showed diffuse hyperplasia of tumor cells with clear boundaries and surrounding normal breast ducts. The adhesion of tumor cells was poor with obvious atypia. Immunohistochemical results showed that the tumor cells were positive for CD20, Bcl6, and MUM-1 but negative for CK (AE1/AE3), ER, PR, CD3, and CD10. Forty percent of the tumor cells were positive for c-Myc, and 80% of tumor cells were positive for Bcl2. The Ki-67 proliferation index was up to 80%. The tumor cells were negative for MYC and BCL2 rearrangements but positive for BCL6 rearrangement by fluorescent in situ hybridization. No abnormality was found in the pathological examination of bone marrow aspiration. Therefore, the male was diagnosed with PB-DLBCL, nongerminal center (non-GCB) phenotype, dual-expression type. The sample were sequenced by a target panel of 121 genes related to lymphoma. Next-generation sequencing revealed six tumor-specific mutated genes (IGH/BCL6, TNFAIP3, PRDM1, CREBBP, DTX1, and FOXO1). The patient was given six cycles of orelabrutinib plus R-CHOP chemotherapy and two cycles of intrathecal injection of cytarabine. The last follow-up was on April 13, 2023 (17 months). No recurrence or metastasis was found in laboratory and imaging examinations.ConclusionWe reported a relatively rare PB-DLBCL in a male, non-GBC phenotype, dual-expression type. It is worth mentioning that this case had IgH/BCL6 fusion, nonsense mutations in TNFAIP3, frameshift mutations in PRDM1, and missense mutations in CREBBP, DTX1, and FOXO1. To the best of our knowledge, this case is the first report of genomic mutational profiles of PB-DLBCL in males.

Molecular associations of response to the new-generation BTK inhibitor zanubrutinib in marginal zone lymphoma.

Using tissue whole exome sequencing (WES) and circulating tumor cell-free DNA (ctDNA), this Australasian Leukaemia & Lymphoma Group translational study sought to characterize primary and acquired molecular determinants of response and resistance of marginal zone lymphoma (MZL) to zanubrutinib for patients treated in the MAGNOLIA clinical trial. WES was performed on baseline tumor samples obtained from 18 patients. For 7 patients, ctDNA sequence was interrogated using a bespoke hybrid-capture next-generation sequencing assay for 48 targeted genes. Somatic mutations were correlated with objective response data and survival analysis using Fisher exact test and Kaplan-Meier (log-rank) method, respectively. Baseline WES identified mutations in 33 of 48 (69%) prioritized genes. NF-κB, NOTCH, or B-cell receptor (BCR) pathway genes were implicated in samples from 16 of 18 patients (89%). KMT2D mutations (n= 11) were most common, followed by FAT1 (n= 9), NOTCH1, NOTCH2, TNFAIP3 (n= 5), and MYD88 (n= 4) mutations. MYD88 or TNFAIP3 mutations correlated with improved progression-free survival (PFS). KMT2D mutations trended to worse PFS. Acquired resistance mutations PLCG2 (R665W/R742P) and BTK (C481Y/C481F) were detected in 2 patients whose disease progressed. A BTK E41K noncatalytic activating mutation was identified before treatment in 1 patient who was zanubrutinib-refractory. MYD88, TNFAIP3, and KMT2D mutations correlate with PFS in patients with relapsed/refractory MZL treated with zanubrutinib. Detection of acquired BTK and PLCG2 mutations in ctDNA while on therapy is feasible and may herald clinical disease progression. This trial was registered at https://anzctr.org.au/ as #ACTRN12619000024145.

The Prevalence of MYD88 L265P and TNFAIP3 Mutations and Their Correlations with Clinico-Hematological Profile in Egyptian Patients with Diffuse Large B Cell Lymphoma.

Activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic active B-cell receptor signaling and a constitutive activation of the NF-KB pathway. MYD88 L265P mutation occurs as a driving force of NF-KB overactivity in ABC-DLBCL. Nonetheless, in cases of DLBCL, the MYD88 L265P mutation has not yet been investigated in association with the tumour necrosis factor alpha induced protein3 (TNFAIP3) mutation. To investigate the frequency of MYD88 and TNFAIP3 mutations in DLBCL and their association to the clinico-hematological profile. We used real-time polymerase chain reaction in order to search for MYD88 L265P and TNFAIP3 mutations in 100 DLBCL patients. MYD88 L265P In 20% of cases, the CT heterozygous genotype was discovered. CT heterozygous genotype was more common in ABC type, stage IV, greater IPI groups, extra-nodal infiltration, and BM infiltration. It was also linked to a shorter OS. TNFAIP3 mutation GA heterozygous genotype was detected in 18% of the patients, with ABC-DLBCL subtype accounting for 77.8%. The GA heterozygous genotype was usually related with stage IV, extranodal infiltration, and a reduced life expectancy. MYD88 L265P and to lesser extent TNFAIP3 mutations are major mutations in ABC- DLBCL and may be predictive factors for poor OS in ABC- DLBCL patients.

The search for an in vitro model for monogenic Behçet’s disease: the influence of A20 protein on functional cell assays

BackgroundA20 protein, encoded by TNFAIP3, is involved in NF-kB pathway regulation. Mutations in TNFAIP3 can lead to A20 haploinsufficiency (A20HA), an important monogenic etiology of Behçet’s disease (BD). Understanding the effect of A20 in immune cells can help to comprehend BD pathophysiology, and pave the road for functional assays for patients with monogenic NF-κB disorders. MethodsTHP-1 (monocytic), HL-60 (neutrophilic) and Daudi (B-lymphoid) cells were transfected with a GFP-tagged plasmid containing either TNFAIP3 or mutated TNFAIP3-L227X genes. Cells were assessed for A20 protein expression, proliferation rates, phagocytic activity and reactive oxygen species (ROS) production. Flow cytometry was used for all assays. ResultsA20 expression (figure 1) was increased in Daudi-TNFAIP3 cells, compared to Daudi [mean of fluorescence intensity (MFI) 241 vs 295, p < 0.01]; THP-1-TNFAIP3 had higher A20 expression (MFI 1396 vs 1169), not statistically significant. A20 expression was reduced in THP-1-TNFAIP3-L227X (MFI 1169 vs 581, p < 0.05) and HL-60-TNFAIP3-L227X (MFI 572 vs 450, p < 0.05), compared to non-transfected cells. THP-1-TNFAIP3-L227X showed higher phagocytic activity than THP-1 and THP-1-TNFAIP3 (MFI 94748 vs 34039.33 vs 18476; p < 0.001) at baseline and after stimulus (MFI 76265 vs 35835 vs 25379; p < 0.05). Similarly, HL-60-TNFAIP3-L227X cells showed higher phagocytic activity than HL-60 (17480 vs 5862; p < 0.01) at baseline and after stimulus (16869 vs 5482; p < 0.01) (figure 2). Constitutional ROS production by THP-1-TNFAIP3 was higher than THP-1 and TNFAIP3-L227X (MFI 291919 vs 98226 vs 230711; p < 0.001). PMA-induced Ki-67 expression of THP-1-TNFAIP3-L227X was increased compared with THP-1 and THP-1-TNFAIP3 (19184 vs 2434 vs 4463; p < 0.01). Ki-67 expression was lower at baseline for Daudi-TNFAIP3-WT, compared to Daudi (MFI 10250 vs 12310; p < 0.01) (figure 3). [Display omitted] [Display omitted] [Display omitted] ConclusionsReduced expression of A20 in TNFAIP3-L227X cells is relevant, considering previous reports of A20HA did not show a dominant negative effect. THP-1-TNFAIP3-L227X presented higher phagocytic activity, a pattern also observed by neutrophilic cells (HL-60-TNFAIP3-L227X), suggesting a specific mutation-induced phenotype. Decreased THP-1-TNFAIP3-L227X oxidative burst suggests a state of cell exhaustion. Lower Ki-67 expression in THP-1-TNFAIP3 and Daudi-TNFAIP3 cells at baseline reinforces the role of A20 as an NF-κB pathway inhibitor.

BCL6 Rearranged Indolent B-Cell Lymphomas Show Mutational Profile Similar to Marginal Zone Lymphomas

Introduction Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. It is a neoplasm of germinal center (GC) B cells with hallmark BCL2 rearrangements (BCL2-R). A distinct subgroup of indolent B cell lymphomas (iBCL), histologically revealing a follicular growth pattern but lacking BCL2-R and with BCL6 rearrangements (BCL6-R) has been described. Historically they have been classified as FL and included under the FL entity in the WHO classification. However, recent studies on diffuse Large B cell lymphomas (DLBCL) (1, 2) have described BCL6-R in ~20% of DLBCL, with a subset of cases showing concurrent gene mutations commonly found in marginal zone lymphomas (MZL) rather than those characteristic of FL or GCB DLBCL. Systematic studies elucidating the clinicopathologic and genetic features of the BCL6-R iBCL are currently lacking thus generating controversy over their appropriate classification and management. Methods The MSKCC cytogenetic lab database was searched from 01/2006 to 07/2021 and a cohort of 131 iBCL with BCL6-R (71 with BCL6-R only and 60 with both BCL6-R & BCL2-R) was obtained (Table 1). The clinical, morphologic, immunophenotypic, and molecular results (50 cases had molecular data) were reviewed. These were compared to 70 BCL2-R conventional FL (cFL; all having molecular data) and 54 BCL2-R and BCL6-R negative iBCL (17 had molecular data). ResultsBCL6-R iBCL showed distinct histological, immunophenotypic, genetic and clinical features. They showed a significantly higher number of Grade 3 FL diagnosis and CD10 negative phenotype. The most commonly mutated genes in BCL6-R only group were KMT2D (48%) and TNFAIP3 (40%); in the BCL6-R & BCL2-R group, KMT2D (72%) and BCL2 (56%); and in the cFL group KMT2D (83%), BCL2 (81%) and CREBBP (66%). Unsupervised clustering (K-Means) performed to cluster the samples based on the mutational status of about 50 genes revealed that majority of the BCL6-R only cases clustered separately from cFLs. They clustered with majority of BCL2-R and BCL6-R negative cases and revealed frequent mutations in TNFAIP3, BCL10, B2M, NOTCH2, SPEN (Fig 1). Majority (2/3) of the BCL6-R & BCL2-R cases clustered with cFL and showed mutations commonly seen in FL (Fig 1). The overall survival (OS) was significantly inferior in the BCL6-R cases (BCL6-R only as well as both BCL6-R & BCL2-R cases) in comparison to cFL even though progression free survival was shortest for cFL. ConclusionBCL6-R only iBCL are biologically distinct, cluster separately from cFL based on mutational profile, and demonstrate an MZL-like mutational profile similar to that is described for the BN2 group of DLBCL (1). These findings suggest that they are a distinct entity, different from cFL and possibly precursor lesions for BN2 DLBCL. In contrast, majority of the BCL6-R & BCL2-R cases cluster with cFL and show mutations commonly seen in FL. Finally, presence of BCL6-R was associated with a worse OS.

Detection of High-Frequency Mutation Genes in HIV-Positive and HIV-Negative Lymphomas

Objective Compare HIV-positive lymphoma with HIV-negative lymphoma mutation genes differences, screen specific mutations genes in HIV-positive lymphoma, analyze high frequency mutation genes in HIV-positive lymphoma patients, and explore the effect of HIV infection on lymphoma gene mutation and disease development. It can provide a theoretical basis for the clincal diagnosis and targeted therapy HIV-positive lymphoma. Methods A total of 7 HIV-positive lymphoma patients and 13 HIV-negative lymphoma patients who were treated in Chongqing University Cancer Hospital from September 2020 to September 2021 were enrolled. Tumor tissue samples were detected by second-generation sequencing technology, and gene mutations in HIV-positive lymphoma patients were analyzed by gene heat map, comparing the gene mutation differences between HIV-positive and HIV-negative lymphoma patients. Results Compared with HIV-negative lymphoma patients, more and more complex gene mutation sites were detected in HIV-positive lymphoma patients. Single nucleotide mutation of MYC gene was more common in HIV-positive lymphoma patients, while deletion of KMT2D gene was more common in HIV-negative lymphoma patients. And TP53 gene is highly mutated in both HIV-positive and HIV-negative lymphoma patients. However, mutations of TET, CCND3, TNFRSF14, ARID1A, TNFAIP3, NFKBIE, CXCR4, STAT3, NOTCH1 and other gene mutation with low mutation abundency occurred only in HIV-positive lymphoma patients. NOTCH2, PRDM1, EZH2, CARD11, EP300, MEF2B, CREBBP and other gene mutations occurred only in HIV-negative lymphoma patients. Conclusion HIV-positive lymphoma patients are more prone to gene mutations, and the gene mutations are more complex and diverse. MYC gene mutations and TP53 gene mutations play an important role in the occurrence and development of HIV-positive lymphoma, while the occurrence and development of HIV-negative lymphoma are more related to KMT2D gene mutations and TP53 gene mutations.

TNFAIP3 mutation causing haploinsufficiency of A20 with a hemophagocytic lymphohistiocytosis phenotype: a report of two cases

BackgroundA20 haploinsufficiency (HA20) is a newly introduced autosomal dominant autoinflammatory disorder, also known as Behcet’s-like disease. Some of the most common symptoms of the disease are recurrent oral, genital, and/or gastrointestinal (GI) ulcers, episodic fever, musculoskeletal symptoms, cutaneous lesions, and recurrent infections. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of multi-organ failure due to excessive immune activation. HLH has been reported in a few HA20 patients. Herein, we report two children with the primary presentation of HLH, with a mutation in TNFAIP3, in favor of HA20.Case presentationsOur first patient was a 4-month-old boy who presented with fever, irritability, pallor, and hepatosplenomegaly. Pancytopenia, elevated ferritin, and decreased fibrinogen levels were found in laboratory evaluation. He was diagnosed with HLH and was treated with methylprednisolone and cyclosporine. Two years later, whole exome sequencing (WES) indicated a mutation in TNFAIP3 at NM_001270507: exon3: c.C386T, p.T129M, consistent with A20 haploinsufficiency. Etanercept, a TNF inhibitor, was prescribed, but the parents were reluctant to initiate the therapy. The patient passed away with the clinical picture of cerebral hemorrhage.The second patient was a 3-month-old boy who presented with a fever and hepatosplenomegaly. Laboratory evaluation found pancytopenia, hyperferritinemia, hypoalbuminemia, hypertriglyceridemia, and hypofibrinogenemia. With the establishment of the HLH diagnosis, he was treated with etoposide, dexamethasone, and cyclosporine, and recovered. WES results revealed a heterozygous de novo variant of TNFAIP3 (c. T824C in exon 6, 6q23.3) that leads to a proline to leucine amino acid change (p. L275P). He was treated with etanercept and has been symptom-free afterward.ConclusionsThis report is a hypothesis for developing of the HLH phenotype in the presence of TNFAIP3 mutation. Our results provide a new perspective on the role of TNFAIP3 mutation in HLH phenotypes, but more extensive studies are required to confirm these preliminary results.

TNFAIP3 mutation is an independent poor overall survival factor for patients with T-cell acute lymphoblastic leukemia.

It is imperative to explore potential biomarkers for predicting clinical outcome and developing targeted therapies for T-cell acute lymphoblastic leukemia (T-ALL). This study aimed to investigate the mutation patterns of tumor necrosis factor-alpha-inducing protein 3 (TNFAIP3, also known as A20) and its role in the prognosis of T-ALL patients. Polymerase chain reaction (PCR) and Sanger sequencing data from T-ALL (n= 49, JNU) and targeted sequencing data from T-ALL (n= 54, NFH) in our clinical center and a publicly available dataset (n= 121, PRJCA002270), were used to detect TNFAIP3 mutation. Three TNFAIP3 single nucleotide polymorphisms (SNPs; g.3033 C > T, g.3910 G > A, and g.3904 A > G) were detected in T-ALL in the JNU dataset, and g.3033 C > T accounted for the highest proportion, reaching 60% (6/10). Interestingly, TNFAIP3 mutation mainly occurred in adults but not pediatric patients in all three datasets (JNU, NFH, and PRJCA002270). T-ALL patients carrying a TNFAIP3 mutation were associated with a trend of poor overall survival (OS) (p= 0.092). Moreover, TNFAIP3 mutation was also an independent factor for OS for T-ALL patients (p= 0.008). Further subgroup analysis suggested that TNFAIP3 mutation predicted poor OS for T-ALL patients who underwent chemotherapy only (p< 0.001), and it was positively correlated with high risk and early T-cell precursor ALL (ETP-ALL) in two independent validation datasets (NFH and PRJCA002270). TNFAIP3 mutation mainly occurs in adult T-ALL patients, and it was associated with adverse clinical outcomes for T-ALL patients; thus, it might be a biomarker for prognostic stratification.

Targeted Mutational Profiling Reveals Clonal Relationships in Metachronous Occurrence of Classic Hodgkin and Mediastinal Large B-Cell Lymphomas.

Classic Hodgkin lymphoma (CHL) patients may infrequently present with a prior or recurrent disease with discordant histology resembling non-Hodgkin lymphomas. These include primary mediastinal large B-cell lymphoma (PMBL), diffuse large B-cell lymphoma (DLBCL), or mediastinal gray-zone lymphoma (MGZL). Such patients are often refractory to standard therapy and their diagnosis is hampered by significant morphologic and immunophenotypic overlap and insufficient molecular data. Among 509 CHL patients seen at an academic medical center, 6 patients had a prior or subsequent diagnosis different from CHL. Paired tissue samples were evaluated by targeted mutational analysis using a 164-gene panel. Our findings show multiple shared variants indicative of a clonal relationship between the CHL and the PMBL, DLBCL, or MGZL diagnoses. Most frequent mutated genes included TNFAIP3 (4 of 6, 66.7%), STAT6 (3 or 6, 50%), ARID1A (3 of 6, 50%), and XPO1 (3 of 5, 60%). Three patients showed the same oncogenic variant within the XPO1 gene (E571K), and mutations in TNFAIP3 and B2M were observed in 2 of the 5 patients with shared variants. In addition, differences in the mutation profile between the lymphoma pairs were also observed, which could represent clonal evolution. Mutational profiling could be of benefit in patients with recurrent/refractory disease with discordant histology, where the clonal relationship could be helpful to inform and guide therapeutic decisions. These findings provide further evidence of a true biological continuum surrounding CHL, PMBL, DLBCL, and MGZL and shed light on underlying genetic events and their clinical impact.

Genomic Mutation Landscape of Primary Breast Lymphoma: Next-Generation Sequencing Analysis.

Primary breast lymphoma (PBL) is a rare subtype of non-Hodgkin's lymphoma (NHL) with rapid progression and high risk of central nervous system metastasis. We have investigated 40 PBL patients retrospectively, and 16 of them were sequenced by a target panel of 112 genes related with lymphoma. Next-generation sequencing (NGS) identified 203 mutations spanning 35 genes and revealed seven potential protein-changing genes (PIM1, MYD88, DTX1, CD79B, KMT2D, TNFAIP3, and ITPKB) with high frequency, referring crucial roles in lymphomagenesis. Our result suggested that PIM1 mutation is correlated with the age and pathological type of PBL patients. Gene TNFAIP3 and KMT2D mutation is only related to the pathological type and primary site, respectively. These high-mutant genes detected in PBL indicated a tendency to shorten overall survival (OS) and progression-free survival (PFS), which may lead to poor prognosis. Furthermore, the nuclear factor kappa-B (NF-κB) pathway and related regulatory factors are essential for the development of targeted therapy as well.

The immune microenvironment features and response to immunotherapy in EBV-associated lymphoepithelioma-like cholangiocarcinoma.

Limited data are available for tumor immune microenvironment (TIME) in Epstein-Barr virus (EBV)-associated lymphoepithelioma-like cholangiocarcinoma (EBV-LELCC), a rare subtype of intrahepatic cholangiocarcinoma (IHCC). We aimed to investigate TIME features in EBV-LELCC and the correlation between the components of TIME and the clinical outcomes. Tumor tissues from five EBV-LELCC cases confirmed through EBER in situ hybridization and five stage-matched conventional IHCC (non-EBV IHCC) cases were collected. These samples were used to evaluate genetic alterations, TIME composition, and PD-L1 expression through ion AmpliSeq comprehensive cancer panel, PanCancer immune profiling panel, immunohistochemistry, and immunofluorescence staining. The correlation between clinical outcomes and TIME components was analyzed in the two EBV-LELCC cases receiving anti-PD-1 treatment. The genetic mutations identified in EBV-LELCC were BARD1, CD19, CD79B, EPHA5, KDM5A, MUC6, MUC16, PTEN, RECQL4, TET1, and TNFAIP3. Both CD79B and TNFAIP3 mutations were involved in the NF-κB signaling pathway. PD-L1 was highly expressed in tumor-infiltrating immune cells, especially the T cells and macrophages. The TIME of EBV-LELCC displayed abundant immune cell infiltration with a stronger adaptive immune response. Increased Th1 cells, NK CD56dim cells, and M1 macrophages, decreased M2 macrophages, exhausted CD8 T cell infiltration, and increased T cell activation signatures in TIME were associated with longer survival. Two patients with metastatic EBV-LELCC had good disease control after anti-PD-1 antibody treatment. A significantly larger TIME component made EBV-LELCCs more sensitive to immune checkpoint blockade (ICB). A better understanding of the composition of TIME in EBV-LELCC is critical for predicting the clinical outcomes of ICB treatment.