CARD11 Mutations Research Articles

The efficacy and safety of ZR2 versus R-CHOP-like for elderly patients with newly diagnosed diffuse large B cell lymphoma: a single-center prospective study in China.

Bruton's tyrosine kinase inhibitors have been demonstrated preliminary efficacy in diffuse large B-cell lymphoma (DLBCL). To compare the safety and efficacy of zanubrutinib plus rituximab and lenalidomide (ZR2) and R-CHOP-like for elderly patients with newly diagnosed DLBCL, we conducted this single-center prospective study. Patients were treated with 6 cycles of ZR2 or R-CHOP-like regimen for the first-line treatment. The primary endpoint was complete response ratio (CRR). The secondary outcome measures were progression-free survival (PFS), overall survival (OS), and adverse events. Between June 15, 2020, and March 11, 2023, 30 patients with ZR2 and 60 patients with R-CHOP-like were enrolled. There were no significant differences observed in CRR (P = 0.878), PFS (P = 0.555) and OS (P = 0.769) between ZR2 and R-CHOP-like group. While, patients in ZR2 group had the following features: significantly older (P = 0.002), more unfit (P < 0.001) and higher prognosis risk scores (P = 0.025). The incidence of grade ≥ 3 anemia (P = 0.008) and pneumonia (P = 0.001) was significantly lower in ZR2 group. Patients with germinal center B-cell-like subtype (GCB), large masses or TP53 mutations had a satisfactory remission rate in ZR2 group (57.1%, 77.8% and 60.0%, respectively). ZR2 and R-CHOP-like regimen had similar efficacy and survival. While, the safety profile for ZR2 was superior. GCB subtype, large masses and TP53 mutations may benefit from ZR2 regimen as well. Patients with EBV-positive and CARD11 mutations may need additional treatment rather than ZR2. Patients with gastrointestinal DLBCL have to be monitored closely by abdominal enhanced CT every cycle. Overall, ZR2 chemo-free regimen might be more appropriate for elderly DLBCL patients.

A new-disease-causing dominant-negative variant in CARD11 gene in a Chinese case with recurrent fever

Immunodeficiency 11B with atopic dermatitis (IMD11B, OMIM:617638) is rare primary immunodeficiency disease caused by germline dominant negative (DN) mutations in the CARD11 gene. Affected patients present with immune dysfunction, recurrent infections and atopic dermatitis. In this study, we sought to identify and characterize the genetic variant in one patient with periodic fever, recurrent infections, and eczema. Trio whole-exome sequencing (WES) was employed in this patient and her parents, and Sanger sequencing validated the potential pathogenic variant. In vitro functional study was performed to evaluate the pathogenicity of genetic variant identified. A very rare missense mutation (c.2324C > T, p.S775L) in CARD11 gene (NM_032415) was identified by WES in the patient but not her parents. Luciferase reporter assays and co-immunoprecipitation demonstrated mutation exerts a dominant-interfering effect on wild-type CARD11, inhibiting the activity of NF-κB. RNA sequencing analysis also confirmed that mutant CARD11 inhibited down-stream transcriptional activity of NF-κB. A review of literature doesn’t found significant genotype–phenotype correlation. We identified a vary rare DN CARD11 mutation, expanding the genetic and phenotypic spectrum of CARD11.

A case of revertant mosaic-like normal-looking spots in a patient with erythroderma with IL36RN and CARD14 heterozygous mutations.

An 89-year-old Japanese woman presented with erythroderma associated with significant scaling. A histological examination showed acanthosis with hyperkeratosis and hyperkeratinization of the hair follicles. Genetic analyses using DNA from the peripheral blood revealed heterozygous mutations in IL36RN (c.115+6T>C) and CARD14 c.2648G>A (p.Arg883His). Based on these findings, we diagnosed her with erythroderma attributable to autoinflammatory keratinization disease. She then developed more than 30 small, round, well-defined, spots on her back and extremities that appeared histologically normal. We suspected that these spots might be revertant mosaicism. Immunohistochemical staining with p65, which is a component of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), revealed nuclear staining in epidermal keratinocytes in erythematous lesions, but not in the normal-looking spots. However, mutations in IL36RN and CARD14 unexpectedly persisted in the epidermis and dermis of the normal-looking spots.

CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation.

Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-κB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138A alteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-κB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1 (mTORC1), which stimulated keratinocyte metabolism, but not for NF-κB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis.

Comparative Efficacy and Safety of Ustekinumab and Secukinumab in the Treatment of Generalized Pustular Psoriasis: A 48-Week Retrospective Cohort Study with Genetic Background Analysis.

Recent studies have shown that novel biologics may provide significant clinical benefits for patients with generalized pustular psoriasis (GPP). Ustekinumab and secukinumab have been approved in Japan for GPP treatment in adult patients. However, the differences in efficacy and safety of these two drugs in GPP are not known. Based on the genetic background, we aimed to compare the efficacy and safety of secukinumab and ustekinumab in patients with GPP. Patients with moderate to severe GPP who were treated with ustekinumab/secukinumab at our department from July 2019 to May 2022 were included in this study and followed up for 48weeks. The difference in efficacy between ustekinumab and secukinumab was evaluated by assessing changes in body temperature, laboratory indices, recovery of skin lesions, and changes in quality of life. Additionally, we collected patients' saliva for genotyping and explored the effect of CARD14 genetic mutations on clinical efficacy. A total of 65 patients (32 adults and 33 children) with moderate to severe GPP were included in this study. 31 patients received ustekinumab therapy, and 34 patients were treated with secukinumab. Secukinumab demonstrated superiority to ustekinumab, as evidenced by a higher GPPASI 90 response at week 2. Additionally, the efficacy of ustekinumab and secukinumab was found to be independent of the presence of the CARD14 mutation. Secukinumab is superior to ustekinumab in rapidly clearing the skin and improving health-related quality of life. Moreover, the responses to ustekinumab/secukinumab in patients were not influenced by CARD14 gene mutations.

Specific Mutation Predict Relapse/Refractory Diffuse Large B-Cell Lymphoma.

The application of rituximab has significantly enhanced the overall survival rates in patients with diffuse large B-cell lymphoma (DLBCL). Regrettably, a significant number of patients still progress to relapse/refractory DLBCL (rrDLBCL). Herein, we employed targeted sequencing of 55 genes to investigate if gene mutations could predict the progression to rrDLBCL. Additionally, we compared the mutation profiles at the time of DLBCL diagnosis with those found in rrDLBCL cases. Our findings highlighted significantly elevated mutation frequencies of TP53, MEF2B and CD58 in diagnostic biopsies from patients who progressed to relapse or refractory disease, with CD58 mutations exclusively observed in the rrDLBCL group. In assessing the predictive power of mutation profiles for treatment responses in primary DLBCL patients, we found that the frequency of CARD11 mutations was substantially higher in non-response group as compared with those who responded to immunochemotherapy. In addition, we revealed mutations in HIST2H2AB, BCL2, NRXN3, FOXO1, HIST1H1C, LYN and TBL1XR1 genes were only detected in initial diagnostic biopsies, mutations in the EBF1 gene were solely detected in the rrDLBCL patients. Collectively, this study elucidates some of the genetic mechanisms contributing to the progression of rrDLBCL and suggests that the presence of CD58 mutations might serve as a powerful predictive marker for relapse/refractory outcomes in primary DLBCL patients.

Distinctive genomic features of human T-lymphotropic virus type 1-related adult T-cell leukemia-lymphoma in Western populations.

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive Human T-cell Leukemia Virus Type 1 (HTLV-1)-driven malignancy. Although Western hemisphere (Afro-Caribbean and South American) patients face worse prognoses, our understanding of ATLL molecular drivers derives mostly from Japanese studies. We performed multi-omic analyses to elucidate the genomic landscape of ATLL in Western cohorts. Recurrent deletion and/or damaging mutations involving FOXO3, ANKRD11, DGKZ, and PTPN6 implicate these genes as potential tumor suppressors. RNA-seq, published functional data and in vitro assays support the roles of ANKRD11 and FOXO3 as regulators of T-cell proliferation and apoptosis in ATLL, respectively. Survival data suggest ANKRD11 mutation may confer a worse prognosis. Japanese and Western cohorts, in addition to acute and lymphomatous subtypes, demonstrated distinct molecular patterns. GATA3 deletion was associated with unfavorable chronic cases. IRF4 and CARD11 mutations frequently emerged in relapses after interferon therapy. Our findings reveal novel putative ATLL driver genes and clinically relevant differences between Japanese and Western ATLL patients.

Hyper IgE Syndromes.

The Hyper IgE Syndromes are rare primary immunodeficiencies characterized by eczema, recurrent skin and respiratory infections and elevated serum IgE levels. Nowadays a geneticmolecular characterization is possible and allows the distinction in various monogenic pathologies, which share some clinical characteristics but also important differences. In addition to long-known STAT3 and DOCK8 gene mutations, in fact, also ZNF341, CARD11, ERBB2IP, IL6R and IL6ST genes mutations can cause the disease. The main clinical manifestations are represented by newborn rash, eczema similar to atopic dermatitis, bacterial and viral skin infections, cold abscesses, respiratory infections with possible pulmonary complications, allergies, gastrointestinal manifestations, malignancies and connective tissue abnormalities. Diagnosis is still a challenge because, especially in the early stages of life, it is difficult to distinguish from other pathologies characterized by eczema and high IgE, such as atopic dermatitis. Several scores and diagnostic pathways have been developed, but it is essential to seek a genetic diagnosis. Treatment is based on prevention and early treatment of infections, meticulous skincare, intravenous immunoglobulins and HSCT, which, in some HIES subtypes, can modify the prognosis. Prognosis is related to the affected gene, but also to early diagnosis, timely treatment of infections and early HSCT.

Abstract PO-016: Test-the-test: Clinical utility of comprehensive whole exome sequencing (WES) and RNA-seq for lymphoma patients

Abstract Due to the rapidly evolving landscape of targeted therapies, there is an unmet need for comprehensive molecular profiling to guide treatment decisions for lymphoma patients. Here, we designed a pilot study to assess the feasibility and turnaround time (TAT) of a comprehensive clinical whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) assay in large B-cell lymphoma (LBCL) patients for clinical decision-making (NCT05464823). Patients aged ≥18 years with histologically documented LBCL requiring therapy were eligible. Formalin-fixed paraffin-embedded tissues underwent WES, RNA-seq, and copy number analysis with concomitant peripheral blood or saliva for germline DNA sequencing. Samples with &amp;lt;20% tumor purity and median coverage of &amp;lt;142x for tumor and/or &amp;lt;95x for normal samples were labeled as quantity not sufficient (QNS). Genomic and transcriptomic data were profiled to uncover clinically relevant biomarkers and match patients with clinical trials on ClinicalTrials.gov. Among 84 patients analyzed to date, 71 received WES and RNA-seq, yielding 67 reports with clinical information (treatment options and clinical trial matching) and 4 QNS reports without actionable findings. The remaining 13 samples were rejected as they failed pathology quality control for next-generation sequencing. Cell of origin (COO, n=62), LymphGen (n=62), and lymphoma microenvironment (LME, n=64) classifications were applied. The median TAT was 8 days for custom reports, and 70.4% of custom reports had a TAT ≤10 days. WES identified frequent TP53 (n=22) and MYD88 (n=11) alterations. COOs were designated as activated (n=26, 41.94%) or germinal center (n=36, 58.06%) LBCL. The LymphGen classifier (Wright et al. Cancer Cell. 2020) showed subtypes EZB MYC- (n=10, 16.13%), MCD (n=8, 12.9%), and A53 (n=6, 11.5%) as the most prevalent. LME classification revealed most LMEs as mesenchymal (n=46, 71.88%). Immune-depleted (n=12, 18.75%), immune-inflamed (n=4, 6.25%), and germinal center-like (n=2, 3.13%) LMEs were less prevalent, but the immune-depleted LME was more common in patients with relapse. Clinically significant findings, such as activated COO subtype (n=27), TP53 loss (n=21), DHITsig+ (n=9), and CARD11 mutations associated with ibrutinib resistance (n=3), were identified in 52 samples. On average, 8 clinical trials were identified per full report. Our results show the clinical utility and acceptable TAT of using a comprehensive WES and RNA-seq assay on a cohort of lymphoma patients. The full test reports included findings on significant alterations, LymphGen and LME subtypes, COOs, treatment options, and clinical trial matches. These robust findings, coupled with rapid TAT, demonstrate the feasibility of using integrated WES and RNA-seq to guide clinical decision-making for lymphoma patients. Citation Format: Dai Chihara, Kumudha Balakrishnan, Gita Masand, Anna Novokreshchenova, Alexander Bagaev, Nikita Kotlov, Ekaterina Postovalova, Eduardo Shugaev-Mendosa, Yuliya Gracheva, Kelley Lauziere, Leznath Kaneunyenye, Brianna McKenna, Amber Berlinski, McKenna Walsh, Linnea Larson, Mariam Kotsinyan, Sheila T Yong, Krystle Nomie, Nathan Fowler, Christopher R. Flowers, Jason Westin. Test-the-test: Clinical utility of comprehensive whole exome sequencing (WES) and RNA-seq for lymphoma patients [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-016.

Clinical implications of CSF-ctDNA positivity in newly diagnosed diffuse large B cell lymphoma.

The clinical implications of CSF-ctDNA positivity in newly diagnosed diffuse large B cell lymphoma (ND-DLBCL) remains largely unexplored. One hundredND-DLBCL patients were consecutively enrolled as training cohort and another 26 ND-DLBCL patients were prospectively enrolled in validation cohort. CSF-ctDNA positivity (CSF(+)) was identified in 25 patients (25.0%) in the training cohort and 7 patients (26.9%) in the validation cohort, extremely higher than CNS involvement rate detected by conventional methods. Patients with mutations of CARD11, JAK2, ID3, and PLCG2 were more predominant with CSF(+) while FAT4 mutations were negatively correlated with CSF(+). The downregulation of PI3K-AKT signaling, focal adhesion, actin cytoskeleton, and tight junction pathways were enriched in CSF(+) ND-DLBCL. Furthermore, pretreatment CSF(+) was significantly associated with poor outcomes. Three risk factors, including high CSF protein level, high plasma ctDNA burden, and involvement of high-risk sites were used to predict the risk of CSF(+) in ND-DLBCL. The sensitivity and specificity of pretreatment CSF-ctDNA to predict CNS relapse were 100% and 77.3%. Taken together, we firstly present the prevalence and the genomic and transcriptomic landscape for CSF-ctDNA(+) DLBCL and highlight the importance of CSF-ctDNA as a noninvasive biomarker in detecting and monitoring of CSF infiltration and predicting CNS relapse in DLBCL.

The First Case of Autosomal Recessive CADINS-like Disease Caused by Novel Compound Heterozygous Hypomorphic Variants in CARD11.

Abstract CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor ligation to signaling governing lymphocyte proliferation and differentiation. We and others have shown that heterozygous, dominant negative (DN) CARD11 mutations disrupting T cell receptor (TCR)-induced NF-κB signaling cause severe atopy in humans, often with recurrent infections and humoral immune deficits. Here we describe a patient with a history of recurrent upper respiratory infections, recalcitrant atopic dermatitis and common variable immunodeficiency, carrying novel compound heterozygous CARD11 variants (G689E, R920L) in regions of the protein (PDZ and L4, respectively) not normally associated with deleterious mutations. By transfecting variant expression plasmids into CARD11-deficient Jurkat T cells harboring an NF-κB-driven GFP reporter, we found that G689E was mildly hypomorphic, whereas R920L was a complete loss-of-function mutation with negligible DN activity against wild-type CARD11. However, TCR-induced NF-κB activity was markedly reduced upon variant co-expression, strongly suggesting this patient represents the first autosomal recessive case of CARD11-associated Atopy with Dominant Interference of NF-κB Signaling (CADINS) disease. Additional experiments examining different amino acid substitutions at these residues highlight key biochemical interactions between the PDZ and L4 domains, with important implications for future CARD11 structure-function and clinical diagnostic studies.

The Molecular Landscape of Primary CNS Lymphomas (PCNSLs) in Children and Young Adults.

Pediatric brain tumors are often noted to be different from their adult counterparts in terms of molecular features. Primary CNS lymphomas (PCNSLs) are mostly found in elderly adults and are uncommon in children and teenagers. There has only been scanty information about the molecular features of PCNSLs at a young age. We examined PCNSLs in 34 young patients aged between 7 and 39 years for gene rearrangements of BCl2, BCL6, CCND1, IRF4, IGH, IGL, IGK, and MYC, homozygous deletions (HD) of CDKN2A, and HLA by FISH. Sequencing was performed using WES, panel target sequencing, or Sanger sequencing due to the small amount of available tissues. The median OS was 97.5 months and longer than that for older patients with PCNSLs. Overall, only 14 instances of gene rearrangement were found (5%), and patients with any gene rearrangement were significantly older (p = 0.029). CDKN2A HD was associated with a shorter OS (p < 0.001). Only 10/31 (32%) showed MYD88 mutations, which were not prognostically significant, and only three of them were L265P mutations. CARD11 mutations were found in 8/24 (33%) cases only. Immunophenotypically, the cases were predominantly GCB, in contrast to older adults (61%). In summary, we showed that molecular findings identified in the PCNSLs of the older patients were only sparingly present in pediatric and young adult patients.

Genetic analysis of different subtypes of aseptic pustulosis in the Chinese population.

Aseptic pustulosis involves inflammatory skin conditions with nonbacterial pustules on erythema, accompanied by neutrophil and eosinophil infiltration in the epidermis. Dysregulation of the interleukin (IL)-36 pathway leads to neutrophil aggregation and pustule formation. Variants in IL36RN, CARD14, AP1S3, MPO, SERPINA3 and BTN3A3 have been identified in generalized pustular psoriasis (GPP) in the past. Some patients with acrodermatitis continua of Hallopeau (ACH), palmoplantar pustulosis and acute generalized exanthematous pustulosis (AGEP) also exhibit mutations in IL36RN, CARD14 and AP1S3, albeit with regional and population-specific variations. This study aims to explore a shared genetic foundation among those with aseptic pustulosis. We performed Sanger sequencing on six genes in 126 patients with aseptic pustulosis. Genetic analysis identified IL36RN variants strongly associated with ACH, AGEP and subcorneal pustular dermatosis (SPD). Immunohistochemistry revealed elevated inflammatory cytokines in all subtypes. This study establishes a significant association between IL36RN variants and ACH, AGEP and SPD, emphasizing the IL-1/IL-36-chemokine-neutrophil axis as a common pathogenic mechanism. Targeting this axis holds promise for therapeutic interventions for aseptic pustulosis.

CARD11 regulates the thymic Treg development in an NF-κB-independent manner.

CARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-κB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg). In this study, we used patients' samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-κB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-κB independent manner. We found CARD11 mutations causing hyper-activated NF-κB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/ FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-κB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-κB independent pathway for thymic Treg lineage commitment.

Abstract 7583: Discovery of a potent small molecule MALT1 protease inhibitor for the treatment of cancer

Abstract The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), as the core component of CBM complex (CARD9/10/11/14-BCL10-MALT1), plays an essential role in activating the NF-κB pathway and regulating the cell proliferation, differentiation and immunity. Inhibiting of MALT1 is considered a promising therapeutic approach for the treatment of several non-Hodgkin B-cell lymphomas, chronic lymphocytic leukemia (CLL), solid tumors and autoimmune diseases. Herein, we report the discovery of a novel potent MALT1 allosteric inhibitor, BPI-530616, which strongly inhibited MALT1 protease activity with subnanomolar IC50, and low off-target activity on other proteases. BPI-530616 can inhibit the proliferation of B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell line, OCI-ly3, bearing CD79b and CARD11 mutations. The cell line is intrinsically resistant to covalent and non-covalent Bruton's Tyrosine Kinase (BTK) inhibitors. Meanwhile, BPI-530616 suppressed the cellular cytokine secretion (e.g.IL-10, IL-2) or MALT1 direct substrate cleavage (e.g. CYLD). In vivo, BPI-530616 dose-dependently suppressed tumor growth and inhibited the cleavage of CYLD in OCI-ly3 xenograft mouse model. To address the role of MALT1 inhibitor in the contest of immune-oncology, a CT26 syngeneic mouse model was set up. Oral administration of BPI-530616 exhibited a good antitumor effect comparable to that of the anti-PD1 antibody, with no observable adverse effects. Additionally, BPI-530616 displayed desirable drug-like properties, including low clearance and good oral bioavailability across multiple pre-clinical species. Taken together, our research has led to the discovery of a novel, potent, selective, and orally bioavailable small molecule MALT1 inhibitor, BPI-530616. It demonstrated anti-tumor efficacy in the DLBCL animal model and showed promising effects in an immune-oncology animal model. Citation Format: Yuzhen Zhou, Bang Fu, Jie Chen, Weidong Cai, Xiao Sun, Yinlong Li, Wei Ren, Qichao Shen, Guolong Du, Xiaojun Zhou, Zhengyao Zou, Haibo Chen, Xiaoyun Liu, Cheng Yang, Xiangyong Liu, Jing Guo, Hao Wu, Lieming Ding, Jiabing Wang, Hong Lan. Discovery of a potent small molecule MALT1 protease inhibitor for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7583.

Abstract 1754: Test-the-test: Clinical utility of comprehensive whole exome sequencing (WES) and RNA-seq for lymphoma patients

Abstract Background: Due to the rapidly evolving landscape of targeted therapies, there is an unmet need for comprehensive molecular profiling to guide treatment decisions for patients with lymphoma. To meet this need, we designed a pilot study to assess the feasibility and turnaround time (TAT) of a comprehensive WES and whole transcriptome sequencing (RNA-seq) assay (BostonGene Tumor PortraitTM test) in large B-cell lymphoma (LBCL) patients for clinical decision-making (NCT05464823). Design: Patients aged ≥18 years with histologically documented LBCL requiring therapy were eligible. Formalin-fixed paraffin-embedded tissues underwent WES, RNA-seq, and copy number analysis (CNA) with concomitant peripheral blood or saliva for germline DNA sequencing. Samples with &amp;lt;20% tumor purity and median coverage of &amp;lt;142x for tumor and/or &amp;lt;95x for normal samples were labeled as quantity not sufficient (QNS). Genomic and transcriptomic data were profiled to uncover clinically relevant biomarkers and match patients with clinical trials on ClinicalTrials.gov. Results: To date, 63 patients received WES and RNA-seq, with 45 full Tumor PortraitTM test clinical reports delivered and 3 partial reports for QNS samples. Cell of origin (COO, n = 41), LymphGen (n = 41), and lymphoma microenvironment (LME, n = 42) classifications were applied. The median TAT was 8 days for advanced reports, and 76% of reports had a TAT ≤9 days. WES identified frequent TP53 (n = 13) and CDK2NA (n = 5) alterations. COOs were designated as activated (n = 16, 39%) or germinal center (n = 25, 61%) LBCL. The LymphGen classifier showed the EZB MYC- (n = 9, 22%), A53 (n = 6, 15%), and MCD (n = 6, 15%) subtypes as the most prevalent. LME classification revealed most LMEs as mesenchymal (n = 30, 71%). Immune-depleted (n = 7, 17%), immune-inflamed (n = 4, 10%), and germinal center-like (n = 1, 2%) LMEs were less prevalent, but the immune-depleted LME was more common in patients with relapse. Clinically significant findings, such as ABC COO (n = 16), TP53 loss (n = 13), DHITsig+ (n = 6), and CARD11 mutations associated with ibrutinib resistance (n = 3), were identified in 33 samples. On average, 8 clinical trials were identified per delivered report. Conclusion: Our results show the clinical utility and acceptable TAT of using a comprehensive WES and RNA-seq assay on a cohort of lymphoma patients. The produced BostonGene Tumor PortraitTM test reports included findings on significant alterations, LymphGen and LME subtypes, COOs, and potential clinical trial matches. These robust findings, coupled with the rapid TAT, demonstrate the feasibility of using integrated WES and RNA-seq to guide clinical decision-making in lymphoma patients. Citation Format: Dai Chihara, Kumudha Balakrishnan, Gita Masand, Anna Novokreshchenova, Alexander Bagaev, Nikita Kotlov, Ekaterina Postovalova, Eduardo Shugaev-Mendosa, Yuliya Gracheva, Anna Love, Krystle Nomie, Nathan Fowler, Christopher R. Flowers, Jason Westin. Test-the-test: Clinical utility of comprehensive whole exome sequencing (WES) and RNA-seq for lymphoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1754.

Somatic mutations in four novel genes contribute to homologous recombination deficiency in breast cancer: a real-world clinical tumor sequencing study.

Breast cancers involving mutations in homologous recombination (HR) genes, most commonly BRCA1 and BRCA2 (BRCA1/2), respond well to PARP inhibitors and platinum-based chemotherapy. However, except for these specific HR genes, it is not clear which other mutations contribute to homologous recombination defects (HRD). Here, we performed next-generation sequencing of tumor tissues and matched blood samples from 119 breast cancer patients using the OncoScreen Plus panel. Genomic mutation characteristics and HRD scores were analyzed. In the HR genes, we found that BRCA1/2 and PLAB2 mutations were related to HRD. HRD was also detected in a subset of patients without germline or somatic mutations in BRCA1/2, PLAB2, or other HR-related genes. Notably, LRP1B, NOTCH3, GATA2, and CARD11 (abbreviated as LNGC) mutations were associated with high HRD scores in breast cancer patients. Furthermore, functional experiments demonstrated that silencing CARD11 and GATA2 impairs HR repair efficiency and enhances the sensitivity of tumor cells to olaparib treatment. In summary, in the absence of mutations in the HR genes, the sensitivity of tumor cells to PARP inhibitors and platinum-based chemotherapy may be enhanced in a subset of breast cancer patients with LNGC somatic mutations.

CARD9 mutations and T cell immune response in patients with chromoblastomycosis

CARD9 mutations and T cell immune response in patients with chromoblastomycosis

Chimeric and mutant CARD9 constructs enable analyses of conserved and diverged autoinhibition mechanisms in the CARD-CC protein family.

Caspase recruitment domain-containing protein (CARD)9, CARD10, CARD11, and CARD14 all belong to the CARD-coiled coil (CC) protein family and originated from a single common ancestral protein early in vertebrate evolution. All four proteins form CARD-CC/BCL10/MALT1 (CBM) complexes leading to nuclear factor-kappa-B (NF-κB) activation after upstream phosphorylation by various protein kinase C (PKC) isoforms. CBM complex signaling is critical for innate and adaptive immunity, but aberrant activation can cause autoimmune or autoinflammatory diseases, or be oncogenic. CARD9 shows a superior auto-inhibition compared with other CARD-CC family proteins, with very low spontaneous activity when overexpressed in HEK293T cells. In contrast, the poor auto-inhibition of other CARD-CC family proteins, especially CARD10 (CARMA3) and CARD14 (CARMA2), is hampering characterization of upstream activators or activating mutations in overexpression studies. We grafted different domains from CARD10, 11, and 14 on CARD9 to generate chimeric CARD9 backbones for functional characterization of activating mutants using NF-κB reporter gene activation in HEK293T cells as readout. CARD11 (CARMA1) activity was not further reduced by grafting on CARD9 backbones. The chimeric CARD9 approach was subsequently validated by using several known disease-associated mutations in CARD10 and CARD14, and additional screening allowed us to identify several previously unknown activating natural variants in human CARD9 and CARD10. Using Genebass as a resource of exome-based disease association statistics, we found that activated alleles of CARD9 correlate with irritable bowel syndrome (IBS), constipation, osteoarthritis, fibromyalgia, insomnia, anxiety, and depression, which can occur as comorbidities.

Test-the-Test: Clinical Utility of Comprehensive Whole Exome Sequencing and RNA-Seq for Patients with Lymphoma

Introduction Due to the rapidly evolving landscape of targeted therapies, there is an unmet need for comprehensive molecular profiling to guide treatment decisions for patients with lymphoma. To meet this need, we designed a pilot study to assess the feasibility and turnaround time (TAT) of comprehensive whole exome (WES) and whole transcriptome (RNA-seq) sequencing (Tumor Portrait test) in patients with lymphoma for future clinical decision-making (NCT05464823). Here, we present interim results for patients with large B-cell lymphoma (LBCL). Methods Patients ≥18 years of age with histologically documented LBCL requiring therapy were eligible. Formalin-fixed paraffin-embedded tissues underwent WES, RNA-seq, and copy number analysis with concomitant peripheral blood or saliva for germline DNA sequencing. During quality control, samples with &amp;lt;20% tumor purity and median coverage of &amp;lt;142x for tumor and/or &amp;lt;95x for normal samples were labeled as quantity not sufficient (QNS)-qualified. Genomic and transcriptomic data were profiled for alterations and molecular features to uncover clinically relevant biomarkers and match patients with clinical trials using data from ClinicalTrials.gov. Results To date, 48 patients received WES and RNA-seq, and cohort data were analyzed for assay performance. Two samples were excluded from the analysis due to follicular lymphoma and chronic lymphocytic leukemia pathology, and seven samples were excluded as QNS. Reports were delivered for 39 patients, which included 37 clinical reports and two advanced reports lacking treatment and clinical trial information for QNS-qualified samples. While the QNS-qualified samples did not meet quality control standards, some data were still obtained and delivered as partial clinical reports. Of the 39 patients who received clinical reports, 34 samples were evaluable for bioinformatics analysis, and two of these samples had either only RNA-seq or WES data available (total n = 34, Figure 1). Cell of origin (COO), LymphGen (Wright et al. Cancer Cell. 2020), and lymphoma microenvironment (LME; Kotlov et al. Cancer Discov. 2021) classifications were applied to 33 patients. The median TAT was 8 days for advanced report delivery, and 85% of reports had a TAT &amp;lt;10 days. WES identified frequent TP53 (n = 11) and KMT2D (n = 10) loss of function and MYD88 (n = 5) gain of function alterations, as anticipated for LBCL. COOs were assigned as activated (ABC; n = 14, 42%) or germinal center (GCB; n = 19, 58%) LBCL. Using the LymphGen classifier, EZB MYC- (n = 7, 22%), A53 (n = 6, 19%), and MCD (n = 5, 16%) were the most prevalent subtypes. LME classification revealed the majority of samples were mesenchymal (ME; n = 22, 67%). Immune-depleted (DE; n = 6, 18%), immune-inflamed (IN; n = 4, 12%), and germinal center-like (GC; n = 1, 3%) subtypes were less prevalent, but the immune-depleted LME occurred more commonly in patients at relapse. Clinically significant findings, such as DHITsig+ (n = 4) and CARD11 mutations indicative of ibrutinib resistance (n = 3), were identified in 26 of the 39 patients ( Table 1), and an average of 8 clinical trials were identified for potential patient enrollment in each delivered report. Conclusions These data indicated that comprehensive WES and RNA-seq molecular profiling can be performed for lymphoma patients with acceptable TAT. This approach identified significant alterations and classified LBCL samples into previously reported lymphoma subtypes. The clinical reports included clinically relevant findings and matched clinical trials, demonstrating the feasibility of incorporating comprehensive molecular profiling in clinical practice for patients with lymphoma.