ABSTRACT
Glioblastoma, IDH-wild type, the most common malignant primary central nervous system tumor, represents a formidable challenge in clinical management due to its poor prognosis and limited therapeutic responses. With an evolving understanding of its underlying biology, there is an urgent need to identify prognostic molecular groups that can be subject to targeted therapy. This study established a cohort of 124 sequential glioblastomas from a tertiary hospital and aimed to find correlations between molecular features and survival outcomes. Comprehensive molecular characterization of the cohort revealed prevalent alterations as previously described, such as TERT promoter mutations and involvement of the PI3K-Akt-mTOR, CK4/6-CDKN2A/B-RB1, and p14ARF-MDM2-MDM4-p53 pathways. MGMT promoter methylation is a significant predictor of improved overall survival, aligned with previous data. Conversely, age showed a marginal association with higher mortality. Multivariate analysis to account for the effect of MGMT promoter methylation and age showed that, in contrast to other published series, this cohort demonstrated improved survival for tumors harboring PTEN mutations, and that there was no observed difference for most other molecular alterations, including EGFR amplification, RB1 loss, or the coexistence of EGFR amplification and deletion/exon skipping (EGFRvIII). Despite limitations in sample size, this study contributes data to the molecular landscape of glioblastomas, prompting further investigations to examine these findings more closely in larger cohorts.
Subject(s)
Brain Neoplasms , Glioblastoma , Isocitrate Dehydrogenase , Humans , Glioblastoma/genetics , Glioblastoma/mortality , Glioblastoma/pathology , Middle Aged , Male , Female , Brain Neoplasms/genetics , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Aged , Adult , Isocitrate Dehydrogenase/genetics , Mutation , Cohort Studies , Prognosis , Biomarkers, Tumor/genetics , DNA Methylation/genetics , Young Adult , Aged, 80 and over , Promoter Regions, Genetic/genetics , Survival AnalysisABSTRACT
Multiple Myeloma (MM) is a plasma cell neoplasm originating in the bone marrow and is the second most common blood cancer in the United States. One challenge in understanding the pathogenesis of MM and improving treatment is a lack of immunocompetent mouse models. We previously developed the IL6Myc mouse that generates plasmacytomas at 100% penetrance that phenotypically resemble aggressive MM. Using comprehensive genomic analysis, we found that the IL6Myc tumors resemble aggressive MM by RNA and protein expression. We also found that IL6Myc tumors accumulated fusions and missense mutations in genes that overlap significantly with human myeloma, indicating that the mouse is good model for studying disease etiology. Lastly, we derived cell lines from IL6Myc tumors that express cell surface markers typical of MM and readily engraft into mice, home to the bone marrow, and induce osteolytic disease. The cell lines may be useful in developing immunotherapies directed against BAFF-R and TACI, though not BCMA, and may also be a good model for studying dexamethasone resistance. These data indicate that the IL6Myc model is useful for studying development of aggressive MM and for developing new treatments against such forms of the disease.
Subject(s)
Multiple Myeloma , Mice , Humans , Animals , Multiple Myeloma/pathology , Bone Marrow/pathologyABSTRACT
Background: Exon 20 (ex20) in-frame insertions or duplications (ins/dup) in epidermal growth factor receptor (EGFR) and its analog erb-b2 receptor tyrosine kinase 2 (ERBB2) are each detected in 1.5% of non-small cell lung cancer (NSCLC). Unlike EGFR p.L858R or ex19 deletions, ex20 ins/dup is associated with de novo resistance to classic EGFR inhibitors, lack of response to immune checkpoint inhibitors, and poor prognosis. US Food and Drug Administration has approved mobocertinib and amivantamab for targeting tumors with this aberration, but the number of comprehensive studies on ex20 ins/dup NSCLC is limited. We identified 18 cases of NSCLCs with EGFR/ERBB2 ex20 ins/dup and correlated the findings with clinical and morphologic information including programed death-ligand 1 (PD-L1) expression. Methods: A total of 536 NSCLC cases tested at our institution between 2014 and 2023 were reviewed. A custom-designed 214-gene next-generation sequencing panel was used for detecting DNA variants, and the FusionPlex CTL panel (ArcherDx) was used for the detection of fusion transcripts from formalin-fixed, paraffin-embedded tissue. Immunohistochemistry (IHC)for PD-L1 was performed using 22C3 or E1L3N clones. Results: Nine EGFR and nine ERBB2 ex20 ins/dup variants were identified from an equal number of men and women, 14 were non- or light smokers, and 15 had stage IV disease. All 18 cases were adenocarcinomas. Seven of the 11 cases with available primary tumors had acinar predominant pattern, two had lepidic predominant pattern, and the remainder had papillary (one case) and mucinous (one case) patterns. Ex20 ins/dup variants were heterogenous in-frame one to four amino acids spanning A767-V774 in EGFR and Y772-P780 in ERBB2 and were clustered in the loop following the C-helix and α C-helix. Twelve cases (67%) had co-existing TP53 variants. Copy number variation in CDK4 amplification was identified in one case. No fusion or microsatellite instability was identified in any case. PD-L1 was positive in two cases, low positive in four cases, and negative in 11 cases. Conclusions: NSCLCs harboring EGFR/ERBB2 ex20 ins/dup are rare and tend to be acinar predominant, negative for PD-L1, more frequent in non- or light smokers, and mutually exclusive with other driver mutations in NSCLC. The correlation of different EGFR/ERBB2 ex20 ins/dup variants and co-existing mutations with response to targeted therapy and the possibility of developing resistant mutations after mobocertinib treatment warrants further investigation.
ABSTRACT
OBJECTIVES: To identify therapeutic targets and correlate with clinical outcomes from mutation profiling of metastatic uveal melanoma (UM) using next-generation sequencing (NGS). METHODS: Melanoma cases that were tested using DNA-based NGS panels of 25 and/or 214 genes were evaluated retrospectively (263 cases) and identified 27 UM cases. BAP1 expression was examined by immunohistochemistry. RESULTS: Mutations in GNA11 (14) and GNAQ (12) were found in 96% (nâ =â 27) of cases of UM, and most had coexisting BAP1 (17) or SF3B1 (4) mutations. Coexisting GNAQ/11-SF3B1 mutations correlated with a longer average time to first metastasis compared with GNAQ/11-BAP1 mutations (99.7 vs 38.5 months, Pâ =â .047). Three patients with BAP1 mutations received trametinib; two are still alive (15 months; 23 months), and one died (32 months). In non-UMs, only 4.2% (nâ =â 236) had BAP1 and 3.8% had SF3B1 mutations; none had coexisting GNAQ/11 mutations. CONCLUSIONS: Coexisting BAP1/SF3B1 and GNAQ/11 mutations were unique to UM. SF3B1 mutations were reported to be UM-specific in melanoma and associated with rare/no metastasis. The finding of mutated SF3B1 in 14.8% (nâ =â 27) of UMs suggests its role should be further evaluated. The correlation of BAP1/SF3B1 mutation with survival also warrants investigation.
Subject(s)
Melanoma , Neoplasms, Second Primary , Uveal Neoplasms , DNA Mutational Analysis , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Genomics , Humans , Melanoma/pathology , Mutation , Phosphoproteins/genetics , Prognosis , RNA Splicing Factors/genetics , Retrospective Studies , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Uveal Neoplasms/geneticsABSTRACT
The molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD) relies on detecting contractions of the unique D4Z4 repeat array at the chromosome 4q35 locus in the presence of a permissive 4q35A haplotype. Long, intact DNA molecules are required for accurate sizing of D4Z4 repeats. We validated the use of optical genome mapping to determine size and haplotype of D4Z4 alleles for FSHD analysis. The cohort included 36 unique DNA specimens from fresh blood samples or archived agarose plugs. High-molecular- weight DNA underwent sequence-specific labeling followed by separation and image analysis with data collection on the Saphyr system. D4Z4 allele sizes were calculated and haplotypes determined from the labeling patterns. Each specimen had previous diagnostic testing using restriction enzyme digests with EcoRI, EcoRI/BlnI, XapI, or HindIII, followed by pulsed field gel electrophoresis and Southern blot analysis with appropriate probes. Optical genome mapping detected 4q35 and 10q26 alleles ranging from 1 to 79 D4Z4 repeats and showed strong correlation with Southern blot allele sizing (R2 = 0.95) and haplotyping (133 of 134; 99.4% haplotype match). Analysis of inter-assay and intra-assay runs showed high reproducibility (0.03 to 0.94 %CV). Subsequent optical genome mapping for routine clinical testing from 315 clinical FSHD cases compared favorably with historical result trends. Optical genome mapping is an accurate and highly reproducible method for chromosomal abnormalities associated with FSHD.
Subject(s)
Chromosome Mapping/methods , Genetic Testing/methods , Genome, Human , Molecular Diagnostic Techniques/methods , Muscular Dystrophy, Facioscapulohumeral/diagnosis , Muscular Dystrophy, Facioscapulohumeral/genetics , Alleles , Chromosome Aberrations , Chromosomes, Human/genetics , Cohort Studies , DNA/genetics , DNA/isolation & purification , Data Accuracy , Haplotypes , Humans , Muscular Dystrophy, Facioscapulohumeral/blood , Reproducibility of Results , Restriction Mapping/methodsABSTRACT
Protective lung tissue-resident memory CD8+T cells (Trm) form after influenza A virus (IAV) infection. We show that IAV infection of mice generates CD69+CD103+and other memory CD8+T cell populations in lung-draining mediastinal lymph nodes (mLNs) from circulating naive or memory CD8+T cells. Repeated antigen exposure, mimicking seasonal IAV infections, generates quaternary memory (4M) CD8+T cells that protect mLN from viral infection better than 1M CD8+T cells. Better protection by 4M CD8+T cells associates with enhanced granzyme A/B expression and stable maintenance of mLN CD69+CD103+4M CD8+T cells, vs the steady decline of CD69+CD103+1M CD8+T cells, paralleling the durability of protective CD69+CD103+4M vs 1M in the lung after IAV infection. Coordinated upregulation in canonical Trm-associated genes occurs in circulating 4M vs 1M populations without the enrichment of canonical downregulated Trm genes. Thus, repeated antigen exposure arms circulating memory CD8+T cells with enhanced capacity to form long-lived populations of Trm that enhance control of viral infections of the mLN.
Subject(s)
CD8-Positive T-Lymphocytes , Lymph Nodes , Animals , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, CD/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cells, Cultured , Female , Influenza A virus/immunology , Lung/cytology , Lung/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Mice , Mice, Inbred C57BL , Orthomyxoviridae Infections/immunology , Transcriptome/geneticsABSTRACT
BACKGROUND: The clinical diagnosis of genetic renal diseases may be limited by the overlapping spectrum of manifestations between diseases or by the advancement of disease where clues to the original process are absent. The objective of this study was to determine whether genetic testing informs diagnosis and facilitates management of kidney disease patients. METHODS: We developed a comprehensive genetic testing panel (KidneySeq) to evaluate patients with various phenotypes including cystic diseases, congenital anomalies of the kidney and urinary tract (CAKUT), tubulointerstitial diseases, transport disorders and glomerular diseases. We evaluated this panel in 127 consecutive patients ranging in age from newborns to 81 years who had samples sent in for genetic testing. RESULTS: The performance of the sequencing pipeline for single-nucleotide variants was validated using CEPH (Centre de'Etude du Polymorphism) controls and for indels using Genome-in-a-Bottle. To test the reliability of the copy number variant (CNV) analysis, positive samples were re-sequenced and analyzed. For patient samples, a multidisciplinary review board interpreted genetic results in the context of clinical data. A genetic diagnosis was made in 54 (43%) patients and ranged from 54% for CAKUT, 53% for ciliopathies/tubulointerstitial diseases, 45% for transport disorders to 33% for glomerulopathies. Pathogenic and likely pathogenic variants included 46% missense, 11% nonsense, 6% splice site variants, 23% insertion-deletions and 14% CNVs. In 13 cases, the genetic result changed the clinical diagnosis. CONCLUSION: Broad genetic testing should be considered in the evaluation of renal patients as it complements other tests and provides insight into the underlying disease and its management.
Subject(s)
Biomarkers/blood , DNA Copy Number Variations , Genetic Testing/methods , High-Throughput Nucleotide Sequencing/methods , Kidney Diseases/diagnosis , Mutation , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Kidney Diseases/blood , Kidney Diseases/genetics , Kidney Diseases/therapy , Male , Middle Aged , Phenotype , Reproducibility of Results , Young AdultABSTRACT
Plasmodium parasite-specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.
Subject(s)
Immunity, Humoral , Malaria/immunology , Plasma Cells/metabolism , Plasmodium falciparum/immunology , Adolescent , Adult , Amino Acids/administration & dosage , Amino Acids/metabolism , Animals , Antibodies, Protozoan/blood , Antibodies, Protozoan/immunology , Antibodies, Protozoan/metabolism , Antimalarials/administration & dosage , DNA, Protozoan/isolation & purification , Disease Models, Animal , Germinal Center/cytology , Germinal Center/immunology , Germinal Center/metabolism , Host-Parasite Interactions/immunology , Humans , Malaria/blood , Malaria/drug therapy , Malaria/parasitology , Mice , Mice, Transgenic , Middle Aged , Nutrients/metabolism , Plasma Cells/immunology , Plasma Cells/parasitology , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Proof of Concept Study , Young AdultABSTRACT
Enhanced host protection against re-infection requires generation of memory T cells of sufficient quantity and functional quality. Unlike well-studied inbred mice, T cell responses of diverse size and quality are generated following infection of humans and outbred mice. Thus, additional models are needed that accurately reflect variation in immune outcomes in genetically diverse populations and to uncover underlying genetic causes. The Collaborative Cross (CC), a large recombinant inbred panel of mice, is an ideal model in this pursuit for the high degree of genetic variation present, because it allows for assessment of genetic factors underlying unique phenotypes. Here, we advance the utility of the CC as a tool to analyze the immune response to viral infection. We describe variability in resting immune cell composition and adaptive immune responses generated among CC strains following systemic virus infection and reveal quantitative trait loci responsible for generation of CD62L+ memory CD8 T cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , T-Lymphocytes/immunology , Animals , Crosses, Genetic , Female , Genetic Variation/genetics , Genotype , Haplotypes/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred Strains , Models, Immunological , Phenotype , Quantitative Trait Loci/genetics , T-Lymphocytes/metabolism , Virus Diseases/geneticsABSTRACT
As a result of an author oversight in the original article [1], the legend of Figure 5A and C is inaccurate and one panel in Figure 5C (FOXM1N H929 cells shown in the top row, left) is wrong.
ABSTRACT
Type 1 IFNs (IFN-I) generally protect mammalian hosts from virus infections, but in some cases, IFN-I is pathogenic. Because IFN-I is protective, it is commonly used to treat virus infections for which no specific approved drug or vaccine is available. The Middle East respiratory syndrome-coronavirus (MERS-CoV) is such an infection, yet little is known about the role of IFN-I in this setting. Here, we show that IFN-I signaling is protective during MERS-CoV infection. Blocking IFN-I signaling resulted in delayed virus clearance, enhanced neutrophil infiltration, and impaired MERS-CoV-specific T cell responses. Notably, IFN-I administration within 1 day after infection (before virus titers peak) protected mice from lethal infection, despite a decrease in IFN-stimulated gene (ISG) and inflammatory cytokine gene expression. In contrast, delayed IFN-ß treatment failed to effectively inhibit virus replication, increased infiltration and activation of monocytes, macrophages, and neutrophils in the lungs, and enhanced proinflammatory cytokine expression, resulting in fatal pneumonia in an otherwise sublethal infection. Together, these results suggest that the relative timing of the IFN-I response and maximal virus replication is key in determining outcomes, at least in infected mice. By extension, IFN-αß or combination therapy may need to be used cautiously to treat viral infections in clinical settings.
Subject(s)
Coronavirus Infections/immunology , Interferon-beta/pharmacology , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Replication , Animals , Cell Separation , Cytokines/immunology , Female , Flow Cytometry , Gene Expression Profiling , Hematopoietic Stem Cells/cytology , Humans , Inflammation , Interferon-beta/immunology , Lung/immunology , Lung/pathology , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Monocytes/immunology , Neutrophils/immunology , Signal TransductionABSTRACT
Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. We and others have shown that there is enrichment or depletion of some gut bacteria in MS patients compared to healthy controls (HC), suggesting an important role of the gut bacteria in disease pathogenesis. Thus, specific gut bacteria that are lower in abundance in MS patients could be used as a potential treatment option for this disease. In particular, we and others have shown that MS patients have a lower abundance of Prevotella compared to HC, whereas the abundance of Prevotella is increased in patients that receive disease-modifying therapies such as Copaxone® (Glatiramer acetate-GA). This inverse correlation between the severity of MS disease and the abundance of Prevotella suggests its potential for use as a therapeutic option to treat MS. Notably we have previously identified a specific strain, Prevotella histicola (P. histicola), that suppresses disease in the animal model of MS, experimental autoimmune encephalomyelitis (EAE) compared with sham treatment. In the present study we analyzed whether the disease suppressing effects of P. histicola synergize with those of the disease-modifying drug Copaxone® to more effectively suppress disease compared to either treatment alone. Treatment with P. histicola was as effective in suppressing disease as treatment with Copaxone®, whereas the combination of P. histicola plus Copaxone® was not more effective than either individual treatment. P. histicola-treated mice had an increased frequency and number of CD4+FoxP3+ regulatory T cells in periphery as well as gut and a decreased frequency of pro-inflammatory IFN-γ and IL17-producing CD4 T cells in the CNS, suggesting P. histicola suppresses disease by boosting anti-inflammatory immune responses and inhibiting pro-inflammatory immune responses. In conclusion, our study indicates that the human gut commensal P. histicola can suppress disease as efficiently as Copaxone® and may provide an alternative treatment option for MS patients.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/therapy , Glatiramer Acetate/therapeutic use , Immunosuppressive Agents/therapeutic use , Prevotella , Animals , Brain/drug effects , Brain/pathology , Encephalomyelitis, Autoimmune, Experimental/microbiology , Encephalomyelitis, Autoimmune, Experimental/pathology , Gastrointestinal Microbiome , HLA-DR3 Antigen/genetics , Humans , Mice, Transgenic , Multiple Sclerosis/microbiology , Multiple Sclerosis/pathology , Multiple Sclerosis/therapy , Spinal Cord/drug effects , Spinal Cord/pathologyABSTRACT
Plasmodium sporozoites inoculated by mosquitoes migrate to the liver and infect hepatocytes prior to release of merozoites that initiate symptomatic blood-stage malaria. Plasmodium parasites are thought to be restricted to hepatocytes throughout this obligate liver stage of development, and how liver-stage-expressed antigens prime productive CD8 T cell responses remains unknown. We found that a subset of liver-infiltrating monocyte-derived CD11c+ cells co-expressing F4/80, CD103, CD207, and CSF1R acquired parasites during the liver stage of malaria, but only after initial hepatocyte infection. These CD11c+ cells found in the infected liver and liver-draining lymph nodes exhibited transcriptionally and phenotypically enhanced antigen-presentation functions and primed protective CD8 T cell responses against Plasmodium liver-stage-restricted antigens. Our findings highlight a previously unrecognized aspect of Plasmodium biology and uncover the fundamental mechanism by which CD8 T cell responses are primed against liver-stage malaria antigens.
Subject(s)
Antigen Presentation , CD8-Positive T-Lymphocytes/immunology , Hepatocytes/parasitology , Immunity, Cellular , Liver/immunology , Malaria/immunology , Monocytes/parasitology , CD11c Antigen/analysis , Liver/parasitology , Monocytes/chemistry , Monocytes/immunology , Plasmodium/immunologyABSTRACT
BACKGROUND: Following up on previous work demonstrating the involvement of the transcription factor forkhead box M1 (FOXM1) in the biology and outcome of a high-risk subset of newly diagnosed multiple myeloma (nMM), this study evaluated whether FOXM1 gene expression may be further upregulated upon tumor recurrence in patients with relapsed multiple myeloma (rMM). Also assessed was the hypothesis that increased levels of FOXM1 diminish the sensitivity of myeloma cells to commonly used myeloma drugs, such as the proteasome inhibitor bortezomib (Bz) and the DNA intercalator doxorubicin (Dox). METHODS: FOXM1 message was evaluated in 88 paired myeloma samples from patients with nMM and rMM, using gene expression microarrays as measurement tool. Sources of differential gene expression were identified and outlier analyses were performed using statistical methods. Two independent human myeloma cell lines (HMCLs) containing normal levels of FOXM1 (FOXM1N) or elevated levels of lentivirus-encoded FOXM1 (FOXM1Hi) were employed to determine FOXM1-dependent changes in cell proliferation, survival, efflux-pump activity, and drug sensitivity. Levels of retinoblastoma (Rb) protein were determined with the assistance of Western blotting. RESULTS: Upregulation of FOXM1 occurred in 61 of 88 (69%) patients with rMM, including 4 patients that exhibited > 20-fold elevated expression peaks. Increased FOXM1 levels in FOXM1Hi myeloma cells caused partial resistance to Bz (1.9-5.6 fold) and Dox (1.5-2.9 fold) in vitro, using FOXM1N myeloma as control. Reduced sensitivity of FOXM1Hi cells to Bz was confirmed in vivo using myeloma-in-mouse xenografts. FOXM1-dependent regulation of total and phosphorylated Rb agreed with a working model of myeloma suggesting that FOXM1 governs both chromosomal instability (CIN) and E2F-dependent proliferation, using a mechanism that involves interaction with NIMA related kinase 2 (NEK2) and cyclin dependent kinase 6 (CDK6), respectively. CONCLUSIONS: These findings enhanced our understanding of the emerging FOXM1 genetic network in myeloma and provided preclinical support for the therapeutic targeting of the FOXM1-NEK2 and CDK4/6-Rb-E2F pathways using small-drug CDK and NEK2 inhibitors. Clinical research is warranted to assess whether this approach may overcome drug resistance in FOXM1Hi myeloma and, thereby, improve the outcome of patients in which the transcription factor is expressed at high levels.
Subject(s)
Antineoplastic Agents/therapeutic use , Drug Tolerance/genetics , Forkhead Box Protein M1/genetics , Multiple Myeloma/drug therapy , Up-Regulation , Animals , Bortezomib/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Doxorubicin/therapeutic use , Drug Resistance/genetics , Forkhead Box Protein M1/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Molecular testing for epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) fusion is routinely performed in patients with stage IV lung adenocarcinoma to assess their eligibility for targeted therapy. Fine-needle aspiration (FNA)-derived material frequently is the only pathologic material available. The identification of genomic aberrations in thyroid nodules from FNA smears may help stratify cancer risk and spare patients from a second surgery. In the current study, the authors tested nucleic acid extracted from the cytology smears of lung and thyroid carcinomas for simultaneous detection of single-nucleotide variant, insertion/deletion, and gene fusion using an RNA-based next-generation sequencing assay. METHODS: A total of 27 cases (17 lung and 10 thyroid carcinomas, the majority of which had known variants) were tested. Areas of interest were scrapped from stained smears using a scalpel. Total nucleic acid was extracted. Gene fusion and mutational analysis was performed using the Comprehensive Thyroid and Lung FusionPlex Assay. Data were analyzed using the analysis pipeline provided by the vendor. Eleven cases with available formalin-fixed, paraffin-embedded (FFPE) tissue were tested in parallel. RESULTS: Gene fusions were detected in 6 cases; common single-nucleotide variants in EGFR, RAS, and BRAF in 14 cases; and in-frame deletions within EGFR in 3 cases. A concordance rate of 100% was observed between FNA and FFPE tissue. CONCLUSIONS: Cytology preparations can be a reliable source for the detection of both DNA and RNA aberrations. The ability to simultaneously detect multiple types of genomic variants is crucial for patients with advanced cancer and maximizes the usefulness of cytology specimens. Cancer Cytopathol 2018;126:158-69. © 2018 American Cancer Society.
Subject(s)
Lung Neoplasms/genetics , Mutation , RNA, Neoplasm/genetics , Sequence Analysis, RNA/methods , Thyroid Neoplasms/genetics , Gene Fusion , Humans , INDEL Mutation , In Situ Hybridization, Fluorescence , Lung Neoplasms/pathology , Polymorphism, Single Nucleotide , Proto-Oncogene Mas , Thyroid Neoplasms/pathologyABSTRACT
The human gut is colonized by a large number of microorganisms (â¼1013 bacteria) that support various physiologic functions. A perturbation in the healthy gut microbiome might lead to the development of inflammatory diseases, such as multiple sclerosis (MS). Therefore, gut commensals might provide promising therapeutic options for treating MS and other diseases. We report the identification of human gut-derived commensal bacteria, Prevotella histicola, which can suppress experimental autoimmune encephalomyelitis (EAE) in a human leukocyte antigen (HLA) class II transgenic mouse model. P. histicola suppresses disease through the modulation of systemic immune responses. P. histicola challenge led to a decrease in pro-inflammatory Th1 and Th17 cells and an increase in the frequencies of CD4+FoxP3+ regulatory T cells, tolerogenic dendritic cells, and suppressive macrophages. Our study provides evidence that the administration of gut commensals may regulate a systemic immune response and may, therefore, have a possible role in treatment strategies for MS.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/therapy , Gastrointestinal Microbiome , Prevotella/immunology , Probiotics/therapeutic use , Animals , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Dendritic Cells/immunology , Encephalomyelitis, Autoimmune, Experimental/microbiology , Forkhead Transcription Factors/metabolism , Genes, MHC Class II , Humans , Macrophages/immunology , Mice , Prevotella/pathogenicity , Th1 Cells/immunology , Th17 Cells/immunologyABSTRACT
Prostaglandin D2 (PGD2), an eicosanoid with both pro- and anti-inflammatory properties, is the most abundantly expressed prostaglandin in the brain. Here we show that PGD2 signaling through the D-prostanoid receptor 1 (DP1) receptor is necessary for optimal microglia/macrophage activation and IFN expression after infection with a neurotropic coronavirus. Genome-wide expression analyses indicated that PGD2/DP1 signaling is required for up-regulation of a putative inflammasome inhibitor, PYDC3, in CD11b+ cells in the CNS of infected mice. Our results also demonstrated that, in addition to PGD2/DP1 signaling, type 1 IFN (IFN-I) signaling is required for PYDC3 expression. In the absence of Pydc3 up-regulation, IL-1ß expression and, subsequently, mortality were increased in infected DP1-/- mice. Notably, survival was enhanced by IL1 receptor blockade, indicating that the effects of the absence of DP1 signaling on clinical outcomes were mediated, at least in part, by inflammasomes. Using bone marrow-derived macrophages in vitro, we confirmed that PYDC3 expression is dependent upon DP1 signaling and that IFN priming is critical for PYDC3 up-regulation. In addition, Pydc3 silencing or overexpression augmented or diminished IL-1ß secretion, respectively. Furthermore, DP1 signaling in human macrophages also resulted in the up-regulation of a putative functional analog, POP3, suggesting that PGD2 similarly modulates inflammasomes in human cells. These findings demonstrate a previously undescribed role for prostaglandin signaling in preventing excessive inflammasome activation and, together with previously published results, suggest that eicosanoids and inflammasomes are reciprocally regulated.
Subject(s)
Coronavirus , Inflammasomes/metabolism , Prostaglandin D2/metabolism , Receptors, Prostaglandin/metabolism , Signal Transduction , Animals , Cyclic AMP/metabolism , Cytokines/metabolism , Humans , Inflammation/metabolism , Interferon Type I/metabolism , Interleukin-1beta/metabolism , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Protein Domains , Receptors, Prostaglandin/antagonists & inhibitors , Up-RegulationABSTRACT
Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the "master regulator" of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.
Subject(s)
Cell Differentiation/genetics , Melanocytes/metabolism , Microphthalmia-Associated Transcription Factor/genetics , Transcription Factor AP-2/genetics , Animals , Base Sequence , Binding Sites/genetics , Cell Line , Cell Line, Tumor , Cells, Cultured , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Humans , Mice, Knockout , Microphthalmia-Associated Transcription Factor/metabolism , Microscopy, Confocal , Mutation , Pigmentation/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Transcription Factor AP-2/metabolism , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Primary aneurysmal bone cyst (ABC) is a neoplastic process due to recurrent translocations involving the USP6 gene. By fluorescence in situ hybridization, up to 69% of primary ABCs harbored USP6 translocations; no USP6 translocation was found in secondary ABC or giant cell tumor of bone (GCT). GCT can recur locally, metastasize to the lungs in some cases, and rarely undergo malignant transformation. Differentiating primary ABC from its mimics is important for treatment and prognosis. We evaluated USP6 fusion and expression in 13 cases of primary and 1 case of secondary ABC, and 9 cases of GCT using nucleic acid extracted from formalin-fixed, paraffin-embedded tissue and a next generation sequencing (NGS)-based assay. USP6 fusions including 7 novel fusions and USP6 transcripts were identified in all 13 primary ABCs. Nine cases with strong evidence of fusions showed high levels of USP6 transcripts by reverse transcription-PCR (RT-PCR). The remaining four had no detectable USP6 expression by a first-round of RT-PCR but the presence of USP6 transcripts was identified by a second-round, nested PCR. The major fusions were confirmed by RT-PCR followed by Sanger sequencing. No USP6 fusion or transcript was detected in any of the GCTs or the case of secondary ABC by NGS or by two rounds of PCR. All USP6 translocations resulted in fusion of the entire USP6 coding sequence with promoters of the fusion gene leading to upregulation of USP6 transcription, which is likely the underlying mechanism for ABC oncogenesis. © 2016 Wiley Periodicals, Inc.