ABSTRACT
PURPOSE: The ClinGen Actionability Working Group (AWG) developed an evidence-based framework to generate actionability reports and scores of gene-condition pairs in the context of secondary findings from genome sequencing. Here we describe the expansion of the framework to include actionability assertions. METHODS: Initial development of the actionability rubric was based on previously scored adult gene-condition pairs and individual expert evaluation. Rubric refinement was iterative and based on evaluation, feedback, and discussion. The final rubric was pragmatically evaluated via integration into actionability assessments for 27 gene-condition pairs. RESULTS: The resulting rubric has a 4-point scale (limited, moderate, strong, and definitive) and uses the highest-scoring outcome-intervention pair of each gene-condition pair to generate a preliminary assertion. During AWG discussions, predefined criteria and factors guide discussion to produce a consensus assertion for a gene-condition pair, which may differ from the preliminary assertion. The AWG has retrospectively generated assertions for all previously scored gene-condition pairs and are prospectively asserting on gene-condition pairs under assessment, having completed over 170 adult and 188 pediatric gene-condition pairs. CONCLUSION: The AWG expanded its framework to provide actionability assertions to enhance the clinical value of their resources and increase their utility as decision aids regarding return of secondary findings.
Subject(s)
Evidence-Based Medicine , Humans , Evidence-Based Medicine/methods , Genetic Testing/methods , Incidental Findings , Whole Genome SequencingSubject(s)
Nail Diseases , Nails, Malformed , Humans , Child , Potassium Permanganate , Nail Diseases/diagnosisABSTRACT
PURPOSE: Synthesis and curation of evidence regarding the clinical actionability of secondary findings (SFs) from genome-scale sequencing are needed to support decision-making on reporting of these findings. To assess actionability of SFs in children and adolescents, the Clinical Genome Resource established the Pediatric Actionability Working Group (AWG). METHODS: The Pediatric AWG modified the framework of the existing Adult AWG, which included production of summary reports of actionability for genes and associated conditions and consensus actionability scores for specific outcome-intervention pairs. Modification of the adult framework for the pediatric setting included accounting for special considerations for reporting presymptomatic or predictive genetic findings in the pediatric context, such as maintaining future autonomy by not disclosing conditions not actionable until adulthood. The Pediatric AWG then applied this new framework to genes and associated conditions with putative actionability. RESULTS: As of September 2021, the Pediatric AWG applied the new framework to 70 actionability topics representing 143 genes. Reports and scores are publicly available at www.clinicalgenome.org. CONCLUSION: The Pediatric AWG continues to curate gene-condition topics and build an evidence-based resource, supporting clinical communities and decision-makers with policy development on the return of SFs in pediatric populations.
Subject(s)
Genetic Testing , Research Report , Adolescent , Adult , Child , Chromosome Mapping , HumansABSTRACT
T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbored PD-L1/PD-L2 copy gain or amplification in 64% of cases, which was associated with increased PD-L1 expression (P = .0111). By directed and unsupervised spatial analyses of multiparametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune "neighborhoods" in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1+ T cells. Furthermore, unbiased clustering of spatially resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in 3 of 5 patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies (NCT03316573; NCT01953692), including 2 complete responses and 1 partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL and also support the potential utility of spatially resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types.
Subject(s)
Histiocytes/immunology , Lymphoma, Large B-Cell, Diffuse/immunology , Programmed Cell Death 1 Receptor/immunology , T-Lymphocytes/immunology , Tumor Escape , B7-H1 Antigen/analysis , B7-H1 Antigen/immunology , Histiocytes/pathology , Humans , Lymphoma, Large B-Cell, Diffuse/pathology , Programmed Cell Death 1 Receptor/analysis , T-Lymphocytes/pathologyABSTRACT
The activated B cell (ABC-like) subtype of diffuse large B cell lymphoma (DLBCL) is characterized by chronic activation of signaling initiated by immunoglobulin µ (IgM). By analyzing the DNA copy number profiles of 1000 DLBCL tumors, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. Using integrative analysis of matched gene expression profiling data, we found that the TCF4 (E2-2) transcription factor gene was the target of these alterations. Overexpression of TCF4 in ABC-like DLBCL cell lines led to its occupancy on immunoglobulin (IGHM) and MYC gene enhancers and increased expression of these genes at the transcript and protein levels. Inhibition of TCF4 activity with dominant-negative constructs was synthetically lethal to ABC-like DLBCL cell lines harboring TCF4 DNA copy gains, highlighting these gains as an attractive potential therapeutic target. Furthermore, the TCF4 gene was one of the top BRD4-regulated genes in DLBCL cell lines. BET proteolysis-targeting chimera (PROTAC) ARV771 extinguished TCF4, MYC, and IgM expression and killed ABC-like DLBCL cells in vitro. In DLBCL xenograft models, ARV771 treatment reduced tumor growth and prolonged survival. This work highlights a genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.
Subject(s)
Immunoglobulins/metabolism , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/metabolism , Transcription Factor 4/genetics , Animals , Blotting, Western , Cell Line , Cell Survival , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , High-Throughput Nucleotide Sequencing , Humans , Male , Mice , Mice, Nude , Signal Transduction/genetics , Signal Transduction/physiology , Xenograft Model Antitumor AssaysABSTRACT
Advances in sequencing technologies permit the analysis of a larger selection of genes for preconception carrier screening. The study was designed as a sequential carrier screen using genome sequencing to analyze 728 gene-disorder pairs for carrier and medically actionable conditions in 131 women and their partners (n = 71) who were planning a pregnancy. We report here on the clinical laboratory results from this expanded carrier screening program. Variants were filtered and classified using the latest American College of Medical Genetics and Genomics (ACMG) guideline; only pathogenic and likely pathogenic variants were confirmed by orthologous methods before being reported. Novel missense variants were classified as variants of uncertain significance. We reported 304 variants in 202 participants. Twelve carrier couples (12/71 couples tested) were identified for common conditions; eight were carriers for hereditary hemochromatosis. Although both known and novel variants were reported, 48% of all reported variants were missense. For novel splice-site variants, RNA-splicing assays were performed to aid in classification. We reported ten copy-number variants and five variants in non-coding regions. One novel variant was reported in F8, associated with hemophilia A; prenatal testing showed that the male fetus harbored this variant and the neonate suffered a life-threatening hemorrhage which was anticipated and appropriately managed. Moreover, 3% of participants had variants that were medically actionable. Compared with targeted mutation screening, genome sequencing improves the sensitivity of detecting clinically significant variants. While certain novel variant interpretation remains challenging, the ACMG guidelines are useful to classify variants in a healthy population.
Subject(s)
Clinical Laboratory Techniques , Genetic Testing/methods , Preconception Care , Whole Genome Sequencing , DNA Copy Number Variations/genetics , Disease/genetics , Female , Genetic Predisposition to Disease , Haplotypes/genetics , Heterozygote , Humans , Introns/genetics , Male , Mutation/genetics , Pregnancy , RNA Splicing/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Purpose Hodgkin Reed-Sternberg (HRS) cells evade antitumor immunity by multiple means, including gains of 9p24.1/ CD274(PD-L1)/ PDCD1LG2(PD-L2) and perturbed antigen presentation. Programmed death 1 (PD-1) receptor blockade is active in classic Hodgkin lymphoma (cHL) despite reported deficiencies of major histocompatibility complex (MHC) class I expression on HRS cells. Herein, we assess bases of sensitivity to PD-1 blockade in patients with relapsed/refractory cHL who were treated with nivolumab (anti-PD-1) in the CheckMate 205 trial. Methods HRS cells from archival tumor biopsies were evaluated for 9p24.1 alterations by fluorescence in situ hybridization and for expression of PD ligand 1 (PD-L1) and the antigen presentation pathway components-ß2-microglobulin, MHC class I, and MHC class II-by immunohistochemistry. These parameters were correlated with clinical responses and progression-free survival (PFS) after PD-1 blockade. Results Patients with higher-level 9p24.1 copy gain and increased PD-L1 expression on HRS cells had superior PFS. HRS cell expression of ß2-microglobulin/MHC class I was not predictive for complete remission or PFS after nivolumab therapy. In contrast, HRS cell expression of MHC class II was predictive for complete remission. In patients with a > 12-month interval between myeloablative autologous stem-cell transplantation and nivolumab therapy, HRS cell expression of MHC class II was associated with prolonged PFS. Conclusion Genetically driven PD-L1 expression and MHC class II positivity on HRS cells are potential predictors of favorable outcome after PD-1 blockade. In cHL, clinical responses to nivolumab were not dependent on HRS cell expression of MHC class I.
Subject(s)
B7-H1 Antigen/biosynthesis , Histocompatibility Antigens Class II/biosynthesis , Hodgkin Disease/drug therapy , Hodgkin Disease/immunology , Nivolumab/therapeutic use , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Antigen Presentation , Antineoplastic Agents, Immunological/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/genetics , B7-H1 Antigen/immunology , Chromosomes, Human, Pair 9 , Cohort Studies , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Hodgkin Disease/genetics , Hodgkin Disease/pathology , Humans , Predictive Value of Tests , Programmed Cell Death 1 Receptor/biosynthesis , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/immunology , Progression-Free Survival , Reed-Sternberg Cells/drug effects , Reed-Sternberg Cells/immunology , Reed-Sternberg Cells/pathology , Treatment Outcome , beta 2-Microglobulin/biosynthesis , beta 2-Microglobulin/genetics , beta 2-Microglobulin/immunologyABSTRACT
Double-hit lymphomas (DHLs) and double-expressor lymphomas (DELs) are associated with resistance to frontline and salvage immunochemotherapy, as well as autologous stem cell transplantation (SCT). We hypothesized that allogeneic SCT (alloSCT) could overcome the chemoresistance associated with DEL/DHL. We retrospectively studied the impact of DEL/DHL status in a multicenter cohort of patients who underwent alloSCT for relapsed/refractory (rel/ref) aggressive B cell non-Hodgkin lymphoma (B-NHL). Seventy-eight patients transplanted at 3 centers in whom tumor tissue was available for immunohistochemistry and fluorescence in situ hybridization were enrolled; 47% had DEL and 13% had DHL. There were no significant differences in 4-year progression-free (PFS) or overall survival (OS) between patients with DEL compared with patients without DEL (PFS 30% versus 39%, P = .24; OS 31% versus 49%, P = .17) or between patients with DHL compared with patients without DHL (PFS 40% versus 34%, P = .62; OS 50% versus 38%, P = .46). The lack of association between DEL or DHL and outcome was confirmed in multivariable models, although inadequate sample size may have limited our ability to detect significant differences. In our cohort alloSCT produced durable remissions in patients with rel/ref aggressive B-NHL irrespective of DEL and DHL status, justifying its consideration in the treatment of patients with rel/ref DEL/DHL.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Lymphoma, B-Cell , Mediastinal Neoplasms , Stem Cell Transplantation , Adult , Aged , Allografts , Disease-Free Survival , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/mortality , Lymphoma, B-Cell/therapy , Male , Mediastinal Neoplasms/genetics , Mediastinal Neoplasms/mortality , Mediastinal Neoplasms/therapy , Middle Aged , Retrospective Studies , Survival RateABSTRACT
Immune checkpoint therapies targeting CTLA-4 and PD-1 have proven effective in cancer treatment. However, the identification of biomarkers for predicting clinical outcomes and mechanisms to overcome resistance remain as critical needs. Angiogenesis is increasingly appreciated as an immune modulator with potential for combinatorial use with checkpoint blockade. Angiopoietin-2 (ANGPT2) is an immune target in patients and is involved in resistance to anti-VEGF treatment with the monoclonal antibody bevacizumab. We investigated the predictive and prognostic value of circulating ANGPT2 in metastatic melanoma patients receiving immune checkpoint therapy. High pretreatment serum ANGPT2 was associated with reduced overall survival in CTLA-4 and PD-1 blockade-treated patients. These treatments also increased serum ANGPT2 in many patients early after treatment initiation, whereas ipilimumab plus bevacizumab treatment decreased serum concentrations. ANGPT2 increases were associated with reduced response and/or overall survival. Ipilimumab increased, and ipilimumab plus bevacizumab decreased, tumor vascular ANGPT2 expression in a subset of patients, which was associated with increased and decreased tumor infiltration by CD68+ and CD163+ macrophages, respectively. In vitro, bevacizumab blocked VEGF-induced ANGPT2 expression in tumor-associated endothelial cells, whereas ANGPT2 increased PD-L1 expression on M2-polarized macrophages. Treatments elicited long-lasting and functional antibody responses to ANGPT2 in a subset of patients receiving clinical benefit. Our findings suggest that serum ANGPT2 may be considered as a predictive and prognostic biomarker for immune checkpoint therapy and may contribute to treatment resistance via increasing proangiogenic and immunosuppressive activities in the tumor microenvironment. Targeting ANGPT2 provides a rational combinatorial approach to improve the efficacy of immune therapy. Cancer Immunol Res; 5(1); 17-28. ©2016 AACR.
Subject(s)
Angiopoietin-2/antagonists & inhibitors , Angiopoietin-2/metabolism , Antineoplastic Agents, Immunological/pharmacology , Biomarkers, Tumor/antagonists & inhibitors , Immunomodulation/drug effects , Neoplasms/immunology , Neoplasms/metabolism , Angiopoietin-2/blood , Antineoplastic Agents, Immunological/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers , CTLA-4 Antigen/antagonists & inhibitors , Humans , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/mortality , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Survival Analysis , Treatment Outcome , Vascular Endothelial Growth Factor A/antagonists & inhibitorsABSTRACT
Purpose Double-hit lymphomas (DHLs) and double-expressor lymphomas (DELs) are subtypes of diffuse large B-cell lymphoma (DLBCL) associated with poor outcomes after standard chemoimmunotherapy. Data are limited regarding outcomes of patients with relapsed or refractory (rel/ref) DEL or DHL who undergo autologous stem-cell transplantation (ASCT). We retrospectively studied the prognostic impact of DEL and DHL status on ASCT outcomes in patients with rel/ref DLBCL. Methods Patients with chemotherapy-sensitive rel/ref DLBCL who underwent ASCT at two institutions and in whom archival tumor material was available were enrolled. Immunohistochemistry for MYC, BCL2, and BCL6 and fluorescence in situ hybridization (FISH) for MYC were performed. In cases with MYC rearrangement or copy gain, FISH for BCL2 and BCL6 was also performed. Results A total of 117 patients were included; 44% had DEL and 10% had DHL. DEL and DHL were associated with inferior progression-free survival (PFS), and DHL was associated with poorer overall survival (OS). The 4-year PFS in patients with DEL compared with those with non-DEL was 48% versus 59% ( P = .049), and the 4-year OS was 56% versus 67% ( P = .10); 4-year PFS in patients with DHL compared with those with non-DHL was 28% versus 57% ( P = .013), and 4-year OS was 25% versus 61% ( P = .002). The few patients with concurrent DEL and DHL had a poor outcome (4-year PFS, 0%). In multivariable models, DEL and DHL were independently associated with inferior PFS, whereas DHL and partial response ( v complete response) at transplant were associated with inferior OS. Conclusion DEL and DHL are both associated with inferior outcomes after ASCT in patients with rel/ref DLBCL. Although ASCT remains a potentially curative approach, these patients, particularly those with DHL, are a high-risk subset who should be targeted for investigational strategies other than standard ASCT.
Subject(s)
Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/therapy , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-6/genetics , Proto-Oncogene Proteins c-myc/genetics , Stem Cell Transplantation , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Disease-Free Survival , Female , Humans , Lymphoma, Large B-Cell, Diffuse/chemistry , Male , Middle Aged , Proto-Oncogene Proteins c-bcl-2/analysis , Proto-Oncogene Proteins c-bcl-6/analysis , Proto-Oncogene Proteins c-myc/analysis , Retrospective Studies , Survival Rate , Transplantation, AutologousABSTRACT
In classical Hodgkin lymphoma (cHL), malignant Hodgkin Reed-Sternberg (HRS) cells evade antitumor immunity by multiple mechanisms, including perturbed antigen presentation and enhanced PD-1 signaling. HRS cell expression of the PD-1 ligands is attributable, in part, to copy number alterations of 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) Amplification of PD-L1/PD-L2 is associated with advanced clinical stage and inferior progression-free survival (PFS) following first-line (induction) therapy. The relationships between altered expression of ß2-microglobulin (ß2M), MHC class I, and MHC class II by HRS cells, PD-L1/PD-L2 amplification, and clinical outcome in cHL are poorly defined. We assessed these variables in diagnostic biopsy specimens from 108 patients with cHL who received uniform treatment and had long-term follow-up and found decreased/absent expression of ß2M/MHC class I in 79% (85/108) and decreased/absent expression of MHC class II in 67% (72/108) of cases. Patients with decreased/absent ß2M/MHC class I had shorter PFS, independent of PD-L1/PD-L2 amplification and advanced stage. Decreased or absent MHC class II was unrelated to outcome. These results suggest that MHC class I-mediated antigen presentation by HRS cells is an important component of the biological response to standard chemo/radiotherapy. The paucity of ß2M/MHC class I expression on HRS cells also prompts speculation regarding alternative mechanisms of action of PD-1 blockade in cHL. Cancer Immunol Res; 4(11); 910-6. ©2016 AACR.
Subject(s)
Chromosome Aberrations , Chromosomes, Human, Pair 9 , Gene Expression , Histocompatibility Antigens Class I/genetics , Hodgkin Disease/genetics , Hodgkin Disease/mortality , beta 2-Microglobulin/genetics , Adolescent , Adult , Aged , Antigen Presentation/immunology , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Female , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Hodgkin Disease/immunology , Hodgkin Disease/therapy , Humans , Male , Middle Aged , Neoplasm Staging , Prognosis , Programmed Cell Death 1 Ligand 2 Protein/genetics , Programmed Cell Death 1 Ligand 2 Protein/metabolism , Young Adult , beta 2-Microglobulin/immunology , beta 2-Microglobulin/metabolismABSTRACT
Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.
Subject(s)
Biomedical Research , Genetic Testing/standards , Genetic Variation/genetics , Genomics/methods , Laboratories/standards , Mutation/genetics , Sequence Analysis, DNA/standards , Data Interpretation, Statistical , Evidence-Based Practice , Exome/genetics , Genome, Human , Guidelines as Topic , High-Throughput Nucleotide Sequencing/methods , Humans , Incidental Findings , Software , United StatesABSTRACT
IMPORTANCE: Patients with squamous cell carcinoma (SCC) of the cervix or vulva have limited therapeutic options, and the potential for immunotherapy for this population has not been evaluated. Recent trials suggest that tumors with a genetic basis for PD-1 (programmed cell death protein 1) ligand expression are highly sensitive to therapeutic antibodies targeting PD-1. OBJECTIVE: To determine the genetic status of CD274 (encoding PD-L1 [programmed cell death 1 ligand 1]) and PDCD1LG2 (encoding PD-L2 [programmed cell death 1 ligand 2]) in SCCs of the cervix and vulva and to correlate the findings with PD-L1 protein expression. DESIGN, SETTING, AND PARTICIPANTS: We performed fluorescence in situ hybridization (FISH) using probes targeting CD274, PDCD1LG2, and the centromeric portion of chromosome 9, and immunohistochemistry (IHC) using an antibody recognizing PD-L1 on formalin-fixed, paraffin-embedded (FFPE) biopsy specimens from 48 cervical SCCs and 23 vulvar SCCs. MAIN OUTCOMES AND MEASURES: Tumors were categorized according to the genetic abnormality in CD274 and PDCD1LG2 (coamplification > cogain > polysomy > disomy) as detected by FISH, and evaluated on a semiquantitative scale (modified H score, the product of the percentage of tumor cells with positive staining and the maximum intensity of positive staining) for PD-L1 protein expression as detected by IHC. RESULTS: Overall, 71 samples of FFPE tissue from cases of cervical SCCs (n = 48) and vulvar SCCs (n = 23) were retrieved from the archives of Brigham and Women's Hospital and included in this study. We observed cogain or coamplification of CD274 and PDCD1LG2 in 32 of 48 cervical SCCs (67%) and 10 of 23 vulvar SCCs (43%). Median PD-L1 protein expression was highest among tumors with CD274 and PDCD1LG2 coamplification and lowest among tumors with disomy. CONCLUSIONS AND RELEVANCE: Recurrent copy number gain of the genes encoding the PD-1 ligands provides a genetic basis for PD-L1 expression in a subset of cervical and vulvar SCCs and identifies a class of patients that are rational candidates for therapies targeting PD-1.
Subject(s)
B7-H1 Antigen/genetics , Carcinoma, Squamous Cell/genetics , Programmed Cell Death 1 Ligand 2 Protein/genetics , Uterine Cervical Neoplasms/genetics , Vulvar Neoplasms/genetics , B7-H1 Antigen/biosynthesis , Female , Gene Dosage , Gene Expression Regulation, Neoplastic/genetics , High-Throughput Nucleotide Sequencing , Humans , Immunohistochemistry , In Situ Hybridization, FluorescenceABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: The root of Bupleurum falcatum L. (BF) has been used in traditional Korean and Chinese medicines for over 2000 years to treat infections, fever, and chronic liver diseases. Among the many active compounds in BF ethanol extract (BFE), saikosaponins exert pharmacological activities including anti-inflammatory effects. Activated microglial cells release a variety of pro-inflammatory substances, leading to neuronal cell death and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The aim of the present study was to investigate the mechanism of the anti-neuroinflammatory effects of BFE using lipopolysaccharide (LPS)-stimulated microglial cells and LPS-intraperitoneal injected C57BL/6 mice. MATERIALS AND METHODS: Dried roots of BF were extracted with 70% ethanol (tenfold volume) on a stirring plate for 24h at room temperature to prepare BFE. Pure saikosaponins (SB3, SB4, and SD) were prepared by solvent extraction and column chromatography fractionation. BV2 murine microglial cells were treated with BFE or saikosaponins for 4h and stimulated with LPS. Generation of nitric oxide (NO), inflammatory cytokines, and reactive oxygen species (ROS) from activated microglial cells were monitored. The effects of BFE on NF-κB activation were determined using RT-PCR, reporter assay, and immunostaining. The in vivo effects of BFE were also assessed by immunohistochemical staining of tissue sections from LPS-injected mouse brains. RESULTS: Treatment with BFE or saikosaponins dose-dependently attenuated LPS-induced production of NO, iNOS mRNA, and ROS by 30-50%. They reduced LPS-mediated increases in the mRNA levels of IL-6, IL-1ß, and TNF-α by approximately 30-70% without affecting cell viability, and decreased LPS-mediated NF-κB activity via reducing p65/RELA mRNA, transcriptional activity, and nuclear localization of NF-κB. BFE also reduced LPS-induced activation of microglia and astrocytes in the hippocampus and substantia nigra of LPS-injected mice. CONCLUSION: Our data suggest that BFE may be effective for reducing neuroinflammation-mediated neurodegeneration through suppressing NF-κB-mediated inflammatory pathways.