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Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage-fusion-bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3-5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.
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Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Neoplasias , Aneuploidia , Cromotripsia , Variações do Número de Cópias de DNA/genética , Haploidia , Recombinação Homóloga/genética , Humanos , Perda de Heterozigosidade/genética , Mutação , Neoplasias/genética , Neoplasias/patologia , Sequenciamento do ExomaRESUMO
ABSTRACT: Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcomes; however, only FLT3-internal tandem duplication (ITD) mutation and adverse karyotype are currently used for risk stratification because of inconsistent results and uncertainty about how other factors should influence treatment, particularly given the strong prognostic effect of postinduction measurable residual disease (MRD). Here, we analyzed a large group of patients with NPM1 mutations (NPM1mut) AML enrolled in prospective trials (National Cancer Research Institute [NCRI] AML17 and AML19, n = 1357) to delineate the impact of baseline molecular and clinical features, postinduction MRD status, and treatment intensity on the outcome. FLT3-ITD (hazard ratio [HR], 1.28; 95% confidence interval [CI], 1.01-1.63), DNMT3A (HR, 1.65; 95% CI, 1.32-2.05), WT1 (HR, 1.74; 95% CI, 1.27-2.38), and non-ABD NPM1mut (HR, 1.64; 95% CI, 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients who achieved MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD-negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin) regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the high-risk molecular subgroups.
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Leucemia Mieloide Aguda , Mutação , Proteínas Nucleares , Nucleofosmina , Tirosina Quinase 3 Semelhante a fms , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Nucleares/genética , Pessoa de Meia-Idade , Feminino , Masculino , Adulto , Idoso , Tirosina Quinase 3 Semelhante a fms/genética , Prognóstico , Adulto Jovem , Neoplasia Residual/genética , DNA Metiltransferase 3A , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas WT1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Adolescente , Resultado do Tratamento , Idoso de 80 Anos ou maisRESUMO
The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity, cellular adhesion, and morphogenesis. Homozygous variants in human CRB1 result in autosomal recessive Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP), with no established genotype-phenotype correlation. The associated protein complexes have key functions in developmental pathways; however, the underlying disease mechanism remains unclear. Using the oko meduzym289/m289 (crb2a-/- ) zebrafish, we performed integrative transcriptomic (RNA-seq data) and methylomic [reduced representation bisulphite sequencing (RRBS)] analysis of whole retina to identify dysregulated genes and pathways. Delayed retinal cell specification was identified in both the crb2a-/- zebrafish and CRB1 patient-derived retinal organoids, highlighting the dysfunction of cell cycle modulation and epigenetic transcriptional control. Differential DNA methylation analysis revealed novel hypermethylated pathways involving biological adhesion, Hippo, and transforming growth factor ß (TGFß) signalling. By integrating gene expression with DNA methylation using functional epigenetic modules (FEM), we identified six key modules involving cell cycle control and disturbance of TGFß, bone morphogenetic protein (BMP), Hippo, and SMAD protein signal transduction pathways, revealing significant interactome hotspots relevant to crb2a function and confirming the epigenetic control of gene regulation in early retinal development, which points to a novel mechanism underlying CRB1-retinopathies. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Polaridade Celular , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Polaridade Celular/genética , Retina/metabolismo , Ciclo Celular , Epigênese Genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Despite the improvements in treatment over the last decades, periodontal disease (PD) affects millions of people around the world and the only treatment available is based on controlling microbial load. Diabetes is known to increase the risk of PD establishment and progression, and recently, glucose metabolism modulation by pharmaceutical or dietarian means has been emphasised as a significant modulator of non-communicable disease development. METHODS: The impact of pharmaceutically controlling glucose metabolism in non-diabetic animals and humans (REBEC, UTN code: U1111-1276-1942) was investigated by repurposing Metformin, as a mean to manage periodontal disease and its associated systemic risk factors. RESULTS: We found that glucose metabolism control via use of Metformin aimed at PD management resulted in significant prevention of bone loss during induced periodontal disease and age-related bone loss in vivo. Metformin also influenced the bacterial species present in the oral environment and impacted the metabolic epithelial and stromal responses to bacterial dysbiosis at a single cell level. Systemically, Metformin controlled blood glucose levels and age-related weight gain when used long-term. Translationally, our pilot randomized control trial indicated that systemic Metformin was safe to use in non-diabetic patients and affected the periodontal tissues. During the medication window, patients showed stable levels of systemic blood glucose, lower circulating hsCRP and lower insulin levels after periodontal treatment when compared to placebo. Finally, patients treated with Metformin had improved periodontal parameters when compared to placebo treated patients. CONCLUSION: This is the first study to demonstrate that systemic interventions using Metformin in non-diabetic individuals aimed at PD prevention have oral-systemic effects constituting a possible novel form of preventive medicine for oral-systemic disease management.
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Diabetes Mellitus Tipo 2 , Metformina , Doenças Periodontais , Animais , Humanos , Metformina/farmacologia , Metformina/uso terapêutico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Glicemia , Doenças Periodontais/tratamento farmacológico , Gerenciamento ClínicoRESUMO
Mosaic mutations present in the germline have important implications for reproductive risk and disease transmission. We previously demonstrated a phenomenon occurring in the male germline, whereby specific mutations arising spontaneously in stem cells (spermatogonia) lead to clonal expansion, resulting in elevated mutation levels in sperm over time. This process, termed "selfish spermatogonial selection," explains the high spontaneous birth prevalence and strong paternal age-effect of disorders such as achondroplasia and Apert, Noonan and Costello syndromes, with direct experimental evidence currently available for specific positions of six genes (FGFR2, FGFR3, RET, PTPN11, HRAS, and KRAS). We present a discovery screen to identify novel mutations and genes showing evidence of positive selection in the male germline, by performing massively parallel simplex PCR using RainDance technology to interrogate mutational hotspots in 67 genes (51.5 kb in total) in 276 biopsies of testes from five men (median age, 83 yr). Following ultradeep sequencing (about 16,000×), development of a low-frequency variant prioritization strategy, and targeted validation, we identified 61 distinct variants present at frequencies as low as 0.06%, including 54 variants not previously directly associated with selfish selection. The majority (80%) of variants identified have previously been implicated in developmental disorders and/or oncogenesis and include mutations in six newly associated genes (BRAF, CBL, MAP2K1, MAP2K2, RAF1, and SOS1), all of which encode components of the RAS-MAPK pathway and activate signaling. Our findings extend the link between mutations dysregulating the RAS-MAPK pathway and selfish selection, and show that the aging male germline is a repository for such deleterious mutations.
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Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Transdução de Sinais , Testículo/metabolismo , Proteínas ras/metabolismo , Idoso , Idoso de 80 Anos ou mais , Variação Genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Emerging and reemerging pathogens are global challenges for public health and the pandemic of Coronavirus disease 2019is a reemerging case of an infectious disease caused by Severe Acute Respiratory Syndrome-Corornavirus-2. Health care worker worldwide are at higher risk worldwide and the situation is the same in Nepal. The knowledge and attitude of health workers will certainly mark the outcome towards this pandemic. So, this study aims to assess the knowledge and attitude of community health workers towards the prevention of COVID-19 virus. METHODS: A descriptive cross-sectional study was carried out among the community health workers of various provinces of Nepal. A semi-structured, self-administered questionnaire was prepared in Google form and circulated to the sampled health workers through various social media platforms like face book, messenger, Instagram and g-mails. A total of 650 invitations were send and among them 420 responded and among them only 399 provided complete response. Responses containing anonymized data was collected analyzed in using SPSS-version-20. The results were interpreted and was checked with various demographic and enabling factors using chi-square test and logistic regression model. Also, ethical approval was taken from NHRC (Nepal Health Research Council (protocol registration number: 360/2020P) prior to the conduction of study. RESULTS: Out the total sample size of 450, we took 399 responses taking into consideration all the inclusion criteria. So, from 399 valid response, 230 (47.6%) were males and 169 (42.4%) were females. 380 (95.2%) employed participants thought that wearing PPE will reduce the chances of getting COVID-19, majority of the participants 80.5% (321) responded that COVID-19 will successfully be controlled and staffs receiving excellent support from palika had high knowledge level. Knowledge level was significantly associated with the enabling factor "support from palika" (P = 0.045).45.9% of the respondents had no availability of COVID-19 response medical items for prevention of COVID-19 at their respective health facilities. Also the logistic regression analysis revealed that the odds of knowledge level was 2 times higher (AOR=1.913 at 95% CI: 1.266-2.891) compared to the female participants (Ref- female). CONCLUSIONS: Proper and adequate knowledge and attitude towards COVID-19 is a paramount in the prevention and control of SARS-COV-2. Health care workers are knowledgeable about COVID-19 and are proactively practicing the preventive measures to minimize the spread of infection but some lack optimistic attitude. Hence, the constantly updated educational programs related to COVID-19 will surely contribute to improving the healthcare workers knowledge and attitude towards COVID-19.
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COVID-19 , Agentes Comunitários de Saúde , Estudos Transversais , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Pessoal de Saúde , Humanos , Masculino , Nepal , SARS-CoV-2 , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: Synovial fluid (SF) derived T-cells are frequently studied as a proxy for investigating the synovial tissue (ST) T-cell infiltrate in inflammatory arthritis. However, since ST is the primary site of inflammatory activity, there is debate as to whether SF provides a true reflection of the ST T-cell population. METHODS: In this study, we used single cell RNA sequencing paired with single cell T-cell receptor (TCR) sequencing to directly compare memory T-cells from paired samples of SF and ST from 6 patients with inflammatory arthritis to investigate their similarity in terms of TCR repertoire and T-cell subset composition. RESULTS: The TCR repertoires of SF and ST T-cells were strikingly similar, particularly for CD8+ T-cells. A median of 49% of the total CD8+ TCR repertoire in SF was shared with ST, compared to 20% shared with blood. Similarly, 47% of the ST CD8+ TCR repertoire was shared with SF compared to 25% with blood. Furthermore, once the effect of collagenase digestion on gene expression by ST T-cells had been accounted for, the frequencies of specific CD8+ and CD4+ T-cell subsets were, in general, similar in SF and ST and were distinct from blood. CONCLUSION: Our results suggest that T-cells migrate and equilibrate between SF and ST and maintain similar phenotypes in both sites. We conclude that SF is an appropriate proxy for investigating the T-cell infiltrate in inflamed synovium, particularly in terms of investigating the TCR repertoire.
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Allogeneic hematopoietic cell transplantation (HCT) is used to treat many blood-based disorders and malignancies, however it can also result in serious adverse events, such as the development of acute graft-versus-host disease (aGVHD). This study aimed to develop a donor-specific epigenetic classifier to reduce incidence of aGVHD by improving donor selection. Genome-wide DNA methylation was assessed in a discovery cohort of 288 HCT donors selected based on recipient aGVHD outcome; this cohort consisted of 144 cases with aGVHD grades III-IV and 144 controls with no aGVHD. We applied a machine learning algorithm to identify CpG sites predictive of aGVHD. Receiver operating characteristic (ROC) curve analysis of these sites resulted in a classifier with an encouraging area under the ROC curve (AUC) of 0.91. To test this classifier, we used an independent validation cohort (n = 288) selected using the same criteria as the discovery cohort. Attempts to validate the classifier failed with the AUC falling to 0.51. These results indicate that donor DNA methylation may not be a suitable predictor of aGVHD in an HCT setting involving unrelated donors, despite the initial promising results in the discovery cohort. Our work highlights the importance of independent validation of machine learning classifiers, particularly when developing classifiers intended for clinical use.
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Biologic therapies targeting the IL-23/IL-17 axis have transformed the treatment of psoriasis. However, the early mechanisms of action of these drugs remain poorly understood. Here, we perform longitudinal single-cell RNA-sequencing in affected individuals receiving IL-23 inhibitor therapy. By profiling skin at baseline, day 3 and day 14 of treatment, we demonstrate that IL-23 blockade causes marked gene expression shifts, with fibroblast and myeloid populations displaying the most extensive changes at day 3. We also identify a transient WNT5A+/IL24+ fibroblast state, which is only detectable in lesional skin. In-silico and in-vitro studies indicate that signals stemming from these WNT5A+/IL24+ fibroblasts upregulate multiple inflammatory genes in keratinocytes. Importantly, the abundance of WNT5A+/IL24+ fibroblasts is significantly reduced after treatment. This observation is validated in-silico, by deconvolution of multiple transcriptomic datasets, and experimentally, by RNA in-situ hybridization. These findings demonstrate that the evolution of inflammatory fibroblast states is a key feature of resolving psoriasis skin.
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Psoríase , Humanos , Psoríase/tratamento farmacológico , Psoríase/genética , Psoríase/metabolismo , Pele/metabolismo , Queratinócitos/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , RNA/metabolismo , Fibroblastos/metabolismo , Análise de Célula ÚnicaRESUMO
Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.
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Linfócitos B , Células Dendríticas , Endodesoxirribonucleases , Microbioma Gastrointestinal , Animais , Feminino , Humanos , Masculino , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Microbioma Gastrointestinal/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/metabolismo , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Current differentiation protocols have not been successful in reproducibly generating fully functional human beta cells in vitro, partly due to incomplete understanding of human pancreas development. Here, we present detailed transcriptomic analysis of the various cell types of the developing human pancreas, including their spatial gene patterns. We integrated single-cell RNA sequencing with spatial transcriptomics at multiple developmental time points and revealed distinct temporal-spatial gene cascades. Cell trajectory inference identified endocrine progenitor populations and branch-specific genes as the progenitors differentiate toward alpha or beta cells. Spatial differentiation trajectories indicated that Schwann cells are spatially co-located with endocrine progenitors, and cell-cell connectivity analysis predicted that they may interact via L1CAM-EPHB2 signaling. Our integrated approach enabled us to identify heterogeneity and multiple lineage dynamics within the mesenchyme, showing that it contributed to the exocrine acinar cell state. Finally, we have generated an interactive web resource for investigating human pancreas development for the research community.
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Pâncreas Exócrino , Transcriptoma , Humanos , Transcriptoma/genética , Pâncreas/metabolismo , Perfilação da Expressão Gênica , Diferenciação Celular/genética , Análise de Célula Única , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
The interplay among different cells in a tissue is essential for maintaining homeostasis. Although disease states have been traditionally attributed to individual cell types, increasing evidence and new therapeutic options have demonstrated the primary role of multicellular functions to understand health and disease, opening new avenues to understand pathogenesis and develop new treatment strategies. We recently described the cellular composition and dynamics of the human oral mucosa; however, the spatial arrangement of cells is needed to better understand a morphologically complex tissue. Here, we link single-cell RNA sequencing, spatial transcriptomics, and high-resolution multiplex fluorescence in situ hybridisation to characterise human oral mucosa in health and oral chronic inflammatory disease. We deconvolved expression for resolution enhancement of spatial transcriptomic data and defined highly specialised epithelial and stromal compartments describing location-specific immune programs. Furthermore, we spatially mapped a rare pathogenic fibroblast population localised in a highly immunogenic region, responsible for lymphocyte recruitment through CXCL8 and CXCL10 and with a possible role in pathological angiogenesis through ALOX5AP. Collectively, our study provides a comprehensive reference for the study of oral chronic disease pathogenesis.
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Perfilação da Expressão Gênica , Interleucina-8 , Transcriptoma , Humanos , Quimiocina CXCL10/genética , Fibroblastos , Linfócitos , Interleucina-8/metabolismoRESUMO
CD69+CD103+ tissue-resident memory T (TRM) cells are important drivers of inflammation. To decipher their role in inflammatory arthritis, we apply single-cell, high-dimensional profiling to T cells from the joints of patients with psoriatic arthritis (PsA) or rheumatoid arthritis (RA). We identify three groups of synovial CD8+CD69+CD103+ TRM cells: cytotoxic and regulatory T (Treg)-like TRM cells are present in both PsA and RA, while CD161+CCR6+ type 17-like TRM cells with a pro-inflammatory cytokine profile (IL-17A+TNFα+IFNγ+) are specifically enriched in PsA. In contrast, only one population of CD4+CD69+CD103+ TRM cells is detected and at similarly low frequencies in both diseases. Type 17-like CD8+ TRM cells have a distinct transcriptomic signature and a polyclonal, but distinct, TCR repertoire. Type 17-like cells are also enriched in CD8+CD103- T cells in PsA compared with RA. These findings illustrate differences in the immunopathology of PsA and RA, with a particular enrichment for type 17 CD8+ T cells in the PsA joint.
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Artrite Psoriásica , Artrite Reumatoide , Humanos , Artrite Psoriásica/metabolismo , Células T de Memória , Subpopulações de Linfócitos T/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Artrite Reumatoide/metabolismo , Memória ImunológicaRESUMO
B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma.
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Linfócitos B , Melanoma , Humanos , Melanoma/genética , Anticorpos , Imunidade Humoral , Autoantígenos/genética , Microambiente TumoralRESUMO
Targeted bisulfite sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. In this study, we benchmarked five commercially available TBS platforms-three hybridization capture-based (Agilent, Roche and Illumina) and two reduced-representation-based (Diagenode and NuGen)-across 11 samples. Two samples were also compared with whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. Although all platforms produced robust and reproducible data, major differences in the number and identity of the CpG sites covered make it difficult to compare datasets generated on different platforms. To overcome this limitation, we applied imputation and show that it improves interoperability from an average of 10.35% (0.8 million) to 97% (7.6 million) common CpG sites. Our study provides guidance on which TBS platform to use for different methylome features and offers an imputation-based harmonization solution that allows comparative, integrative analysis.
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Epigenoma , Sequenciamento de Nucleotídeos em Larga Escala , Ilhas de CpG/genética , Metilação de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodosRESUMO
PURPOSE: The immune tumor microenvironment and the potential therapeutic opportunities for immunotherapy in small intestinal neuroendocrine tumors (siNET) have not been fully defined. EXPERIMENTAL DESIGN: Herein, we studied 40 patients with primary and synchronous metastatic siNETs, and matched blood and normal tissue obtained during surgery. We interrogated the immune checkpoint landscape using multi-parametric flow cytometry. In addition, matched FFPE tissue was obtained for multi-parametric IHC to determine the relative abundance and distribution of T-cell infiltrate. Tumor mutational burden (TMB) was also assessed and correlated with immune infiltration. RESULTS: Effector tumor-infiltrating lymphocytes (TIL) had a higher expression of PD-1 in the tumor microenvironment compared with the periphery. In addition, CD8+ TILs had a significantly higher co-expression of PD-1/ICOS and PD-1/CTLA-4 (cytotoxic T lymphocyte antigen-4) and higher levels of PD-1 expression compared with normal tissue. IHC revealed that the majority of cases have ≤10% intra-tumoral T cells but a higher number of peri-tumoral T cells, demonstrating an "exclusion" phenotype. Finally, we confirmed that siNETs have a low TMB compared with other tumor types in the TCGA database but did not find a correlation between TMB and CD8/Treg ratio. CONCLUSIONS: Taken together, these results suggest that a combination therapy approach will be required to enhance the immune response, using PD-1 as a checkpoint immunomodulator backbone in combination with other checkpoint targeting molecules (CTLA-4 or ICOS), or with drugs targeting other pathways to recruit "excluded" T cells into the tumor microenvironment to treat patients with siNETs.
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Neoplasias Intestinais , Tumores Neuroendócrinos , Biomarcadores Tumorais/metabolismo , Linfócitos T CD8-Positivos , Antígeno CTLA-4 , Humanos , Neoplasias Intestinais/patologia , Linfócitos do Interstício Tumoral , Tumores Neuroendócrinos/patologia , Receptor de Morte Celular Programada 1 , Microambiente Tumoral/genéticaRESUMO
B cells generate antibodies that are essential for immune protection, but their subgroups are poorly defined. Here, we perform undirected deep profiling of B cells in matched human lymphoid tissues from deceased transplant organ donors and blood. In addition to identifying unanticipated features of tissue-based B cell differentiation, we resolve two subsets of marginal zone B (MZB) cells differing in cell surface and transcriptomic profiles, clonal relationships to other subsets, enrichment of genes in the NOTCH pathway, distribution bias within splenic marginal zone microenvironment, and immunoglobulin repertoire diversity and hypermutation frequency. Each subset is present in spleen, gut-associated lymphoid tissue, mesenteric lymph nodes, and blood. MZB cells and the lineage from which they are derived are depleted in lupus nephritis. Here, we show that this depletion is of only one MZB subset. The other remains unchanged as a proportion of total B cells compared with health. Thus, it is important to factor MZB cell heterogeneity into studies of human B cell responses and pathology.
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Linfócitos B , Tecido Linfoide , Humanos , Ativação Linfocitária , Contagem de Linfócitos , BaçoRESUMO
Single-cell profiling of circulating tumor cells (CTCs) as part of a minimally invasive liquid biopsy presents an opportunity to characterize and monitor tumor heterogeneity and evolution in individual patients. In this study, we aimed to compare single-cell copy number variation (CNV) data with tissue and define the degree of intra- and inter-patient genomic heterogeneity. We performed next-generation sequencing (NGS) whole-genome CNV analysis of 125 single CTCs derived from seven patients with neuroendocrine neoplasms (NEN) alongside matched white blood cells (WBC), formalin-fixed paraffin-embedded (FFPE), and fresh frozen (FF) samples. CTC CNV profiling demonstrated recurrent chromosomal alterations in previously reported NEN copy number hotspots, including the prognostically relevant loss of chromosome 18. Unsupervised hierarchical clustering revealed CTCs with distinct clonal lineages as well as significant intra- and inter-patient genomic heterogeneity, including subclonal alterations not detectable by bulk analysis and previously unreported in NEN. Notably, we also demonstrated the presence of genomically distinct CTCs according to the enrichment strategy utilized (EpCAM-dependent vs size-based). This work has significant implications for the identification of therapeutic targets, tracking of evolutionary change, and the implementation of CTC-biomarkers in cancer.
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Células Neoplásicas Circulantes , Tumores Neuroendócrinos , Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA , Genômica , Humanos , Células Neoplásicas Circulantes/patologia , Tumores Neuroendócrinos/genética , Sequenciamento Completo do GenomaRESUMO
B cells emerge from the bone marrow as transitional (TS) B cells that differentiate through T1, T2, and T3 stages to become naive B cells. We have identified a bifurcation of human B cell maturation from the T1 stage forming IgMhi and IgMlo developmental trajectories. IgMhi T2 cells have higher expression of α4ß7 integrin and lower expression of IL-4 receptor (IL4R) compared with the IgMlo branch and are selectively recruited into gut-associated lymphoid tissue. IgMhi T2 cells also share transcriptomic features with marginal zone B cells (MZBs). Lineage progression from T1 cells to MZBs via an IgMhi trajectory is identified by pseudotime analysis of scRNA-sequencing data. Reduced frequency of IgMhi gut-homing T2 cells is observed in severe SLE and is associated with reduction of MZBs and their putative IgMhi precursors. The collapse of the gut-associated MZB maturational axis in severe SLE affirms its existence in health.
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Diferenciação Celular/imunologia , Trato Gastrointestinal/imunologia , Imunoglobulina M/metabolismo , Nefrite Lúpica/imunologia , Tecido Linfoide/imunologia , Células Precursoras de Linfócitos B/imunologia , Adulto , Idoso , Doadores de Sangue , Estudos de Casos e Controles , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Feminino , Humanos , Cadeias beta de Integrinas/metabolismo , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Nefrite Lúpica/sangue , Nefrite Lúpica/patologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma , Adulto JovemRESUMO
Undifferentiated sarcomas (USARCs) of adults are diverse, rare, and aggressive soft tissue cancers. Recent sequencing efforts have confirmed that USARCs exhibit one of the highest burdens of structural aberrations across human cancer. Here, we sought to unravel the molecular basis of the structural complexity in USARCs by integrating DNA sequencing, ploidy analysis, gene expression, and methylation profiling. We identified whole genome duplication as a prevalent and pernicious force in USARC tumorigenesis. Using mathematical deconvolution strategies to unravel the complex copy-number profiles and mutational timing models we infer distinct evolutionary pathways of these rare cancers. In addition, 15% of tumors exhibited raised mutational burdens that correlated with gene expression signatures of immune infiltration, and good prognosis.