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Follicular lymphoma (FL) exhibits considerable variability in biological features and clinical trajectories across patients. To dissect the diversity of FL, we utilized a Bernoulli mixture model to identify genetic subtypes in 713 pre-treatment tumor tissue samples. Our analysis revealed the existence of five subtypes with unique genetic profiles that correlated with clinicopathological characteristics. The clusters were enriched in specific mutations as follows: CS (CREBBP and STAT6), TT (TNFAIP3 and TP53), GM (GNA13 and MEF2B), Q (quiescent, for low mutation burden), and AR (mutations of mTOR pathway-related genes). The subtype Q was enriched for patients with stage I disease and associated with a lower proliferative history than the other subtypes. The AR subtype was unique in its enrichment for IgM-expressing FL cases and was associated with advanced-stage and more than 4 nodal sites. The existence of subtypes was validated in an independent cohort of 418 samples from the GALLIUM trial. Notably, patients assigned to the TT subtype consistently experienced inferior progression-free survival when treated with immunochemotherapy. Our findings offer insight into core pathways distinctly linked with each FL cluster and are expected to be informative in the era of targeted therapies.
Assuntos
Linfoma Folicular , Humanos , Linfoma Folicular/genética , Linfoma Folicular/patologia , Feminino , Masculino , Mutação , Pessoa de Meia-Idade , Idoso , Biomarcadores Tumorais/genética , PrognósticoRESUMO
Fluorescence in situ hybridization (FISH) using break-apart probes is recommended for identifying high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). Unbalanced MYC break-apart patterns, where the red or green signal is lost, are commonly reported as an equivocal result by clinical laboratories. In a cohort of 297 HGBCL-DH-BCL2, 13% of tumors had unbalanced MYC break-apart patterns with loss of red (LR: 2%) or green (LG: 11%) signal. To determine the significance of these patterns, MYC rearrangements were characterized by sequencing in 130 HGBCL-DH-BCL2, including 3 LR and 14 LG tumors. A MYC rearrangement was identified for 71% of tumors with LR or LG patterns, with the majority involving immunoglobulin loci or other recurrent MYC rearrangement partners. The architecture of these rearrangements consistently preserved the rearranged MYC allele, with the MYC gene predicted to be on the derivative chromosome containing the signal that is still present in nearly all cases. MYC protein expression, MYC mRNA expression, and the proportion of tumors expressing the dark zone signature was not significantly different between balanced and unbalanced groups. These results support a recommendation that unbalanced MYC break-apart FISH patterns be reported as positive for MYC rearrangement in the context of diagnosing HGBCL-DH-BCL2.
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BACKGROUND: The encoding of cell intrinsic drug resistance states in breast cancer reflects the contributions of genomic and non-genomic variations and requires accurate estimation of clonal fitness from co-measurement of transcriptomic and genomic data. Somatic copy number (CN) variation is the dominant mutational mechanism leading to transcriptional variation and notably contributes to platinum chemotherapy resistance cell states. Here, we deploy time series measurements of triple negative breast cancer (TNBC) single-cell transcriptomes, along with co-measured single-cell CN fitness, identifying genomic and transcriptomic mechanisms in drug-associated transcriptional cell states. RESULTS: We present scRNA-seq data (53,641 filtered cells) from serial passaging TNBC patient-derived xenograft (PDX) experiments spanning 2.5 years, matched with genomic single-cell CN data from the same samples. Our findings reveal distinct clonal responses within TNBC tumors exposed to platinum. Clones with high drug fitness undergo clonal sweeps and show subtle transcriptional reversion, while those with weak fitness exhibit dynamic transcription upon drug withdrawal. Pathway analysis highlights convergence on epithelial-mesenchymal transition and cytokine signaling, associated with resistance. Furthermore, pseudotime analysis demonstrates hysteresis in transcriptional reversion, indicating generation of new intermediate transcriptional states upon platinum exposure. CONCLUSIONS: Within a polyclonal tumor, clones with strong genotype-associated fitness under platinum remained fixed, minimizing transcriptional reversion upon drug withdrawal. Conversely, clones with weaker fitness display non-genomic transcriptional plasticity. This suggests CN-associated and CN-independent transcriptional states could both contribute to platinum resistance. The dominance of genomic or non-genomic mechanisms within polyclonal tumors has implications for drug sensitivity, restoration, and re-treatment strategies.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Análise de Célula Única , Transcriptoma , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Humanos , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Camundongos , Variações do Número de Cópias de DNA , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genéticaRESUMO
In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole-genome sequencing (WGS). Here we now introduce MRD-EDGE, a machine-learning-guided WGS ctDNA single-nucleotide variant (SNV) and copy-number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGESNV uses deep learning and a ctDNA-specific feature space to increase SNV signal-to-noise enrichment in WGS by ~300× compared to previous WGS error suppression. MRD-EDGECNV also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1 Gb to 200 Mb, vastly expanding its applicability within solid tumors. We harness the improved performance to identify MRD following surgery in multiple cancer types, track changes in TF in response to neoadjuvant immunotherapy in lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal-to-noise enrichment in MRD-EDGESNV enables plasma-only (non-tumor-informed) disease monitoring in advanced melanoma and lung cancer, yielding clinically informative TF monitoring for patients on immune-checkpoint inhibition.
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DNA Tumoral Circulante , Variações do Número de Cópias de DNA , Aprendizado de Máquina , Neoplasia Residual , Carga Tumoral , Humanos , DNA Tumoral Circulante/genética , DNA Tumoral Circulante/sangue , Neoplasia Residual/genética , Sequenciamento Completo do Genoma , Neoplasias/genética , Neoplasias/sangue , Neoplasias/terapia , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologiaRESUMO
ABSTRACT: Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements ("double hit"; HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of and mechanisms driving IG vs non-IG MYC rearrangements have not been elucidated. Here, we used custom targeted capture and/or whole-genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, although BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because 1 IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B-cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.
Assuntos
Rearranjo Gênico , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-myc , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Linfoma de Células B/genética , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologiaRESUMO
The role for routine whole genome and transcriptome analysis (WGTA) for poor prognosis pediatric cancers remains undetermined. Here, we characterize somatic mutations, structural rearrangements, copy number variants, gene expression, immuno-profiles and germline cancer predisposition variants in children and adolescents with relapsed, refractory or poor prognosis malignancies who underwent somatic WGTA and matched germline sequencing. Seventy-nine participants with a median age at enrollment of 8.8 y (range 6 months to 21.2 y) are included. Germline pathogenic/likely pathogenic variants are identified in 12% of participants, of which 60% were not known prior. Therapeutically actionable variants are identified by targeted gene report and whole genome in 32% and 62% of participants, respectively, and increase to 96% after integrating transcriptome analyses. Thirty-two molecularly informed therapies are pursued in 28 participants with 54% achieving a clinical benefit rate; objective response or stable disease ≥6 months. Integrated WGTA identifies therapeutically actionable variants in almost all tumors and are directly translatable to clinical care of children with poor prognosis cancers.
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Variações do Número de Cópias de DNA , Perfilação da Expressão Gênica , Neoplasias , Humanos , Criança , Neoplasias/genética , Neoplasias/terapia , Feminino , Adolescente , Masculino , Pré-Escolar , Prognóstico , Perfilação da Expressão Gênica/métodos , Lactente , Transcriptoma , Adulto Jovem , Sequenciamento Completo do Genoma , Mutação em Linhagem Germinativa , Mutação , Genoma Humano/genética , Predisposição Genética para DoençaRESUMO
Immune checkpoint inhibitors (ICIs) are increasingly used in the treatment of many tumor types, and durable responses can be observed in select populations. However, patients may exhibit significant immune-related adverse events (irAEs) that may lead to morbidity. There is limited information on whether the presence of specific germline mutations may highlight those at elevated risk of irAEs. We evaluated 117 patients with metastatic solid tumors or hematologic malignancies who underwent genomic analysis through the ongoing Personalized OncoGenomics (POG) program at BC Cancer and received an ICI during their treatment history. Charts were reviewed for irAEs. Whole genome sequencing of a fresh biopsy and matched normal specimens (blood) was performed at the time of POG enrollment. Notably, we found that MHC class I alleles in the HLA-B27 family, which have been previously associated with autoimmune conditions, were associated with grade 3 hepatitis and pneumonitis (q = 0.007) in patients treated with combination PD-1/PD-L1 and CTLA-4 inhibitors, and PD-1 inhibitors in combination with IDO-1 inhibitors. These data highlight that some patients may have a genetic predisposition to developing irAEs.
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Inibidores de Checkpoint Imunológico , Neoplasias , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Inibidores de Checkpoint Imunológico/uso terapêutico , Masculino , Neoplasias/tratamento farmacológico , Feminino , Pessoa de Meia-Idade , Idoso , Mutação em Linhagem Germinativa , Adulto , Idoso de 80 Anos ou maisRESUMO
Human papillomavirus (HPV) integration has been implicated in transforming HPV infection into cancer, but its genomic consequences have been difficult to study using short-read technologies. To resolve the dysregulation associated with HPV integration, we performed long-read sequencing on 63 cervical cancer genomes. We identified six categories of integration events based on HPV-human genomic structures. Of all HPV integrants, defined as two HPV-human breakpoints bridged by an HPV sequence, 24% contained variable copies of HPV between the breakpoints, a phenomenon we termed heterologous integration. Analysis of DNA methylation within and in proximity to the HPV genome at individual integration events revealed relationships between methylation status of the integrant and its orientation and structure. Dysregulation of the human epigenome and neighboring gene expression in cis with the HPV-integrated allele was observed over megabase-ranges of the genome. By elucidating the structural, epigenetic, and allele-specific impacts of HPV integration, we provide insight into the role of integrated HPV in cervical cancer.
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Follicular lymphoma (FL) accounts for â¼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning-derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.
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Linfoma Folicular , Linfoma Difuso de Grandes Células B , Humanos , Linfoma Folicular/patologia , Mutação , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologiaRESUMO
Germline structural variants (SVs) are challenging to resolve by conventional genetic testing assays. Long-read sequencing has improved the global characterization of SVs, but its sensitivity at cancer susceptibility loci has not been reported. Nanopore long-read genome sequencing was performed for nineteen individuals with pathogenic copy number alterations in BRCA1, BRCA2, CHEK2 and PALB2 identified by prior clinical testing. Fourteen variants, which spanned single exons to whole genes and included a tandem duplication, were accurately represented. Defining the precise breakpoints of SVs in BRCA1 and CHEK2 revealed unforeseen allelic heterogeneity and informed the mechanisms underlying the formation of recurrent deletions. Integrating read-based and statistical phasing further helped define extended haplotypes associated with founder alleles. Long-read sequencing is a sensitive method for characterizing private, recurrent and founder SVs underlying breast cancer susceptibility. Our findings demonstrate the potential for nanopore sequencing as a powerful genetic testing assay in the hereditary cancer setting.
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Neoplasias da Mama , Sequenciamento por Nanoporos , Nanoporos , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Predisposição Genética para Doença , Testes Genéticos/métodosRESUMO
Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.
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Linfoma de Burkitt , Infecções por Vírus Epstein-Barr , Linfoma Difuso de Grandes Células B , Criança , Humanos , Adulto , Linfoma de Burkitt/patologia , Herpesvirus Humano 4 , Linfoma Difuso de Grandes Células B/patologia , MutaçãoRESUMO
Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called "double-hit signature" (DHITsig)-a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.
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Linfoma Difuso de Grandes Células B , Proteínas Proto-Oncogênicas c-myc , Humanos , Hibridização in Situ Fluorescente , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/epidemiologia , Linfoma Difuso de Grandes Células B/genética , PrognósticoRESUMO
Oncogenic KRAS mutations are absent in approximately 10% of patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) and may represent a subgroup of mPDAC with therapeutic options beyond standard-of-care cytotoxic chemotherapy. While distinct gene fusions have been implicated in KRAS wildtype mPDAC, information regarding other types of mutations remain limited, and gene expression patterns associated with KRAS wildtype mPDAC have not been reported. Here, we leverage sequencing data from the PanGen trial to perform comprehensive characterization of the molecular landscape of KRAS wildtype mPDAC and reveal increased frequency of chr1q amplification encompassing transcription factors PROX1 and NR5A2. By leveraging data from colorectal adenocarcinoma and cholangiocarcinoma samples, we highlight similarities between cholangiocarcinoma and KRAS wildtype mPDAC involving both mutation and expression-based signatures and validate these findings using an independent dataset. These data further establish KRAS wildtype mPDAC as a unique molecular entity, with therapeutic opportunities extending beyond gene fusion events.
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Adenocarcinoma , Neoplasias dos Ductos Biliares , Carcinoma Ductal Pancreático , Colangiocarcinoma , Neoplasias Pancreáticas , Adenocarcinoma/patologia , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Carcinoma Ductal Pancreático/patologia , Colangiocarcinoma/genética , Humanos , Mutação , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Neoplasias PancreáticasRESUMO
In this study, we evaluate the impact of whole genome and transcriptome analysis (WGTA) on predictive molecular profiling and histologic diagnosis in a cohort of advanced malignancies. WGTA was used to generate reports including molecular alterations and site/tissue of origin prediction. Two reviewers analyzed genomic reports, clinical history, and tumor pathology. We used National Comprehensive Cancer Network (NCCN) consensus guidelines, Food and Drug Administration (FDA) approvals, and provincially reimbursed treatments to define genomic biomarkers associated with approved targeted therapeutic options (TTOs). Tumor tissue/site of origin was reassessed for most cases using genomic analysis, including a machine learning algorithm (Supervised Cancer Origin Prediction Using Expression [SCOPE]) trained on The Cancer Genome Atlas data. WGTA was performed on 652 cases, including a range of primary tumor types/tumor sites and 15 malignant tumors of uncertain histogenesis (MTUH). At the time WGTA was performed, alterations associated with an approved TTO were identified in 39 (6%) cases; 3 of these were not identified through routine pathology workup. In seven (1%) cases, the pathology workup either failed, was not performed, or gave a different result from the WGTA. Approved TTOs identified by WGTA increased to 103 (16%) when applying 2021 guidelines. The histopathologic diagnosis was reviewed in 389 cases and agreed with the diagnostic consensus after WGTA in 94% of non-MTUH cases (n = 374). The remainder included situations where the morphologic diagnosis was changed based on WGTA and clinical data (0.5%), or where the WGTA was non-contributory (5%). The 15 MTUH were all diagnosed as specific tumor types by WGTA. Tumor board reviews including WGTA agreed with almost all initial predictive molecular profile and histopathologic diagnoses. WGTA was a powerful tool to assign site/tissue of origin in MTUH. Current efforts focus on improving therapeutic predictive power and decreasing cost to enhance use of WGTA data as a routine clinical test.
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Neoplasias , Algoritmos , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Humanos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Fusobacterium nucleatum is a ubiquitous opportunistic pathogen with an emerging role as an oncomicrobe in colorectal cancer and other cancer settings. F. nucleatum can adhere to and invade host cells in a manner that varies across F. nucleatum strains and host cell phenotypes. Here, we performed pairwise cocultures between three F. nucleatum strains and two immortalized primary host cell types (human colonic epithelial [HCE] cells and human carotid artery endothelial [HCAE] cells) followed by transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate transcriptional and epigenetic host cell responses. We observed that F. nucleatum-induced host cell transcriptional modulation involves strong upregulation of genes related to immune migration and inflammatory processes, such as TNF, CXCL8, CXCL1, and CCL20. Furthermore, we identified genes strongly upregulated in a cell line-specific manner. In HCE cells, overexpressed genes included UBD and DUOX2/DUOXA2, associated with p53 degradation-mediated proliferation and intestinal reactive oxygen species (ROS) production, respectively. In HCAE cells, overexpressed genes included EFNA1 and LIF, two genes commonly upregulated in colorectal cancer and associated with poor patient outcomes, and PTGS2 (COX2), a gene associated with the protective effect of aspirin in the colorectal cancer setting. Interestingly, we also observed downregulation of numerous histone modification genes upon F. nucleatum exposure. We used the ChIP-seq data to annotate chromatin states genome wide and found significant chromatin remodeling following F. nucleatum exposure in HCAE cells, with increased frequencies of active enhancer and low-signal/quiescent states. Thus, our results highlight increased inflammation and chemokine gene expression as conserved host cell responses to F. nucleatum exposure and extensive host cell epigenomic changes specific to host cell type. IMPORTANCE Fusobacterium nucleatum is a bacterium normally found in the healthy oral cavity but also has an emerging role in colorectal cancer and other cancer settings. The host-microbe interactions of F. nucleatum and its involvement in tumor initiation, progression, and treatment resistance are not fully understood. We explored host cell changes that occur in response to F. nucleatum. We identified key genes differentially expressed in response to various conditions of F. nucleatum exposure and determined that the conserved host cell response to F. nucleatum was dominated by increased inflammation and chemokine gene expression. Additionally, we found extensive host cell epigenomic changes as a novel aspect of host modulation associated with F. nucleatum exposure. These results extend our understanding of F. nucleatum as an emerging pathogen and highlight the importance of considering strain heterogeneity and host cell phenotypic variation when exploring pathogenic mechanisms of F. nucleatum.
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Epigenoma , Infecções por Fusobacterium/genética , Infecções por Fusobacterium/microbiologia , Fusobacterium nucleatum/fisiologia , Transcriptoma , Linhagem Celular , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Oxidases Duais/genética , Oxidases Duais/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Fusobacterium/metabolismo , Fusobacterium nucleatum/genética , Interações Hospedeiro-Patógeno , HumanosRESUMO
Pancreatic neuroendocrine neoplasms (PNENs) are biologically and clinically heterogeneous. Here, we use a multi-omics approach to uncover the molecular factors underlying this heterogeneity. Transcriptomic analysis of 84 PNEN specimens, drawn from two cohorts, is substantiated with proteomic profiling and identifies four subgroups: Proliferative, PDX1-high, Alpha cell-like and Stromal/Mesenchymal. The Proliferative subgroup, consisting of both well- and poorly differentiated specimens, is associated with inferior overall survival probability. The PDX1-high and Alpha cell-like subgroups partially resemble previously described subtypes, and we further uncover distinctive metabolism-related features in the Alpha cell-like subgroup. The Stromal/Mesenchymal subgroup exhibits molecular characteristics of YAP1/WWTR1(TAZ) activation suggestive of Hippo signaling pathway involvement in PNENs. Whole-exome sequencing reveals subgroup-enriched mutational differences, supported by activity inference analysis, and identifies hypermorphic proto-oncogene variants in 14.3% of sequenced PNENs. Our study reveals differences in cellular signaling axes that provide potential directions for PNEN patient stratification and treatment strategies.
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Biomarcadores Tumorais , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteoma , Transcriptoma , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Proliferação de Células , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Masculino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/terapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Prognóstico , Proteômica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/metabolismoRESUMO
RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.
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Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.
Assuntos
Variações do Número de Cópias de DNA , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Mama Triplo Negativas/genética , Animais , Linhagem Celular Tumoral , Cisplatino/farmacologia , Células Clonais/patologia , Feminino , Aptidão Genética , Humanos , Camundongos , Modelos Estatísticos , Transplante de Neoplasias , Proteína Supressora de Tumor p53/genética , Sequenciamento Completo do GenomaRESUMO
As more clinically-relevant genomic features of myeloid malignancies are revealed, it has become clear that targeted clinical genetic testing is inadequate for risk stratification. Here, we develop and validate a clinical transcriptome-based assay for stratification of acute myeloid leukemia (AML). Comparison of ribonucleic acid sequencing (RNA-Seq) to whole genome and exome sequencing reveals that a standalone RNA-Seq assay offers the greatest diagnostic return, enabling identification of expressed gene fusions, single nucleotide and short insertion/deletion variants, and whole-transcriptome expression information. Expression data from 154 AML patients are used to develop a novel AML prognostic score, which is strongly associated with patient outcomes across 620 patients from three independent cohorts, and 42 patients from a prospective cohort. When combined with molecular risk guidelines, the risk score allows for the re-stratification of 22.1 to 25.3% of AML patients from three independent cohorts into correct risk groups. Within the adverse-risk subgroup, we identify a subset of patients characterized by dysregulated integrin signaling and RUNX1 or TP53 mutation. We show that these patients may benefit from therapy with inhibitors of focal adhesion kinase, encoded by PTK2, demonstrating additional utility of transcriptome-based testing for therapy selection in myeloid malignancy.