RESUMEN
Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.
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Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias/inmunología , Linfocitos T/inmunología , Biomarcadores de Tumor/metabolismo , Antígenos CD8/metabolismo , Quimiocina CXCL13/metabolismo , Cromosomas Humanos Par 9/genética , Estudios de Cohortes , Ciclina D1/genética , Variaciones en el Número de Copia de ADN/genética , Exoma/genética , Amplificación de Genes , Humanos , Evasión Inmune/efectos de los fármacos , Análisis Multivariante , Mutación/genética , Neoplasias/patología , Polimorfismo de Nucleótido Simple/genética , Receptores CCR5/metabolismo , Linfocitos T/efectos de los fármacos , Carga Tumoral/genéticaRESUMEN
Clear cell renal cell carcinoma (ccRCC) is characterized by near-universal loss of the short arm of chromosome 3, deleting several tumor suppressor genes. We analyzed whole genomes from 95 biopsies across 33 patients with clear cell renal cell carcinoma. We find hotspots of point mutations in the 5' UTR of TERT, targeting a MYC-MAX-MAD1 repressor associated with telomere lengthening. The most common structural abnormality generates simultaneous 3p loss and 5q gain (36% patients), typically through chromothripsis. This event occurs in childhood or adolescence, generally as the initiating event that precedes emergence of the tumor's most recent common ancestor by years to decades. Similar genomic changes drive inherited ccRCC. Modeling differences in age incidence between inherited and sporadic cancers suggests that the number of cells with 3p loss capable of initiating sporadic tumors is no more than a few hundred. Early development of ccRCC follows well-defined evolutionary trajectories, offering opportunity for early intervention.
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Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Progresión de la Enfermedad , Neoplasias Renales/genética , Neoplasias Renales/patología , Mutación , Regiones no Traducidas 5' , Adulto , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 5 , Femenino , Dosificación de Gen , Genoma Humano , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Telomerasa/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.
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Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/patología , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Biomarcadores de Tumor , Cromosomas , Evolución Clonal , Progresión de la Enfermedad , Evolución Molecular , Femenino , Heterogeneidad Genética , Variación Genética , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Modelos Estadísticos , Mutación , Metástasis de la Neoplasia , Fenotipo , Filogenia , Pronóstico , Estudios Prospectivos , Análisis de Secuencia de ADNRESUMEN
Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases.
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Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/patología , Mutación , Metástasis de la Neoplasia , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Biopsia , Mapeo Cromosómico , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 9 , Progresión de la Enfermedad , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Fenotipo , Estudios Prospectivos , Trombosis , Resultado del TratamientoRESUMEN
Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. VIDEO ABSTRACT.
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Carcinoma de Pulmón de Células no Pequeñas/inmunología , Antígenos HLA/genética , Neoplasias Pulmonares/inmunología , Escape del Tumor , Adulto , Anciano , Anciano de 80 o más Años , Presentación de Antígeno , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Estudios de Cohortes , Femenino , Antígenos HLA/inmunología , Humanos , Pérdida de Heterocigocidad , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Masculino , Persona de Mediana Edad , Mutación , Polimorfismo de Nucleótido SimpleRESUMEN
Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy1. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study2,3. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis.
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Evolución Molecular , Genoma Humano , Neoplasias Pulmonares , Metástasis de la Neoplasia , Transcriptoma , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Genómica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Metástasis de la Neoplasia/genética , Transcriptoma/genética , Alelos , Aprendizaje Automático , Genoma Humano/genéticaRESUMEN
Metastatic disease is responsible for the majority of cancer-related deaths1. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse.
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Carcinoma de Pulmón de Células no Pequeñas , Evolución Clonal , Células Clonales , Evolución Molecular , Neoplasias Pulmonares , Metástasis de la Neoplasia , Humanos , Carcinoma de Pulmón de Células no Pequeñas/patología , Células Clonales/patología , Estudios de Cohortes , Progresión de la Enfermedad , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia/diagnóstico , Metástasis de la Neoplasia/patología , Recurrencia Local de NeoplasiaRESUMEN
A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 µm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1ß. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for PM2.5 air pollutants and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.
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Adenocarcinoma del Pulmón , Contaminantes Atmosféricos , Contaminación del Aire , Transformación Celular Neoplásica , Neoplasias Pulmonares , Animales , Ratones , Adenocarcinoma del Pulmón/inducido químicamente , Adenocarcinoma del Pulmón/genética , Contaminantes Atmosféricos/efectos adversos , Contaminantes Atmosféricos/análisis , Contaminación del Aire/efectos adversos , Contaminación del Aire/análisis , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Exposición a Riesgos Ambientales , Receptores ErbB/genética , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Material Particulado/efectos adversos , Material Particulado/análisis , Tamaño de la Partícula , Estudios de Cohortes , Macrófagos Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/patologíaRESUMEN
Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.
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Inestabilidad Cromosómica/genética , Evolución Molecular , Cariotipo , Metástasis de la Neoplasia/genética , Neoplasias/genética , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 8/genética , Células Clonales/metabolismo , Células Clonales/patología , Ciclina E/genética , Variaciones en el Número de Copia de ADN/genética , Femenino , Humanos , Pérdida de Heterocigocidad/genética , Masculino , Mutagénesis , Metástasis de la Neoplasia/patología , Neoplasias/patología , Proteínas Oncogénicas/genéticaRESUMEN
Women in developing countries still face enormous challenges when accessing reproductive health care. Access to voluntary family planning empowers women allowing them to complete their education and join the paid workforce. This effectively helps to end poverty, hunger and promotes good health for all. According to the United Nations (UN) organization, in 2022, an estimated 257 million women still lacked access to safe and effective family planning methods globally. One of the main barriers is the associated cost of modern contraceptive methods. Funded by the Bill & Melinda Gates Foundation, Almac Group worked on the development of a novel biocatalytic route to etonogestrel and levonorgestrel, two modern contraceptive APIs, with the goal of substantially decreasing the cost of production and so enabling their use in developing nations. This present work combines the selection and engineering of a carbonyl reductase (CRED) enzyme from Almac's selectAZyme™ panel, with process development, to enable efficient and economically viable bioreduction of ethyl secodione to (13R,17S)-secol, the key chirality introducing intermediate en route to etonogestrel and levonorgestrel API. CRED library screening returned a good hit with an Almac CRED from Bacillus weidmannii, which allowed for highly stereoselective bioreduction at low enzyme loading of less than 1% w/w under screening assay conditions. However, the only co-solvent tolerated was DMSO up to â¼30% v/v, and it was impossible to achieve reaction completion with any enzyme loading at substrate titres of 20 g L-1 and above, due to the insolubility of the secodione. This triggered a rapid enzyme engineering program fully based on computational mutant selection. A small panel of 93 CRED mutants was rationally designed to increase the catalytic activity as well as thermal and solvent stability. The best mutant, Mutant-75, enabled a reaction at 45 °C to go to completion at 90 g L-1 substrate titre in a buffer/DMSO/heptane reaction medium fed over 6 h with substrate DMSO stock solution, with a low enzyme loading of 3.5% w/w wrt substrate. In screening assay conditions, Mutant-75 also showed a 2.2-fold activity increase. Our paper shows which computations and rational decisions enabled this outcome.
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This corrects the article DOI: 10.1038/nature22364.
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The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Linaje de la Célula/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Evolución Molecular , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/diagnóstico , Biopsia/métodos , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Rastreo Celular , Células Clonales/metabolismo , Células Clonales/patología , Análisis Mutacional de ADN , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Detección Precoz del Cáncer/métodos , Humanos , Límite de Detección , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Reacción en Cadena de la Polimerasa Multiplex , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Cuidados Posoperatorios/métodos , Reproducibilidad de los Resultados , Carga TumoralRESUMEN
The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg36-Ser37, but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser37-Leu38). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.
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Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Receptor PAR-2/antagonistas & inhibidores , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Condrosarcoma/genética , Condrosarcoma/metabolismo , Condrosarcoma/patología , Humanos , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 8 de la Matriz/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
BACKGROUND: Among patients with non-small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC. METHODS: In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival. RESULTS: We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10-4), which remained significant in multivariate analysis. CONCLUSIONS: Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601 .).
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Carcinoma de Pulmón de Células no Pequeñas/genética , Inestabilidad Cromosómica , Heterogeneidad Genética , Neoplasias Pulmonares/genética , Mutación , Recurrencia Local de Neoplasia/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Variaciones en el Número de Copia de ADN , Supervivencia sin Enfermedad , Evolución Molecular , Exoma , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Masculino , Filogenia , Pronóstico , Estudios Prospectivos , Factores de Riesgo , Análisis de Secuencia de ADN/métodosRESUMEN
Osteoarthritis (OA) is a degenerative condition caused by dysregulation of multiple molecular signalling pathways. Such dysregulation results in damage to cartilage, a smooth and protective tissue that enables low friction articulation of synovial joints. Matrix metalloproteinases (MMPs), especially MMP-13, are key enzymes in the cleavage of type II collagen which is a vital component for cartilage integrity. Transforming growth factor beta (TGFß) can protect against pro-inflammatory cytokine-mediated MMP expression. With age there is a change in the ratio of two TGFß type I receptors (Alk1/Alk5), a shift that results in TGFß losing its protective role in cartilage homeostasis. Instead, TGFß promotes cartilage degradation which correlates with the spontaneous development of OA in murine models. However, the mechanism by which TGFß protects against pro-inflammatory responses and how this changes with age has not been extensively studied. As TGFß signalling is complex, we used systems biology to combine experimental and computational outputs to examine how the system changes with age. Experiments showed that the repressive effect of TGFß on chondrocytes treated with a pro-inflammatory stimulus required Alk5. Computational modelling revealed two independent mechanisms were needed to explain the crosstalk between TGFß and pro-inflammatory signalling pathways. A novel meta-analysis of microarray data from OA patient tissue was used to create a Cytoscape network representative of human OA and revealed the importance of inflammation. Combining the modelled genes with the microarray network provided a global overview into the crosstalk between the different signalling pathways involved in OA development. Our results provide further insights into the mechanisms that cause TGFß signalling to change from a protective to a detrimental pathway in cartilage with ageing. Moreover, such a systems biology approach may enable restoration of the protective role of TGFß as a potential therapy to prevent age-related loss of cartilage and the development of OA.
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Envejecimiento/fisiología , Transducción de Señal/fisiología , Biología de Sistemas/métodos , Factor de Crecimiento Transformador beta/metabolismo , Envejecimiento/genética , Línea Celular , Condrocitos/metabolismo , Perfilación de la Expresión Génica , Humanos , Osteoartritis/metabolismo , Transducción de Señal/genéticaRESUMEN
Mutations in the coiled-coil and DNA-binding domains of STAT1 lead to delayed STAT1 dephosphorylation and subsequently gain-of-function. The associated clinical phenotype is broad and can include chronic mucocutaneous candidiasis (CMC) and/or combined immunodeficiency (CID). We report a case of CMC/CID in a 10-year-old boy due to a novel mutation in the small ubiquitin molecule (SUMO) consensus site at the C-terminal region of STAT1 leading to gain-of-function by impaired sumoylation. Immunodysregulatory features of disease improved after Janus kinase inhibitor (jakinib) treatment. Functional testing after treatment confirmed reversal of the STAT1 hyper-phosphorylation and downstream transcriptional activity. IL-17 and IL-22 production was, however, not restored with jakinib therapy (ruxolitinib), and the patient remained susceptible to opportunistic infection. In conclusion, a mutation in the SUMO consensus site of STAT1 can lead to gain-of-function that is reversible with jakinib treatment. However, full immunocompetence was not restored, suggesting that this treatment strategy might serve well as a bridge to definitive therapy such as hematopoietic stem cell transplant rather than a long-term treatment option.
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Candidiasis Mucocutánea Crónica/genética , Enfermedades de Inmunodeficiencia Primaria/genética , Pirazoles/uso terapéutico , Factor de Transcripción STAT1/genética , Candidiasis Mucocutánea Crónica/diagnóstico , Candidiasis Mucocutánea Crónica/tratamiento farmacológico , Niño , Mutación con Ganancia de Función , Humanos , Quinasas Janus/antagonistas & inhibidores , Masculino , Nitrilos , Enfermedades de Inmunodeficiencia Primaria/diagnóstico , Enfermedades de Inmunodeficiencia Primaria/tratamiento farmacológico , Pirazoles/farmacología , Pirimidinas , Sumoilación/genética , Resultado del TratamientoRESUMEN
Cancer chromosomal instability (CIN) results in an increased rate of change of chromosome number and structure and generates intratumour heterogeneity. CIN is observed in most solid tumours and is associated with both poor prognosis and drug resistance. Understanding a mechanistic basis for CIN is therefore paramount. Here we find evidence for impaired replication fork progression and increased DNA replication stress in CIN(+) colorectal cancer (CRC) cells relative to CIN(-) CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three new CIN-suppressor genes (PIGN (also known as MCD4), MEX3C (RKHD2) and ZNF516 (KIAA0222)) encoded on chromosome 18q that are subject to frequent copy number loss in CIN(+) CRC. Chromosome 18q loss was temporally associated with aneuploidy onset at the adenoma-carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage, reduces the frequency of chromosome segregation errors after CIN-suppressor gene silencing, and attenuates segregation errors and DNA damage in CIN(+) cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity.
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Inestabilidad Cromosómica/genética , Neoplasias Colorrectales/genética , Replicación del ADN/genética , Aneuploidia , Línea Celular Tumoral , Inestabilidad Cromosómica/efectos de los fármacos , Segregación Cromosómica/efectos de los fármacos , Segregación Cromosómica/genética , Cromosomas Humanos Par 18/efectos de los fármacos , Cromosomas Humanos Par 18/genética , Neoplasias Colorrectales/patología , Variaciones en el Número de Copia de ADN/genética , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Replicación del ADN/efectos de los fármacos , Eliminación de Gen , Silenciador del Gen , Genes Supresores de Tumor , Humanos , Mitosis/efectos de los fármacos , Nucleósidos/farmacología , Fosfotransferasas/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Irreversible breakdown of cartilage extracellular matrix (ECM) by the collagenase matrix metalloproteinase 13 (MMP13) represents a key event in osteoarthritis (OA) progression. Although inflammation is most commonly associated with inflammatory joint diseases, it also occurs in OA and is thus relevant to the prevalent tissue destruction. Here, inflammation generates a cFOS AP-1 early response that indirectly affects MMP13 gene expression. To ascertain a more direct effect on prolonged MMP13 production we examined the potential molecular events occurring between the rapid, transient expression of cFOS and the subsequent MMP13 induction. Importantly, we show MMP13 mRNA expression is mirrored by nascent hnRNA transcription. Employing ChIP assays, cFOS recruitment to the MMP13 promoter occurs at an early stage prior to gene transcription and that recruitment of transcriptional initiation markers also correlated with MMP13 expression. Moreover, protein synthesis inhibition following early FOS expression resulted in a significant decrease in MMP13 expression thus indicating a role for different regulatory factors modulating expression of the gene. Subsequent mRNA transcriptome analyses highlighted several genes induced soon after FOS that could contribute to MMP13 expression. Specific small interfering RNA-mediated silencing highlighted that ATF3 was as highly selective for MMP13 as cFOS. Moreover, ATF3 expression was AP-1(cFOS/cJUN)-dependent and expression levels were maintained after the early transient cFOS response. Furthermore, ATF3 bound the proximal MMP13 AP-1 motif in stimulated chondrocytes at time points that no longer supported binding of FOS Consequently, these findings support roles for both cFOS (indirect) and ATF3 (direct) in effecting MMP13 transcription in human chondrocytes.
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Factor de Transcripción Activador 3/metabolismo , Condrocitos/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Metaloproteinasa 13 de la Matriz/biosíntesis , Elementos de Respuesta/fisiología , Transcriptoma/fisiología , Factor de Transcripción Activador 3/genética , Células Cultivadas , Humanos , Metaloproteinasa 13 de la Matriz/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Pre-clinical non-small cell lung cancer (NSCLC) models are poorly representative of the considerable inter- and intra-tumor heterogeneity of the disease in patients. Primary cell-based in vitro models of NSCLC are therefore desirable for novel therapy development and personalized cancer medicine. Methods have been described to generate rapidly proliferating epithelial cell cultures from multiple human epithelia using 3T3-J2 feeder cell culture in the presence of Y-27632, a RHO-associated protein kinase (ROCK) inhibitor, in what are known as "conditional reprograming conditions" (CRC) or 3T3 + Y. In some cancer studies, variations of this methodology have allowed primary tumor cell expansion across a number of cancer types but other studies have demonstrated the preferential expansion of normal epithelial cells from tumors in such conditions. Here, we report our experience regarding the derivation of primary NSCLC cell cultures from 12 lung adenocarcinoma patients enrolled in the Tracking Cancer Evolution through Therapy (TRACERx) clinical study and discuss these in the context of improving the success rate for in vitro cultivation of cells from NSCLC tumors.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Técnicas de Cocultivo/métodos , Células Epiteliales/citología , Neoplasias Pulmonares/patología , Células 3T3 , Anciano , Anciano de 80 o más Años , Amidas/farmacología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Proliferación Celular , Células Epiteliales/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/genética , Masculino , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Piridinas/farmacología , Mucosa Respiratoria/citología , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
BACKGROUND: The focus of tumour-specific antigen analyses has been on single nucleotide variants (SNVs), with the contribution of small insertions and deletions (indels) less well characterised. We investigated whether the frameshift nature of indel mutations, which create novel open reading frames and a large quantity of mutagenic peptides highly distinct from self, might contribute to the immunogenic phenotype. METHODS: We analysed whole-exome sequencing data from 5777 solid tumours, spanning 19 cancer types from The Cancer Genome Atlas. We compared the proportion and number of indels across the cohort, with a subset of results replicated in two independent datasets. We assessed in-silico tumour-specific neoantigen predictions by mutation type with pan-cancer analysis, together with RNAseq profiling in renal clear cell carcinoma cases (n=392), to compare immune gene expression across patient subgroups. Associations between indel burden and treatment response were assessed across four checkpoint inhibitor datasets. FINDINGS: We observed renal cell carcinomas to have the highest proportion (0·12) and number of indel mutations across the pan-cancer cohort (p<2·2â×â10-16), more than double the median proportion of indel mutations in all other cancer types examined. Analysis of tumour-specific neoantigens showed that enrichment of indel mutations for high-affinity binders was three times that of non-synonymous SNV mutations. Furthermore, neoantigens derived from indel mutations were nine times enriched for mutant specific binding, as compared with non-synonymous SNV derived neoantigens. Immune gene expression analysis in the renal clear cell carcinoma cohort showed that the presence of mutant-specific neoantigens was associated with upregulation of antigen presentation genes, which correlated (r=0·78) with T-cell activation as measured by CD8-positive expression. Finally, analysis of checkpoint inhibitor response data revealed frameshift indel count to be significantly associated with checkpoint inhibitor response across three separate melanoma cohorts (p=4·7â×â10-4). INTERPRETATION: Renal cell carcinomas have the highest pan-cancer proportion and number of indel mutations. Evidence suggests indels are a highly immunogenic mutational class, which can trigger an increased abundance of neoantigens and greater mutant-binding specificity. FUNDING: Cancer Research UK, UK National Institute for Health Research (NIHR) at the Royal Marsden Hospital National Health Service Foundation Trust, Institute of Cancer Research and University College London Hospitals Biomedical Research Centres, the UK Medical Research Council, the Rosetrees Trust, Novo Nordisk Foundation, the Prostate Cancer Foundation, the Breast Cancer Research Foundation, the European Research Council.