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BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.
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Células Madre Pluripotentes Inducidas , Mucosa Respiratoria , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/citología , Diferenciación Celular/fisiología , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Organoides/metabolismoRESUMEN
Air liquid interfaced (ALI) epithelial barriers are essential for homeostatic functions such as nutrient transport and immunological protection. Dysfunction of such barriers are implicated in a variety of autoimmune and inflammatory disorders and, as such, sensors capable of monitoring barrier health are integral for disease modelling, diagnostics and drug screening applications. To date, gold-standard electrical methods for detecting barrier resistance require rigid electrodes bathed in an electrolyte, which limits compatibility with biological architectures and is non-physiological for ALI. This work presents a flexible all-planar electronic device capable of monitoring barrier formation and perturbations in human respiratory and intestinal cells at ALI. By interrogating patient samples with electrochemical impedance spectroscopy and simple equivalent circuit models, disease-specific and patient-specific signatures are uncovered. Device readouts are validated against commercially available chopstick electrodes and show greater conformability, sensitivity and biocompatibility. The effect of electrode size on sensing efficiency is investigated and a cut-off sensing area is established, which is one order of magnitude smaller than previously reported. This work provides the first steps in creating a physiologically relevant sensor capable of mapping local and real-time changes of epithelial barrier function at ALI, which will have broad applications in toxicology and drug screening applications.
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Electrónica , Humanos , ElectrodosRESUMEN
Cigarette smoking has many serious negative health consequences. The relationship between smoking and SARS-CoV-2 infection is controversial, specifically whether smokers are at increased risk of infection. We investigated the impact of cigarette smoke on ACE2 isoform expression and SARS-CoV-2 infection in differentiated primary human bronchial epithelial cells at the air-liquid-interface (ALI). We assessed the expression of ACE2 in response to CSE and therapeutics reported to modulate ACE2. We exposed ALI cultures to cigarette smoke extract (CSE) and then infected them with SARS-CoV-2. We measured cellular infection using flow cytometry and whole-transwell immunofluorescence. We found that CSE increased expression of full-length ACE2 (flACE2) but did not alter the expression of a Type I-interferon sensitive truncated isoform (dACE2) that lacks the capacity to bind SARS-CoV-2. CSE did not have a significant impact on key mediators of the innate immune response. Importantly, we show that, despite the increase in flACE2, CSE did not alter airway cell infection after CSE exposure. We found that nicotine does not significantly alter flACE2 expression but that NRF2 agonists do lead to an increase in flACE2 expression. This increase was not associated with an increase in SARS-CoV-2 infection. Our results are consistent with the epidemiological data suggesting that current smokers do not have an excess of SARS-CoV-2 infection. but that those with chronic respiratory or cardiovascular disease are more vulnerable to severe COVID-19. They suggest that, in differentiated conducting airway cells, flACE2 expression levels may not limit airway SARS-CoV-2 infection.
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Background: Quantitative proteomics is able to provide a comprehensive, unbiased description of changes to cells caused by viral infection, but interpretation may be complicated by differential changes in infected and uninfected 'bystander' cells, or the use of non-physiological cellular models. Methods: In this paper, we use fluorescence-activated cell sorting (FACS) and quantitative proteomics to analyse cell-autonomous changes caused by authentic SARS-CoV-2 infection of respiratory epithelial cells, the main target of viral infection in vivo. First, we determine the relative abundance of proteins in primary human airway epithelial cells differentiated at the air-liquid interface (basal, secretory and ciliated cells). Next, we specifically characterise changes caused by SARS-CoV-2 infection of ciliated cells. Finally, we compare temporal proteomic changes in infected and uninfected 'bystander' Calu-3 lung epithelial cells and compare infection with B.29 and B.1.1.7 (Alpha) variants. Results: Amongst 5,709 quantified proteins in primary human airway ciliated cells, the abundance of 226 changed significantly in the presence of SARS-CoV-2 infection (q <0.05 and >1.5-fold). Notably, viral replication proceeded without inducing a type-I interferon response. Amongst 6,996 quantified proteins in Calu-3 cells, the abundance of 645 proteins changed significantly in the presence of SARS-CoV-2 infection (q < 0.05 and > 1.5-fold). In contrast to the primary cell model, a clear type I interferon (IFN) response was observed. Nonetheless, induction of IFN-inducible proteins was markedly attenuated in infected cells, compared with uninfected 'bystander' cells. Infection with B.29 and B.1.1.7 (Alpha) variants gave similar results. Conclusions: Taken together, our data provide a detailed proteomic map of changes in SARS-CoV-2-infected respiratory epithelial cells in two widely used, physiologically relevant models of infection. As well as identifying dysregulated cellular proteins and processes, the effectiveness of strategies employed by SARS-CoV-2 to avoid the type I IFN response is illustrated in both models.
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BACKGROUND: There are limited effective prophylactic/early treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Viral entry requires spike protein binding to the angiotensin-converting enzyme-2 receptor and cleavage by transmembrane serine protease 2 (TMPRSS2), a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is in clinical trials in early SARS-CoV-2 infection. METHODS: We used differentiated primary human airway epithelial cells at the air-liquid interface to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection. RESULTS: We first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-CoV-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, oral camostat is rapidly metabolised in the circulation, with poor airway bioavailability. We therefore modelled local airway administration by applying camostat to the apical surface of differentiated airway cultures. We demonstrated that a brief exposure to topical camostat effectively restricts SARS-CoV-2 infection. CONCLUSION: These experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.
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Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/administración & dosificación , Administración Tópica , Andrógenos/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/farmacología , COVID-19/prevención & control , COVID-19/virología , Células Cultivadas , Células Epiteliales , Ésteres/farmacología , Expresión Génica , Células Caliciformes/inmunología , Células Caliciformes/metabolismo , Guanidinas/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Serina Endopeptidasas/genética , Transducción de Señal , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates the rapid development of new therapies against coronavirus disease 2019 (COVID-19) infection. Here, we present the identification of 200 approved drugs, appropriate for repurposing against COVID-19. We constructed a SARS-CoV-2-induced protein network, based on disease signatures defined by COVID-19 multiomics datasets, and cross-examined these pathways against approved drugs. This analysis identified 200 drugs predicted to target SARS-CoV-2-induced pathways, 40 of which are already in COVID-19 clinical trials, testifying to the validity of the approach. Using artificial neural network analysis, we classified these 200 drugs into nine distinct pathways, within two overarching mechanisms of action (MoAs): viral replication (126) and immune response (74). Two drugs (proguanil and sulfasalazine) implicated in viral replication were shown to inhibit replication in cell assays. This unbiased and validated analysis opens new avenues for the rapid repurposing of approved drugs into clinical trials.
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Reposicionamiento de Medicamentos , SARS-CoV-2/fisiología , Antivirales/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/patología , COVID-19/virología , Humanos , Redes Neurales de la Computación , Proguanil/farmacología , Proguanil/uso terapéutico , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Sulfasalazina/farmacología , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Face masks and personal respirators are used to curb the transmission of SARS-CoV-2 in respiratory droplets; filters embedded in some personal protective equipment could be used as a non-invasive sample source for applications, including at-home testing, but information is needed about whether filters are suited to capture viral particles for SARS-CoV-2 detection. In this study, we generated inactivated virus-laden aerosols of 0.3-2 microns in diameter (0.9 µm mean diameter by mass) and dispersed the aerosolized viral particles onto electrostatic face mask filters. The limit of detection for inactivated coronaviruses SARS-CoV-2 and HCoV-NL63 extracted from filters was between 10 to 100 copies/filter for both viruses. Testing for SARS-CoV-2, using face mask filters and nasopharyngeal swabs collected from hospitalized COVID-19-patients, showed that filter samples offered reduced sensitivity (8.5% compared to nasopharyngeal swabs). The low concordance of SARS-CoV-2 detection between filters and nasopharyngeal swabs indicated that number of viral particles collected on the face mask filter was below the limit of detection for all patients but those with the highest viral loads. This indicated face masks are unsuitable to replace diagnostic nasopharyngeal swabs in COVID-19 diagnosis. The ability to detect nucleic acids on face mask filters may, however, find other uses worth future investigation.
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COVID-19/patología , Máscaras/virología , Nasofaringe/virología , SARS-CoV-2/aislamiento & purificación , Adulto , Aerosoles , Anciano , COVID-19/virología , Femenino , Hospitalización , Humanos , Límite de Detección , Masculino , Persona de Mediana Edad , Tamaño de la Partícula , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/fisiología , Electricidad Estática , Carga Viral , Adulto JovenRESUMEN
SOX2 is a pleiotropic nuclear transcription factor with major roles in stem cell biology and in development. Over the last 10 years SOX2 has also been implicated as a lineage-specific oncogene, notably in squamous carcinomas but also neurological tumours, particularly glioblastoma. Squamous carcinomas (SQCs) comprise a common group of malignancies for which there are no targeted therapeutic interventions. In this article we review the molecular epidemiological and laboratory evidence linking SOX2 with squamous carcinogenesis, explore in detail the multifaceted impact of SOX2 in SQC, describe areas of uncertainty and highlight areas for potential future research.
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Carcinoma de Células Escamosas/patología , Neoplasias/patología , Factores de Transcripción SOXB1/metabolismo , Animales , Carcinogénesis , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Factores de Transcripción SOXB1/genética , Transducción de SeñalRESUMEN
Patients diagnosed with lung squamous cell carcinoma (LUSC) have limited targeted therapies. We report here the identification and characterisation of BCL11A, as a LUSC oncogene. Analysis of cancer genomics datasets revealed BCL11A to be upregulated in LUSC but not in lung adenocarcinoma (LUAD). Experimentally we demonstrate that non-physiological levels of BCL11A in vitro and in vivo promote squamous-like phenotypes, while its knockdown abolishes xenograft tumour formation. At the molecular level we found that BCL11A is transcriptionally regulated by SOX2 and is required for its oncogenic functions. Furthermore, we show that BCL11A and SOX2 regulate the expression of several transcription factors, including SETD8. We demonstrate that shRNA-mediated or pharmacological inhibition of SETD8 selectively inhibits LUSC growth. Collectively, our study indicates that BCL11A is integral to LUSC pathology and highlights the disruption of the BCL11A-SOX2 transcriptional programme as a novel candidate for drug development.
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Carcinoma de Células Escamosas/genética , Proteínas Portadoras/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción SOXB1/metabolismo , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/genética , Técnicas de Silenciamiento del Gen , Sitios Genéticos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Oncogenes , Organoides/patología , Unión Proteica , Proteínas RepresorasRESUMEN
RATIONALE: Improving the early detection and chemoprevention of lung cancer are key to improving outcomes. The pathobiology of early squamous lung cancer is poorly understood. We have shown that amplification of sex-determining region Y-box 2 (SOX2) is an early and consistent event in the pathogenesis of this disease, but its functional oncogenic potential remains uncertain. We tested the impact of deregulated SOX2 expression in a novel organotypic system that recreates the molecular and microenvironmental context in which squamous carcinogenesis occurs. OBJECTIVES: (1) To develop an in vitro model of bronchial dysplasia that recapitulates key molecular and phenotypic characteristics of the human disease; (2) to test the hypothesis that SOX2 deregulation is a key early event in the pathogenesis of bronchial dysplasia; and (3) to use the model for studies on pathogenesis and chemoprevention. METHODS: We engineered the inducible activation of oncogenes in immortalized bronchial epithelial cells. We used three-dimensional tissue culture to build an organotypic model of bronchial dysplasia. MEASUREMENTS AND MAIN RESULTS: We recapitulated human bronchial dysplasia in vitro. SOX2 deregulation drives dysplasia, and loss of tumor promoter 53 is a cooperating genetic event that potentiates the dysplastic phenotype. Deregulated SOX2 alters critical genes implicated in hallmarks of cancer progression. Targeted inhibition of AKT prevents the initiation of the dysplastic phenotype. CONCLUSIONS: In the appropriate genetic and microenvironmental context, acute deregulation of SOX2 drives bronchial dysplasia. This confirms its oncogenic potential in human cells and affords novel insights into the impact of SOX2 deregulation. This model can be used to test therapeutic agents aimed at chemoprevention.
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Displasia Broncopulmonar/genética , Displasia Broncopulmonar/fisiopatología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/fisiopatología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatología , Factores de Transcripción SOXB1/genética , Técnicas de Cultivo de Célula , Humanos , Modelos BiológicosRESUMEN
Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M mutation causes over 50% of resistance; MET amplification is also common. Preclinical data suggest synergy between MET and EGFR inhibitors. We hypothesized that EGFR-MET dimerization determines response to MET inhibition, depending on EGFR mutation status, independently of MET copy number. We tested this hypothesis by generating isogenic cell lines from NCI-H1975 cells, which co-express L858R and T790M EGFR mutations, namely H1975L858R/T790M (EGFR TKI resistant); H1975L858R (sensitized) and H1975WT (wild-type). We assessed cell proliferation in vitro and tumor growth/stroma formation in derived xenograft models in response to a MET TKI (SGX523) and correlated with EGFR-MET dimerization assessed by Förster Resonance Energy Transfer (FRET). SGX523 significantly reduced H1975L858R/T790M cell proliferation, xenograft tumor growth and decreased ERK phosphorylation. The same was not seen in H1975L858R or H1975WT cells. SGX523 only reduced stroma formation in H1975L858R. SGX523 reduced EGFR-MET dimerization in H1975L858R/T790M but induced dimer formation in H1975L858R with no effect in H1975WT. Our data suggests that MET inhibition by SGX523 and EGFR-MET heterodimerisation are determined by EGFR genotype. As tumor behaviour is modulated by this interaction, this could determine treatment efficacy.
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Adenocarcinoma/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3-stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083-96. ©2017 AACR.
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Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Linfocitos/inmunología , Linfocitos/patología , Receptores Nucleares Huérfanos/inmunología , Animales , Línea Celular Tumoral , Quimiocina CCL21/inmunología , Quimiocina CXCL13/inmunología , Femenino , Humanos , Inmunidad Innata , Metástasis Linfática , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Metástasis de la Neoplasia , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND: Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission. METHODS: We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction. RESULTS: Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status. CONCLUSIONS: The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.).
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Leucemia Mieloide Aguda/genética , Mutación , Proteínas Nucleares/genética , Secuencia de Bases , ADN de Neoplasias/análisis , Exoma , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Neoplasia Residual/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Pronóstico , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , TranscriptomaRESUMEN
INTRODUCTION: There is much interest in the use of noninvasive biomarkers in the management of lung cancer, particularly with respect to early diagnosis and monitoring the response to intervention. Cell-free tumor DNA in patients with cancer has been shown to hold potential as a noninvasive biomarker, in which the response to treatment may be evaluated using a blood test only. Multiple technologies have been suggested as being appropriate to measure cell-free tumor DNA. Microdroplet digital polymerase chain reaction (mdPCR) has a number of attributes that suggest it may be a useful tool for detecting clinically relevant genetic events. It offers precise and accurate quantitation of mutant alleles, including rare variants. METHODS: We evaluate the performance of mdPCR in the analysis of DNA extracted from reference standards, tumor biopsies, and patient plasma. RESULTS: The potential of mdPCR to detect clinically relevant mutations is demonstrated, in both formalin-fixed paraffin-embedded material and plasma. Furthermore, we show that mdPCR can be used to track changes in peripheral blood biomarkers in response to treatment and to detect the emergence of drug-resistant clones. CONCLUSIONS: MdPCR has potential as a tool to detect and quantify tumor-derived mutational events in cell-free DNA from patients with lung cancer.
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Adenocarcinoma/química , Adenocarcinoma/genética , Biomarcadores de Tumor/análisis , Análisis Mutacional de ADN/métodos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Reacción en Cadena de la Polimerasa/métodos , Adenocarcinoma/sangre , Adenocarcinoma del Pulmón , Biomarcadores de Tumor/sangre , Femenino , Humanos , Neoplasias Pulmonares/sangre , MasculinoRESUMEN
BACKGROUND: Squamous cell carcinoma of the lung is a common cancer with 95% mortality at 5â years. These cancers arise from preinvasive lesions, which have a natural history of development progressing through increasing severity of dysplasia to carcinoma in situ (CIS), and in some cases, ending in transformation to invasive carcinoma. Synchronous preinvasive lesions identified at autopsy have been previously shown to be clonally related. METHODS: Using autofluorescence bronchoscopy that allows visual observation of preinvasive lesions within the upper airways, together with molecular profiling of biopsies using gene sequencing and loss-of-heterozygosity analysis from both preinvasive lesions and from intervening normal tissue, we have monitored individual lesions longitudinally and documented their visual, histological and molecular relationship. RESULTS: We demonstrate that rather than forming a contiguous field of abnormal tissue, clonal CIS lesions can develop at multiple anatomically discrete sites over time. Further, we demonstrate that patients with CIS in the trachea have invariably had previous lesions that have migrated proximally, and in one case, into the other lung over a period of 12â years. CONCLUSIONS: Molecular information from these unique biopsies provides for the first time evidence that field cancerisation of the upper airways can occur through cell migration rather than via local contiguous cellular expansion as previously thought. Our findings urge a clinical strategy of ablating high-grade premalignant airway lesions with subsequent attentive surveillance for recurrence in the bronchial tree.
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Carcinoma in Situ , Carcinoma de Células Escamosas , Movimiento Celular , Neoplasias Pulmonares , Mutación , Lesiones Precancerosas , Neoplasias de la Tráquea , Adulto , Carcinoma in Situ/genética , Carcinoma in Situ/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Genes p53 , Humanos , Pérdida de Heterocigocidad , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Neoplasias de la Tráquea/genética , Neoplasias de la Tráquea/patologíaRESUMEN
Copy number alterations are frequently found in colorectal cancer (CRC), and recurrent gains or losses are likely to correspond to regions harbouring genes that promote or impede carcinogenesis respectively. Gain of chromosome 13q is common in CRC but, because the region of gain is frequently large, identification of the driver gene(s) has hitherto proved difficult. We used array comparative genomic hybridization to analyse 124 primary CRCs, demonstrating that 13q34 is a region of gain in 35% of CRCs, with focal gains in 4% and amplification in a further 1.6% of cases. To reduce the number of potential driver genes to consider, it was necessary to refine the boundaries of the narrowest copy number changes seen in this series and hence define the minimal copy region (MCR). This was performed using molecular copy-number counting, identifying IRS2 as the only complete gene, and therefore the likely driver oncogene, within the refined MCR. Analysis of available colorectal neoplasia data sets confirmed IRS2 gene gain as a common event. Furthermore, IRS2 protein and mRNA expression in colorectal neoplasia was assessed and was positively correlated with progression from normal through adenoma to carcinoma. In functional in vitro experiments, we demonstrate that deregulated expression of IRS2 activates the oncogenic PI3 kinase pathway and increases cell adhesion, both characteristics of invasive CRC cells. Together, these data identify IRS2 as a likely driver oncogene in the prevalent 13q34 region of gain/amplification and suggest that IRS2 over-expression may provide an additional mechanism of PI3 kinase pathway activation in CRC.
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Adenocarcinoma/genética , Cromosomas Humanos Par 13 , Neoplasias Colorrectales/genética , Dosificación de Gen/genética , Proteínas Sustrato del Receptor de Insulina/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Pólipos del Colon/genética , Pólipos del Colon/metabolismo , Pólipos del Colon/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Inestabilidad Genómica/genética , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , Transducción de Señal/genética , Células Tumorales CultivadasRESUMEN
The efficient delivery of personalized medicine is a key goal of healthcare over the next decade. It is likely that PCR strategies will play an important role in the delivery of this goal. Digital PCR has certain advantages over more traditional PCR protocols. In this article we will discuss the current status of digital PCR, highlighting its advantages and focusing on how it can be utilized in biomarker development and analysis, including the use of individualized biomarkers. We will explore recent developments in this field including examples of how digital PCR may integrate with next generation sequencing to deliver truly personalized medicine.
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Técnicas de Diagnóstico Molecular , Reacción en Cadena de la Polimerasa/métodos , Biomarcadores/metabolismo , Variaciones en el Número de Copia de ADN , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Farmacogenética , Medicina de Precisión , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
Rearrangements of the genome can be detected by microarray methods and massively parallel sequencing, which identify copy-number alterations and breakpoint junctions, but these techniques are poorly suited to reconstructing the long-range organization of rearranged chromosomes, for example, to distinguish between translocations and insertions. The single-DNA-molecule technique HAPPY mapping is a method for mapping normal genomes that should be able to analyse genome rearrangements, i.e. deviations from a known genome map, to assemble rearrangements into a long-range map. We applied HAPPY mapping to cancer cell lines to show that it could identify rearrangement of genomic segments, even in the presence of normal copies of the genome. We could distinguish a simple interstitial deletion from a copy-number loss at an inversion junction, and detect a known translocation. We could determine whether junctions detected by sequencing were on the same chromosome, by measuring their linkage to each other, and hence map the rearrangement. Finally, we mapped an uncharacterized reciprocal translocation in the T-47D breast cancer cell line to about 2 kb and hence cloned the translocation junctions. We conclude that HAPPY mapping is a versatile tool for determining the structure of rearrangements in the human genome.
Asunto(s)
Aberraciones Cromosómicas , Mapeo Cromosómico/métodos , Neoplasias/genética , Línea Celular Tumoral , Deleción Cromosómica , Variaciones en el Número de Copia de ADN , Ligamiento Genético , Genoma Humano , Humanos , Translocación GenéticaRESUMEN
The term 'field cancerization' is used to describe an epithelial surface that has a propensity to develop cancerous lesions, and in the case of the aerodigestive tract this is often as a result of chronic exposure to carcinogens in cigarette smoke 1, 2. The clinical endpoint is the development of multiple tumours, either simultaneously or sequentially in the same epithelial surface. The mechanisms underlying this process remain unclear; one possible explanation is that the epithelium is colonized by a clonal population of cells that are at increased risk of progression to cancer. We now address this possibility in a short case series, using individual genomic events as molecular biomarkers of clonality. In squamous lung cancer the most common genomic aberration is 3q amplification. We use a digital PCR technique to assess the clonal relationships between multiple biopsies in a longitudinal bronchoscopic study, using amplicon boundaries as markers of clonality. We demonstrate that clonality can readily be defined by these analyses and confirm that field cancerization occurs at a pre-invasive stage and that pre-invasive lesions and subsequent cancers are clonally related. We show that while the amplicon boundaries can be shared between different biopsies, the degree of 3q amplification and the internal structure of the 3q amplicon varies from lesion to lesion. Finally, in this small cohort, the degree of 3q amplification corresponds to clinical progression.