RESUMEN
The transcription factor Nrf2 plays a crucial role in the anti-oxidative stress response, protection of DNA from injury and DNA repair mechanisms. Nrf2 activity reduces cancer initiation, but how Nrf2 affects whole-genome alterations upon carcinogenic stimulus remains unexplored. Although recent genome-wide analysis using next-generation sequencing revealed landscapes of nucleotide mutations and copy number alterations in various human cancers, genomic changes in murine cancer models have not been thoroughly examined. We elucidated the relationship between Nrf2 expression levels and whole exon mutation patterns using an ethyl-carbamate (urethane)-induced lung carcinogenesis model employing Nrf2-deficient and Keap1-kd mice, the latter of which express high levels of Nrf2. Exome analysis demonstrated that single nucleotide and trinucleotide mutation patterns and the Kras mutational signature differed significantly and were dependent on the expression level of Nrf2. The Nrf2-deficient tumors exhibited fewer copy number alterations relative to the Nrf2-wt and Keap1-kd tumors. The observed trend in genomic alterations likely prevented the Nrf2-deficient tumors from progressing into malignancy. For the first time, we present whole-exome sequencing results for chemically-induced lung tumors in the Nrf2 gain or loss of function mouse models. Our results demonstrate that different Nrf2 expression levels lead to distinct gene mutation patterns that underly different oncogenic mechanisms in each tumor genotype.
Asunto(s)
Neoplasias Pulmonares , Factor 2 Relacionado con NF-E2 , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/genética , Modelos Animales de Enfermedad , Genómica , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Mutación , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Nucleótidos/efectos adversos , Nucleótidos/metabolismo , UretanoRESUMEN
The neuroendocrine carcinoma of the gastrointestinal system (GIS-NEC) is a rare but highly malignant neoplasm. We analyzed 115 cases using whole-genome/exome sequencing, transcriptome sequencing, DNA methylation assays, and/or ATAC-seq and found GIS-NECs to be genetically distinct from neuroendocrine tumors (GIS-NET) in the same location. Clear genomic differences were also evident between pancreatic NECs (Panc-NEC) and nonpancreatic GIS-NECs (Nonpanc-NEC). Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic features. Alterations in TP53 and RB1 proved common in GIS-NECs, and most Nonpanc-NECs with intact RB1 demonstrated mutually exclusive amplification of CCNE1 or MYC. Alterations of the Notch gene family were characteristic of Nonpanc-NECs. Transcription factors for neuroendocrine differentiation, especially the SOX2 gene, appeared overexpressed in most GIS-NECs due to hypermethylation of the promoter region. This first comprehensive study of genomic alterations in GIS-NECs uncovered several key biological processes underlying genesis of this very lethal form of cancer. SIGNIFICANCE: GIS-NECs are genetically distinct from GIS-NETs. GIS-NECs arising in different organs show similar histopathologic features and share some genomic features, but considerable differences exist between Panc-NECs and Nonpanc-NECs. In addition, Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic and epigenomic features. This article is highlighted in the In This Issue feature, p. 587.
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Carcinoma Neuroendocrino , Tumores Neuroendocrinos , Carcinoma Neuroendocrino/genética , Exoma , Humanos , Recién Nacido , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/patología , Páncreas/patología , Secuenciación del ExomaRESUMEN
BACKGROUND: Intra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. Mouse models that recapitulate cancer development are useful for controlled serial time sampling. RESULTS: We performed single-cell exome and transcriptome sequencing of 200 cells to investigate how ITH is generated in a mouse colorectal cancer model. In the model, a single normal intestinal cell is grown into organoids that mimic the intestinal crypt structure. Upon RNAi-mediated downregulation of a tumor suppressor gene APC, the transduced organoids were serially transplanted into mice to allow exposure to in vivo microenvironments, which play relevant roles in cancer development. The ITH of the transcriptome increased after the transplantation, while that of the exome decreased. Mutations generated during organoid culture did not greatly change at the bulk-cell level upon the transplantation. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes after the transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages. CONCLUSIONS: This study revealed an evolutionary dynamics of single-cell RNA and DNA heterogeneity in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.
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Neoplasias Colorrectales , Animales , Neoplasias Colorrectales/genética , ADN , Exoma/genética , Heterogeneidad Genética , Ratones , ARN , Análisis de Secuencia de ARN , Microambiente TumoralRESUMEN
Next-generation sequencing (NGS) of tumor tissue (ie, clinical sequencing) can guide clinical management by providing information about actionable gene aberrations that have diagnostic and therapeutic significance. Here, we undertook a hospital-based prospective study (TOP-GEAR project, 2nd stage) to investigate the feasibility and utility of NGS-based analysis of 114 cancer-associated genes (the NCC Oncopanel test). We examined 230 cases (comprising more than 30 tumor types) of advanced solid tumors, all of which were matched with nontumor samples. Gene profiling data were obtained for 187 cases (81.3%), 111 (59.4%) of which harbored actionable gene aberrations according to the Clinical Practice Guidelines for Next Generation Sequencing in Cancer Diagnosis and Treatment (Edition 1.0) issued by 3 major Japanese cancer-related societies. Twenty-five (13.3%) cases have since received molecular-targeted therapy according to their gene aberrations. These results indicate the utility of tumor-profiling multiplex gene panel testing in a clinical setting in Japan. This study is registered with UMIN Clinical Trials Registry (UMIN 000011141).
Asunto(s)
Biomarcadores de Tumor , Perfilación de la Expresión Génica , Genes Relacionados con las Neoplasias , Neoplasias/genética , Adulto , Anciano , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN , Femenino , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Mutación , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Pronóstico , Resultado del TratamientoRESUMEN
Advanced cancer genomics technologies are now being employed in clinical sequencing, where next-generation sequencers are used to simultaneously identify multiple types of DNA alterations for prescription of molecularly targeted drugs. However, no computational tool is available to accurately detect DNA alterations in formalin-fixed paraffin-embedded (FFPE) samples commonly used in hospitals. Here, we developed a computational tool tailored to the detection of single nucleotide variations, indels, fusions, and copy number alterations in FFPE samples. Elaborated multilayer noise filters reduced the inherent noise while maintaining high sensitivity, as evaluated in tumor-unmatched normal samples using orthogonal technologies. This tool, cisCall, should facilitate clinical sequencing in everyday diagnostics. It is available at https://www.ciscall.org .
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Biología Computacional/métodos , Variaciones en el Número de Copia de ADN/genética , Formaldehído/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Adhesión en Parafina , Línea Celular Tumoral , HumanosRESUMEN
Lynch syndrome is a cancer predisposition syndrome caused by germline mutations in mismatch repair (MMR) genes. MMR deficiency is a ubiquitous feature of Lynch syndrome-associated colorectal adenocarcinomas; however, it remains unclear when the MMR-deficient phenotype is acquired during tumorigenesis. To probe this issue, the present study examined genetic alterations and MMR statuses in Lynch syndrome-associated colorectal adenomas and adenocarcinomas, in comparison with sporadic adenomas. Among the Lynch syndrome-associated colorectal tumors, 68 of 86 adenomas (79%) and all adenocarcinomas were MMR-deficient, whereas all the sporadic adenomas were MMR-proficient, as determined by microsatellite instability testing and immunohistochemistry for MMR proteins. Sequencing analyses identified APC or CTNNB1 mutations in the majority of sporadic adenomas (58/84, 69%) and MMR-proficient Lynch syndrome-associated adenomas (13/18, 72%). However, MMR-deficient Lynch syndrome-associated adenomas had less APC or CTNNB1 mutations (25/68, 37%) and frequent frameshift RNF43 mutations involving mononucleotide repeats (45/68, 66%). Furthermore, frameshift mutations affecting repeat sequences constituted 14 of 26 APC mutations (54%) in MMR-deficient adenomas whereas these frameshift mutations were rare in MMR-proficient adenomas in patients with Lynch syndrome (1/12, 8%) and in sporadic adenomas (3/52, 6%). Lynch syndrome-associated adenocarcinomas exhibited mutation profiles similar to those of MMR-deficient adenomas. Considering that WNT pathway activation sufficiently drives colorectal adenoma formation, the distinct mutation profiles of WNT pathway genes in Lynch syndrome-associated adenomas suggest that MMR deficiency commonly precedes adenoma formation.
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Adenoma/genética , Carcinogénesis/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Reparación de la Incompatibilidad de ADN/genética , Adenocarcinoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Vía de Señalización Wnt/genética , Adulto JovenRESUMEN
Gastrointestinal stromal tumors represent the most common mesenchymal tumor of the digestive tract, driven by gain-of-function mutations in KIT. Despite its proven benefits, half of the patients treated with imatinib show disease progression within 2 years due to secondary resistance mutations in KIT. It remains unclear how the genomic and transcriptomic features change during the acquisition of imatinib resistance. Here, we performed exome sequencing and microarray transcription analysis for four imatinib-resistant cell lines and one cell line briefly exposed to imatinib. We also performed exome sequencing of clinical tumor samples. The cell line briefly exposed to imatinib exhibited few single-nucleotide variants and copy-number alterations, but showed marked upregulation of genes related to detoxification and downregulation of genes involved in cell cycle progression. Meanwhile, resistant cell lines harbored numerous genomic changes: amplified genes related to detoxification and deleted genes with cyclin-dependent kinase activity. Some variants in the resistant samples were traced back to the drug-sensitive samples, indicating the presence of ancestral subpopulations. The subpopulations carried variants associated with cell death. Pre-existing cancer cells with genetic alterations promoting apoptosis resistance may serve as a basis whereby cancer cells with critical mutations, such as secondary KIT mutations, can establish full imatinib resistance. © 2017 The Authors Genes, Chromosomes and Cancer Published by Wiley Periodicals, Inc.
Asunto(s)
Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos/genética , Tumores del Estroma Gastrointestinal/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genómica/métodos , Mesilato de Imatinib/farmacología , Antineoplásicos/farmacología , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/patología , Humanos , Mutación/genética , Pronóstico , Proteínas Proto-Oncogénicas c-kit/genética , Células Tumorales CultivadasRESUMEN
Mucinous gastric carcinoma (MGC) is a unique subtype of gastric cancer with a poor survival outcome. Comprehensive molecular profiles and putative therapeutic targets of MGC remain undetermined. We subjected 16 tumour-normal tissue pairs to whole-exome sequencing (WES) and an expanded set of 52 tumour-normal tissue pairs to subsequent targeted sequencing. The latter focused on 114 genes identified by WES. Twenty-two histologically differentiated MGCs (D-MGCs) and 46 undifferentiated MGCs (U-MGCs) were analysed. Chromatin modifier genes, including ARID1A (21%), MLL2 (19%), MLL3 (15%), and KDM6A (7%), were frequently mutated (47%) in MGC. We also identified mutations in potential therapeutic target genes, including MTOR (9%), BRCA2 (9%), BRCA1 (7%), and ERBB3 (6%). RHOA mutation was detected only in 4% of U-MGCs and in no D-MGCs. MYH9 was recurrently (13%) mutated in MGC, with all these being of the U-MGC subtype (p = 0.023). Three U-MGCs harboured MYH9 nonsense mutations. MYH9 knockdown enhanced cell migration and induced intracytoplasmic mucin and cellular elongation. BCOR mutation was associated with improved survival. In U-MGCs, the MLH1 expression status and combined mutation status (TP53/BCL11B or TP53/MLL2) were prognostic factors. A comparative analysis of driver genes revealed that the mutation profile of D-MGC was similar to that of intestinal-type gastric cancer, whereas U-MGC was a distinct entity, harbouring a different mutational profile to intestinal- and diffuse-type gastric cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Adenocarcinoma Mucinoso/genética , Mutación , Neoplasias Gástricas/genética , Adenocarcinoma Mucinoso/patología , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Gástricas/patologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumors. In the present study, we determined the mutational status of KRAS in plasma cfDNA using multiplex picoliter-droplet digital PCR in 259 patients with PDAC. We constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples in 48 patients who had ≥1% mutant allele frequencies of KRAS in plasma cfDNA. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA. We also analyzed somatic copy number alterations based on the targeted sequencing data using our in-house algorithm, and potentially targetable amplifications were detected. Assessment of mutations and copy number alterations in plasma cfDNA may provide a prognostic and diagnostic tool to assist decisions regarding optimal therapeutic strategies for PDAC patients.