RESUMEN
Variability in the accuracy of somatic mutation detection may affect the discovery of alterations and the therapeutic management of cancer patients. To address this issue, we developed a somatic mutation discovery approach based on machine learning that outperformed existing methods in identifying experimentally validated tumor alterations (sensitivity of 97% versus 90 to 99%; positive predictive value of 98% versus 34 to 92%). Analysis of paired tumor-normal exome data from 1368 TCGA (The Cancer Genome Atlas) samples using this method revealed concordance for 74% of mutation calls but also identified likely false-positive and false-negative changes in TCGA data, including in clinically actionable genes. Determination of high-quality somatic mutation calls improved tumor mutation load-based predictions of clinical outcome for melanoma and lung cancer patients previously treated with immune checkpoint inhibitors. Integration of high-quality machine learning mutation detection in clinical next-generation sequencing (NGS) analyses increased the accuracy of test results compared to other clinical sequencing analyses. These analyses provide an approach for improved identification of tumor-specific mutations and have important implications for research and clinical management of cancer patients.
Asunto(s)
Aprendizaje Automático , Mutación/genética , Exoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoterapia , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Programas Informáticos , Secuenciación del ExomaRESUMEN
Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Genes erbB-1 , Genes ras , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Inmunológicos/farmacología , Evolución Clonal , Neoplasias Colorrectales/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
PURPOSE: Targeted inhibition of EGFR with the mAbs cetuximab or panitumumab is a valuable treatment for RAS wild-type colorectal cancers. The efficacy of EGFR blockade is limited by the emergence of acquired resistance often attributed to secondary KRAS mutations. Remarkably, tumor biopsies from resistant patients show that only a fraction of the resilient cells carry KRAS mutations. We hypothesized that a paracrine cross-talk driven by the resistant subpopulation may provide in trans protection of surrounding sensitive cells. EXPERIMENTAL DESIGN: Conditioned medium assays and three-dimensional cocultures were used to assess paracrine networks between cetuximab-sensitive and -resistant cells. Production of EGFR ligands by cells sensitive to cetuximab and panitumumab was measured. The ability of recombinant EGFR ligands to protect sensitive cells from cetuximab was assessed. Biochemical activation of the EGFR signaling pathway was measured by Western blotting. RESULTS: Colorectal cancer cells sensitive to EGFR blockade can successfully grow despite cetuximab treatment when in the company of their resistant derivatives. Media conditioned by resistant cells protect sensitive parental cells from cetuximab. EGFR blockade triggers increased secretion of TGFα and amphiregulin. Increased secretion of ligands by resistant cells can sustain EGFR/ERK signaling in sensitive cells. CONCLUSIONS: Colorectal cancer cells that develop resistance to cetuximab and panitumumab secrete TGFα and amphiregulin, which protect the surrounding cells from EGFR blockade. This paracrine protective mechanism might be therapeutically exploitable.
Asunto(s)
Anfirregulina/metabolismo , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Comunicación Paracrina , Factor de Crecimiento Transformador alfa/metabolismo , Anfirregulina/farmacología , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cetuximab , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Medios de Cultivo Condicionados/farmacología , Genes ras , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mutación , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
Cetuximab and panitumumab are anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies used as therapies for metastatic colorectal cancer patients. Intrinsic mechanisms of resistance, such as RAS mutations, can prevent patients from having a response with clinical benefit. The clinical efficacy of EGFR targeted antibodies is limited by the development of acquired (secondary) resistance, which typically occurs within 3-12 months from the start of therapy. Preclinical models and analyses of clinical samples have uncovered some of the alterations that confer a selective advantage to tumor cells when under the pressure of anti-EGFR therapy. Molecular profiling of clinical specimens confirmed that genetic alterations of genes in the EGFR-RAS-RAF-MEK signaling pathway and of receptor tyrosine kinases are mechanisms of acquired resistance to anti-EGFR antibodies. The escape from anti-EGFR blockade appears to converge on the (re)activation of MEK-ERK or AKT as revealed in preclinical studies. Circulating tumor DNA and patient derived xenografts have proven useful tools to monitor patients for resistance to anti-EGFR therapy and test combination therapies to overcome or reverse resistance.
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Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Receptores ErbB/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas c-akt/metabolismo , Recto/efectos de los fármacos , Recto/metabolismo , Recto/patología , Proteínas ras/metabolismoRESUMEN
Defective DNA repair may contribute to early age and late stage at time of diagnosis and mutations in critical tumor suppressor genes, such as TP53 in breast cancer. Using DNA samples from 436 breast cancer cases (374 Caucasians and 62 African-Americans), we tested these associations with 18 non-synonymous single-nucleotide polymorphisms (nsSNPs) in four DNA repair pathways: (i) base excision repair: ADPRT V762A, APE1 D148E, XRCC1 R194W/R280H/R399Q and POLD1 R119H; (ii) double-strand break repair: NBS1 E185Q and XRCC3 T241M; (iii) mismatch repair: MLH1 I219V, MSH3 R940Q/T1036A and MSH6 G39E and (iv) nucleotide excision repair: ERCC2 D312N/K751Q, ERCC4 R415Q, ERCC5 D1104H and XPC A499V/K939Q. Younger age at diagnosis (<50) was associated with ERCC2 312 DN/NN genotypes [odds ratio (OR) = 1.76; 95% confidence interval (CI) = 1.10, 2.81] and NBS1 185 QQ genotype (OR = 3.09; 95% CI = 1.47, 6.49). The XPC 939 QQ genotype was associated with TP53 mutations (OR = 5.80; 95% CI = 2.23, 15.09). There was a significant trend associating younger age at diagnosis (<50) with increasing numbers of risk genotypes for ERCC2 312 DN/NN, MSH6 39 EE and NBS1 185 QQ (P(trend) < 0.001). A similar significant trend was also observed associating TP53 mutations with increasing numbers of risk genotypes for XRCC1 399 QQ, XPC 939 QQ, ERCC4 415 QQ and XPC 499 AA (P(trend) < 0.001). Our pilot data suggest that nsSNPs of multiple DNA repair pathways are associated with younger age at diagnosis and TP53 mutations in breast cancer and larger studies are warranted to further evaluate these associations.
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Neoplasias de la Mama/genética , Reparación del ADN , Genes p53 , Mutación , Polimorfismo Genético , Adulto , Factores de Edad , Anciano , Índice de Masa Corporal , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Femenino , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
DNA methylation, an essential regulator of transcription and chromatin structure, is established and maintained by the coordinated action of three DNA methyltransferases: DNMT1, DNMT3A and DNMT3B, and the inactive accessory factor DNMT3L. Disruptions in DNMT3B function are linked to carcinogenesis and genetic disease. DNMT3B is also highly alternatively spliced in a tissue- and disease-specific manner. The impact of intra-DNMT3 interactions and alternative splicing on the function of DNMT3 family members remains unclear. In the present work, we focused on DNMT3B. Using a panel of in vitro assays, we examined the consequences of DNMT3B splicing and mutations on its ability to bind DNA, interact with itself and other DNMT3's, and methylate DNA. Our results show that, while the C-terminal catalytic domain is critical for most DNMT3B functions, parts of the N-terminal region, including the PWWP domain, are also important. Alternative splicing and domain deletions also influence DNMT3B's cellular localization. Furthermore, our data reveal the existence of extensive DNMT3B self-interactions that differentially impact on its activity. Finally, we show that catalytically inactive isoforms of DNMT3B are capable of modulating the activity of DNMT3A-DNMT3L complexes. Our studies therefore suggest that seemingly 'inactive' DNMT3B isoforms may influence genomic methylation patterns in vivo.
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Empalme Alternativo , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Animales , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN Metiltransferasa 3A , Células HEK293 , Humanos , Síndromes de Inmunodeficiencia/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Mutación Puntual , Estructura Terciaria de Proteína , Eliminación de Secuencia , Secuencias Repetidas en Tándem , ADN Metiltransferasa 3BRESUMEN
DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the NH(2)-terminal regulatory domain. This variant, which we term DNMT3B3Delta5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Delta5. DNMT3B3Delta5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Delta5 versus DNMT3B3, revealing that DNMT3B3Delta5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Delta5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results show that DNMT3B3Delta5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B.
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ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Neoplasias/genética , Neoplasias/metabolismo , Células Madre Pluripotentes/metabolismo , Empalme Alternativo/genética , Animales , Secuencia de Bases/genética , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/aislamiento & purificación , Epigénesis Genética/genética , Exones/genética , Inestabilidad Genómica/genética , Humanos , Ratones , Células Madre Pluripotentes/citología , Isoformas de Proteínas , Estructura Terciaria de Proteína/genética , Ensayo de Tumor de Célula Madre , ADN Metiltransferasa 3BRESUMEN
DNA methylation is a heritable and stable epigenetic mark associated with transcriptional repression. Changes in the patterns and levels of global and regional DNA methylation regulate development and contribute directly to disease states such as cancer. Recent findings provide intriguing insights into the epigenetic crosstalk between DNA methylation, histone modifications, and small interfering RNAs in the control of cell development and carcinogenesis. In this review, we summarize the recent studies in DNA methylation primarily focusing on the interplay between different epigenetic modifications and their potential role in gene silencing in development and disease. Although the molecular mechanisms involved in the epigenetic crosstalk are not fully understood, unraveling their precise regulation is important not only for understanding the underpinnings of cellular development and cancer, but also for the design of clinically relevant and efficient therapeutics using stem cells and anticancer drugs that target tumor initiating cells.
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Metilación de ADN , Crecimiento y Desarrollo/genética , Neoplasias/genética , Animales , Diferenciación Celular , Epigénesis Genética , Silenciador del Gen , Humanos , Modelos Genéticos , Células Madre Pluripotentes/fisiología , Proteínas del Grupo Polycomb , Proteínas Represoras/metabolismoRESUMEN
Polymorphisms in the cytochrome P450 1B1 (CYP1B1) and glutathione S-transferase (GST) drug metabolic enzymes, which are responsible for metabolic activation/detoxification of estrogen and environmental carcinogens, were analyzed for their association with breast cancer risk in 541 cases and 635 controls from a North Carolina population. Each polymorphism, altering the catalytic function of their respective enzymes, was analyzed in Caucasian and African-American women. As reported in previous studies, individual polymorphisms did not significantly impact breast cancer risk in either Caucasian or African-American women. However, African-American women exhibited a trend towards a protective effect when they had at least one CYP1B1 119S allele (OR=0.53; 95% CI=0.20-1.40) and increased risk for those women harboring at least one CYP1B1 432V allele (OR=5.52; 95% CI=0.50-61.37). Stratified analyses demonstrated significant interactions in younger (age < or =60) Caucasian women with the CYP1B1 119SS genotype (OR=3.09; 95% CI=1.22-7.84) and younger African-American women with the GSTT1 null genotype (OR=4.07; 95% CI=1.12-14.80). A notable trend was also found in Caucasian women with a history of smoking and at least one valine allele at GSTP1 114 (OR=2.12; 95% CI=1.02-4.41). In Caucasian women, the combined GSTP1 105IV/VV and CYP1B1 119AA genotypes resulted in a near 2-fold increase in risk (OR=1.96; 95% CI=1.04-3.72) and the three way combination of GSTP1 105IV/VV, CYP1B1 119AS/SS and GSTT1 null genotypes resulted in an almost 4-fold increase in risk (OR=3.97; 95% CI=1.27-12.40). These results suggest the importance of estrogen/carcinogen metabolic enzymes in the etiology of breast cancer, especially in women before the age of 60, as well as preventative measures such as smoking cessation.
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Hidrocarburo de Aril Hidroxilasas/genética , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Gutatión-S-Transferasa pi/genética , Glutatión Transferasa/genética , Polimorfismo Genético , Adulto , Anciano , Alelos , Estudios de Casos y Controles , Citocromo P-450 CYP1B1 , Femenino , Humanos , Persona de Mediana Edad , Oportunidad RelativaRESUMEN
Genome-wide DNA methylation patterns are frequently deregulated in cancer. There is considerable interest in targeting the methylation machinery in tumor cells using nucleoside analogs of cytosine, such as 5-aza-2'-deoxycytidine (5-azadC). 5-azadC exerts its antitumor effects by reactivation of aberrantly hypermethylated growth regulatory genes and cytoxicity resulting from DNA damage. We sought to better characterize the DNA damage response of tumor cells to 5-azadC and the role of DNA methyltransferases 1 and 3B (DNMT1 and DNMT3B, respectively) in modulating this process. We demonstrate that 5-azadC treatment results in growth inhibition and G(2) arrest-hallmarks of a DNA damage response. 5-azadC treatment led to formation of DNA double-strand breaks, as monitored by formation of gamma-H2AX foci and comet assay, in an ATM (ataxia-telangiectasia mutated)-dependent manner, and this damage was repaired following drug removal. Further analysis revealed activation of key strand break repair proteins including ATM, ATR (ATM-Rad3-related), checkpoint kinase 1 (CHK1), BRCA1, NBS1, and RAD51 by Western blotting and immunofluorescence. Significantly, DNMT1-deficient cells demonstrated profound defects in these responses, including complete lack of gamma-H2AX induction and blunted p53 and CHK1 activation, while DNMT3B-deficient cells generally showed mild defects. We identified a novel interaction between DNMT1 and checkpoint kinase CHK1 and showed that the defective damage response in DNMT1-deficient cells is at least in part due to altered CHK1 subcellular localization. This study therefore greatly enhances our understanding of the mechanisms underlying 5-azadC cytotoxicity and reveals novel functions for DNMT1 as a component of the cellular response to DNA damage, which may help optimize patient responses to this agent in the future.
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Azacitidina/análogos & derivados , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Daño del ADN/genética , Metilación de ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genoma Humano/genética , Proteínas de la Ataxia Telangiectasia Mutada , Azacitidina/farmacología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , ADN/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Unión al ADN/metabolismo , Decitabina , Marcadores Genéticos , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Unión Proteica , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , ADN Metiltransferasa 3BRESUMEN
Polymorphisms in phase I and phase II enzymes may enhance the occurrence of mutations at critical tumor suppressor genes, such as p53, and increase breast cancer risk by either increasing the activation or detoxification of carcinogens and/or endogenous estrogens. We analyzed polymorphisms in CYP1B1, GSTM1, GSTT1, and GSTP1 and p53 mutations in 323 breast tumor samples. Approximately 11% of patients exhibited mutations in p53. Women with mutations had a significantly younger age of diagnosis (P = 0.01) and a greater incidence of tumors classified as stage II or higher (P = 0.002). More women with mutations had a history of smoking (55%) compared to women without mutations (39%). Although none of the genotypes alone were associated with p53 mutations, positive smoking history was associated with p53 mutations in women with the GSTM1 null allele [OR = 3.54; 95% CI = 0.97-12.90 P = 0.06] compared to women with the wild-type genotype and smoking history [OR = 0.62, 95% CI = 0.19-2.07], although this association did not reach statistical significance. To test for gene-gene interactions, our exploratory analysis in the Caucasian cases suggested that individuals with the combined GSTP1 105 VV, CYP1B1 432 LV/VV, and GSTM1 positive genotype were more likely to harbor mutations in p53 [OR = 4.94; 95% CI = 1.11-22.06]. Our results suggest that gene-smoking and gene-gene interactions may impact the prevalence of p53 mutations in breast tumors. Elucidating the etiology of breast cancer as a consequence of common genetic polymorphisms and the genotoxic effects of smoking will enable us to improve the design of prevention strategies, such as lifestyle modifications, in genetically susceptible subpopulations.
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Neoplasias de la Mama/genética , Genes p53 , Mutación , Polimorfismo Genético , Fumar/genética , Adulto , Anciano , Secuencia de Bases , Neoplasias de la Mama/enzimología , Cartilla de ADN , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Recessive dystrophic epidermolysis bullosa (RDEB) is an autosomal recessive disorder characterized by the loss of collagen type VII, an intrinsic component of the anchoring fibrils, which attach the epidermis to the dermis. Of the genetic blistering disorders, RDEB has the highest rate of morbidity and mortality, with morbidity arising from fusion of digits in a mitten-glove deformity and growth retardation associated with anemia. The leading cause of death in RDEB is cutaneous squamous cell carcinoma, which causes death through invasion and metastasis. In order to better understand the pathogenesis of these rare but aggressive squamous cell carcinoma (SCC), we analyzed them for mutations in p53 and loss of p16ink4a. Three tumors demonstrated mutations in the p53 tumor suppressor gene. We also analyzed SCC from patients with RDEB for the presence of p16ink4a hypermethylation, and found two tumors that have loss of p16ink4a through hypermethylation. This is the first description of specific abnormalities in tumor suppressor genes in RDEB associated SCC, and demonstrates that alterations in both p53 and p16ink4a can contribute to RDEB associated SCC.