RESUMO
We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.
Assuntos
Metilação de DNA/efeitos dos fármacos , Interferon Tipo I/imunologia , Melanoma/imunologia , Melanoma/terapia , Animais , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilases de Modificação do DNA/antagonistas & inibidores , Retrovirus Endógenos/genética , Feminino , Humanos , Imunoterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , RNA de Cadeia Dupla/metabolismoRESUMO
PURPOSE: Although age is a recognized independent prognostic risk factor, its relative importance among molecular subtypes of Breast cancer (BCA) is not well documented. The aim of this study was to evaluate the prognostic role of age at diagnosis among different immunohistochemical subtypes of BCA. METHODS: We conducted a retrospective study of women with invasive BCA undergoing surgery at the Johns Hopkins Hospital, excluding patients presenting with stage IV breast cancer. Patients were stratified into three age groups: ≤ 40, 41-60, and > 60 years, and multivariable analysis was performed using Cox regression. We also identified differentially expressed genes (DEG) between age groups among BCA subtypes in the public TCGA dataset. Finally, we identified key driver genes within the DEGs using a weighted gene co-expression network analysis. RESULTS: Luminal A breast cancer patients had significantly lower 5 year disease-free survival (DFS) and distant metastasis-free survival (DMFS) in the ≤ 40 year age group compared to the 41-60 year age group, while the other molecular subtypes showed no significant association of DFS or DMFS with age. Age was a stronger outcome predictor than tumor grade or proliferative index in Luminal A BCA patients, but not other subtypes. BCA TCGA gene expression data were divided into two groups (≤ 40 years, > 40 years). We identified 374 DEGs in the Luminal A BCA subset, which were enriched in seven pathways and two modules of co-expressed genes. No age group-specific DEGs were identified in non-Luminal A subtypes. CONCLUSIONS: Age at diagnosis may be an important prognostic factor in Luminal A BCA.
Assuntos
Neoplasias da Mama/mortalidade , Adulto , Fatores Etários , Idoso , Neoplasias da Mama/química , Neoplasias da Mama/classificação , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Pessoa de Meia-Idade , Prognóstico , Receptor ErbB-2/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Estudos RetrospectivosRESUMO
Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.
Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Detecção Precoce de Câncer/métodos , Fezes/química , Proteínas HMGA/genética , Metaboloma , Polipose Adenomatosa do Colo/genética , Animais , Ácidos e Sais Biliares/metabolismo , Cromatografia Líquida de Alta Pressão , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismoRESUMO
Recent advances in molecular analyses of ovarian cancer have revealed a wealth of promising tumour-specific biomarkers, including protein, DNA mutations and methylation; however, reliably detecting such alterations at satisfactorily high sensitivity and specificity through low-cost methods remains challenging, especially in early-stage diseases. Here we present PapDREAM, a new approach that enables detection of rare, ovarian-cancer-specific aberrations of DNA methylation from routinely-collected cervical Pap specimens. The PapDREAM approach employs a microfluidic platform that performs highly parallelized digital high-resolution melt to analyze locus-specific DNA methylation patterns on a molecule-by-molecule basis at or near single CpG-site resolution at a fraction (< 1/10th) of the cost of next-generation sequencing techniques. We demonstrate the feasibility of the platform by assessing intermolecular heterogeneity of DNA methylation in a panel of methylation biomarker loci using DNA derived from Pap specimens obtained from a cohort of 43 women, including 18 cases with ovarian cancer and 25 cancer-free controls. PapDREAM leverages systematic multidimensional bioinformatic analyses of locus-specific methylation heterogeneity to improve upon Pap-specimen-based detection of ovarian cancer, demonstrating a clinical sensitivity of 50% at 99% specificity in detecting ovarian cancer cases with an area under the receiver operator curve of 0.90. We then establish a logistic regression model that could be used to identify high-risk patients for subsequent clinical follow-up and monitoring. The results of this study support the utility of PapDREAM as a simple, low-cost screening method with the potential to integrate with existing clinical workflows for early detection of ovarian cancer. KEY POINTS: We present a microfluidic platform for detection and analysis of rare, heterogeneously methylated DNA within Pap specimens towards detection of ovarian cancer. The platform achieves high sensitivity (fractions <0.00005%) at a suitably low cost (â¼$25) for routine screening applications. Furthermore, it provides molecule-by-molecule quantitative analysis to facilitate further study on the effect of heterogeneous methylation on cancer development.
Assuntos
Metilação de DNA , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/diagnóstico , Metilação de DNA/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Pessoa de Meia-Idade , DNA/genética , DNA/análise , Sensibilidade e Especificidade , Adulto , Teste de Papanicolaou/métodos , Teste de Papanicolaou/estatística & dados numéricosRESUMO
UNLABELLED: We have created a statistically grounded tool for determining the correlation of genomewide data with other datasets or known biological features, intended to guide biological exploration of high-dimensional datasets, rather than providing immediate answers. The software enables several biologically motivated approaches to these data and here we describe the rationale and implementation for each approach. Our models and statistics are implemented in an R package that efficiently calculates the spatial correlation between two sets of genomic intervals (data and/or annotated features), for use as a metric of functional interaction. The software handles any type of pointwise or interval data and instead of running analyses with predefined metrics, it computes the significance and direction of several types of spatial association; this is intended to suggest potentially relevant relationships between the datasets. AVAILABILITY AND IMPLEMENTATION: The package, GenometriCorr, can be freely downloaded at http://genometricorr.sourceforge.net/. Installation guidelines and examples are available from the sourceforge repository. The package is pending submission to Bioconductor.
Assuntos
Bases de Dados Genéticas , Genômica/métodos , Armazenamento e Recuperação da Informação , Modelos Genéticos , Modelos Estatísticos , Software , Animais , Cromossomos , Epigenômica , Loci Gênicos , Genoma , Humanos , Internet , RNA de Transferência/genética , Estatísticas não Paramétricas , Interface Usuário-ComputadorRESUMO
High mobility group A1 (HMGA1) chromatin regulators are upregulated in diverse tumors where they portend adverse outcomes, although how they function in cancer remains unclear. Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by dense desmoplastic stroma composed predominantly of cancer-associated fibroblasts and fibrotic tissue. Here, we uncover an epigenetic program whereby HMGA1 upregulates FGF19 during tumor progression and stroma formation. HMGA1 deficiency disrupts oncogenic properties in vitro while impairing tumor inception and progression in KPC mice and subcutaneous or orthotopic models of PDAC. RNA sequencing revealed HMGA1 transcriptional networks governing proliferation and tumor-stroma interactions, including the FGF19 gene. HMGA1 directly induces FGF19 expression and increases its protein secretion by recruiting active histone marks (H3K4me3, H3K27Ac). Surprisingly, disrupting FGF19 via gene silencing or the FGFR4 inhibitor BLU9931 recapitulates most phenotypes observed with HMGA1 deficiency, decreasing tumor growth and formation of a desmoplastic stroma in mouse models of PDAC. In human PDAC, overexpression of HMGA1 and FGF19 defines a subset of tumors with extremely poor outcomes. Our results reveal what we believe is a new paradigm whereby HMGA1 and FGF19 drive tumor progression and stroma formation, thus illuminating FGF19 as a rational therapeutic target for a molecularly defined PDAC subtype.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Carcinogênese/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Inativação Gênica , Proteína HMGA1a/genética , Proteína HMGA1a/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
Current molecular liquid biopsy assays to detect recurrence or monitor response to treatment require sophisticated technology, highly trained personnel, and a turnaround time of weeks. We describe the development and technical validation of an automated Liquid Biopsy for Breast Cancer Methylation (LBx-BCM) prototype, a DNA methylation detection cartridge assay that is simple to perform and quantitatively detects nine methylated markers within 4.5 h. LBx-BCM demonstrated high interassay reproducibility when analyzing exogenous methylated DNA (75-300 DNA copies) spiked into plasma (Coefficient of Variation, CV = 7.1 - 10.9%) and serum (CV = 19.1 - 36.1%). It also demonstrated high interuser reproducibility (Spearman r = 0.887, P < 0.0001) when samples of metastatic breast cancer (MBC, N = 11) and normal control (N = 4) were evaluated independently by two users. Analyses of interplatform reproducibility indicated very high concordance between LBx-BCM and the reference assay, cMethDNA, among 66 paired plasma samples (MBC N = 40, controls N = 26; Spearman r = 0.891; 95% CI = 0.825 - 0.933, P< 0.0001). LBx-BCM achieved a ROC AUC = 0.909 (95% CI = 0.836 - 0.982), 83% sensitivity and 92% specificity; cMethDNA achieved a ROC AUC = 0.896 (95% CI = 0.817 - 0.974), 83% sensitivity and 92% specificity in test set samples. The automated LBx-BCM cartridge prototype is fast, with performance levels equivalent to the highly sensitive, manual cMethDNA method. Future prospective clinical studies will evaluate LBx-BCM detection sensitivity and its ability to monitor therapeutic response during treatment for advanced breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/diagnóstico , Reprodutibilidade dos Testes , Metilação de DNA/genética , DNA , Biópsia LíquidaRESUMO
In 2002, Ker-Chau Li introduced the liquid association measure to characterize three-way interactions between genes, and developed a computationally efficient estimator that can be used to screen gene expression microarray data for such interactions. That study, and others published since then, have established the biological validity of the method, and clearly demonstrated it to be a useful tool for the analysis of genomic data sets. To build on this work, we have sought a parametric family of multivariate distributions with the flexibility to model the full range of trivariate dependencies encompassed by liquid association. Such a model could situate liquid association within a formal inferential theory. In this article, we describe such a family of distributions, a trivariate, conditional normal model having Gaussian univariate marginal distributions, and in fact including the trivariate Gaussian family as a special case. Perhaps the most interesting feature of the distribution is that the parameterization naturally parses the three-way dependence structure into a number of distinct, interpretable components. One of these components is very closely aligned to liquid association, and is developed as a measure we call modified liquid association. We develop two methods for estimating this quantity, and propose statistical tests for the existence of this type of dependence. We evaluate these inferential methods in a set of simulations and illustrate their use in the analysis of publicly available experimental data.
Assuntos
Biometria/métodos , Interpretação Estatística de Dados , Ligação Genética/genética , Predisposição Genética para Doença/epidemiologia , Predisposição Genética para Doença/genética , Variação Genética/genética , Modelos Estatísticos , Algoritmos , Simulação por Computador , Análise Discriminante , Métodos Epidemiológicos , Humanos , Estatística como AssuntoRESUMO
BACKGROUND: While molecular testing is a promising strategy for preoperative assessment of cytologically indeterminate thyroid nodules, thyroid fine needle aspiration biopsy (FNA) presents unique challenges for molecular assays, including contaminating peripheral blood mononuclear cells (PBMC) and variable numbers of evaluable epithelial thyroid cells. Moreover, the newly recognized entity, noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), has added an additional challenge to the currently available molecular diagnostic platforms. New diagnostic tools are still needed to correctly distinguish benign and malignant thyroid nodules preoperatively. METHODS: Twenty-two transcript splice variants from 12 genes we previously identified as discriminating benign from malignant thyroid nodules were characterized in 80 frozen thyroid tumors from 8 histological subtypes. Isoforms detectable in PBMC were excluded, and the 5 most discriminating isoforms were further validated by real-time quantitative PCR (qPCR) on intraoperative FNA samples from 59 malignant tumors, 55 benign nodules, and 23 NIFTP samples. The qPCR threshold cycle values for each transcript were normalized to the thyrocyte-specific thyroid peroxidase isoform 1 (TPO1) and z-transformed. Receiver operating characteristic (ROC) analyses of the composite transcript scores were used to evaluate classification of thyroid FNAs by the 5-gene isoform expression panel. RESULTS: A molecular signature was developed by combining expression levels of specific isoforms of CDH3, FNDC4, HMGA2, KLK7, and PLAG1. FNAs containing at least 12-36 thyrocytes were sufficient for this assay. The 5-gene composite score achieved an area under the ROC curve (AUC) of 0.86 for distinguishing malignant from benign nodules, with a specificity of 91%, sensitivity of 75%, negative predictive value of 91%, and positive predictive value of 74%. CONCLUSION: Our newly developed 5-gene isoform expression panel distinguishes benign from malignant thyroid tumors and, may help distinguish benign from malignant thyroid nodules in the context of the new NIFTP subtype.
Assuntos
Biomarcadores Tumorais/metabolismo , Leucócitos Mononucleares/patologia , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biópsia por Agulha Fina , Feminino , Seguimentos , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Prognóstico , Estudos Retrospectivos , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/cirurgia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/cirurgia , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/cirurgia , Adulto JovemRESUMO
BACKGROUND: Definitive diagnosis of primary central nervous system lymphoma (PCNSL) requires invasive surgical brain biopsy, causing treatment delays. In this paper, we identified and validated tumor-specific markers that can distinguish PCNSL from other CNS tumors in tissues. In a pilot study, we tested these newly identified markers in plasma. RESULTS: The Methylation Outlier Detector program was used to identify markers in TCGA dataset of 48 diffuse large B-cell lymphoma (DLBCL) and 656 glioblastomas and lower-grade gliomas. Eight methylated markers clearly distinguished DLBCL from gliomas. Marker performance was verified (ROC-AUC of ≥ 0.989) in samples from several GEO datasets (95 PCNSL; 2112 other primary CNS tumors of 11 types). Next, we developed a novel, efficient assay called Tailed Amplicon Multiplexed-Methylation-Specific PCR (TAM-MSP), which uses two of the methylation markers, cg0504 and SCG3 triplexed with ACTB. FFPE tissue sections (25 cases each) of PCNSL and eight types of other primary CNS tumors were analyzed using TAM-MSP. TAM-MSP distinguished PCNSL from the other primary CNS tumors with 100% accuracy (AUC = 1.00, 95% CI 0.95-1.00, P < 0.001). The TAM-MSP assay also detected as few as 5 copies of fully methylated plasma DNA spiked into 0.5 ml of healthy plasma. In a pilot study of plasma from 15 PCNSL, 5 other CNS tumors and 6 healthy individuals, methylation in cg0504 and SCG3 was detectable in 3/15 PCNSL samples (20%). CONCLUSION: The Methylation Outlier Detector program identified methylated markers that distinguish PCNSL from other CNS tumors with accuracy. The high level of accuracy achieved by these markers was validated in tissues by a novel method, TAM-MSP. These studies lay a strong foundation for a liquid biopsy-based test to detect PCNSL-specific circulating tumor DNA.
Assuntos
Neoplasias do Sistema Nervoso Central/diagnóstico , Neoplasias do Sistema Nervoso Central/genética , Metilação de DNA/genética , Epigenômica/métodos , Linfoma/diagnóstico , Linfoma/genética , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Preoperative staging of suspicious axillary lymph nodes (ALNs) allows patients to be triaged to ALN dissection or to sentinel lymph node biopsy (SLNB). Ultrasound-guided fine needle aspiration (FNA) and cytology of ALN is moderately sensitive but its clinical utility relies heavily on the cytologist's experience. We proposed that the 5-h automated GeneXpert system-based prototype breast cancer detection assay (BCDA) that quantitatively measures DNA methylation in ten tumor-specific gene markers could provide a facile, accurate test for detecting cancer in FNA of enlarged lymph nodes. We validated the assay in ALN-FNA samples from a prospective study of patients (N = 230) undergoing SLNB. In a blinded analysis of 218 evaluable LN-FNAs from 108 malignant and 110 benign LNs by histology, BCDA displayed a sensitivity of 90.7% and specificity of 99.1%, achieving an area under the ROC curve, AUC of 0.958 (95% CI: 0.928-0.989; P < 0.0001). Next, we conducted a study of archival FNAs of ipsilateral palpable LNs (malignant, N = 72, benign, N = 53 by cytology) collected in the outpatient setting prior to neoadjuvant chemotherapy (NAC). Using the ROC-threshold determined in the prospective study, compared to cytology, BCDA achieved a sensitivity of 94.4% and a specificity of 92.5% with a ROC-AUC = 0.977 (95% CI: 0.953-1.000; P < 0.0001). Our study shows that the automated assay detects cancer in suspicious lymph nodes with a high level of accuracy within 5 h. This cancer detection assay, scalable for analysis to scores of LN FNAs, could assist in determining eligibility of patients to different treatment regimens.
RESUMO
Genetic and epigenetic changes drive carcinogenesis, and their integrated analysis provides insights into mechanisms of cancer development. Computational methods have been developed to measure copy number variation (CNV) from methylation array data, including ChAMP-CNV, CN450K, and, introduced here, Epicopy. Using paired single nucleotide polymorphism (SNP) and methylation array data from the public The Cancer Genome Atlas repository, we optimized CNV calling and benchmarked the performance of these methods. We optimized the thresholds of all three methods and showed comparable performance across methods. Using Epicopy as a representative analysis of Illumina450K array, we show that Illumina450K-derived CNV methods achieve a sensitivity of 0.7 and a positive predictive value of 0.75 in identifying CNVs, which is similar to results achieved when comparing competing SNP microarray platforms with each other.
Assuntos
Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Metilação de DNA , Neoplasias/genética , Algoritmos , Epigênese Genética , Genoma Humano , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
PURPOSE: An unmet need in low-resource countries is an automated breast cancer detection assay to prioritize women who should undergo core breast biopsy and pathologic review. Therefore, we sought to identify and validate a panel of methylated DNA markers to discriminate between cancer and benign breast lesions using cells obtained by fine-needle aspiration (FNA).Experimental Design: Two case-control studies were conducted comparing cancer and benign breast tissue identified from clinical repositories in the United States, China, and South Africa for marker selection/training (N = 226) and testing (N = 246). Twenty-five methylated markers were assayed by Quantitative Multiplex-Methylation-Specific PCR (QM-MSP) to select and test a cancer-specific panel. Next, a pilot study was conducted on archival FNAs (49 benign, 24 invasive) from women with mammographically suspicious lesions using a newly developed, 5-hour, quantitative, automated cartridge system. We calculated sensitivity, specificity, and area under the receiver-operating characteristic curve (AUC) compared with histopathology for the marker panel. RESULTS: In the discovery cohort, 10 of 25 markers were selected that were highly methylated in breast cancer compared with benign tissues by QM-MSP. In the independent test cohort, this panel yielded an AUC of 0.937 (95% CI = 0.900-0.970). In the FNA pilot, we achieved an AUC of 0.960 (95% CI = 0.883-1.0) using the automated cartridge system. CONCLUSIONS: We developed and piloted a fast and accurate methylation marker-based automated cartridge system to detect breast cancer in FNA samples. This quick ancillary test has the potential to prioritize cancer over benign tissues for expedited pathologic evaluation in poorly resourced countries.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Metilação de DNA/genética , Neoplasias/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/isolamento & purificação , Biópsia por Agulha Fina , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Detecção Precoce de Câncer , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/patologia , Projetos Piloto , Regiões Promotoras Genéticas/genéticaRESUMO
PURPOSE: High-grade serous ovarian carcinoma (HGSOC) typically remains undiagnosed until advanced stages when peritoneal dissemination has already occurred. Here, we sought to identify HGSOC-specific alterations in DNA methylation and assess their potential to provide sensitive and specific detection of HGSOC at its earliest stages. EXPERIMENTAL DESIGN: MethylationEPIC genome-wide methylation analysis was performed on a discovery cohort comprising 23 HGSOC, 37 non-HGSOC malignant, and 36 histologically unremarkable gynecologic tissue samples. The resulting data were processed using selective bioinformatic criteria to identify regions of high-confidence HGSOC-specific differential methylation. Quantitative methylation-specific real-time PCR (qMSP) assays were then developed for 8 of the top-performing regions and analytically validated in a cohort of 90 tissue samples. Lastly, qMSP assays were used to assess and compare methylation in 30 laser-capture microdissected (LCM) fallopian tube epithelia samples obtained from cancer-free and serous tubal intraepithelial carcinoma (STIC) positive women. RESULTS: Bioinformatic selection identified 91 regions of robust, HGSOC-specific hypermethylation, 23 of which exhibited an area under the receiver-operator curve (AUC) value ≥ 0.9 in the discovery cohort. Seven of 8 top-performing regions demonstrated AUC values between 0.838 and 0.968 when analytically validated by qMSP in a 90-patient cohort. A panel of the 3 top-performing genes (c17orf64, IRX2, and TUBB6) was able to perfectly discriminate HGSOC (AUC 1.0). Hypermethylation within these loci was found exclusively in LCM fallopian tube epithelia from women with STIC lesions, but not in cancer-free fallopian tubes. CONCLUSIONS: A panel of methylation biomarkers can be used to accurately identify HGSOC, even at precursor stages of the disease.
Assuntos
Biomarcadores Tumorais , Metilação de DNA , Perfilação da Expressão Gênica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transcriptoma , Biópsia , Estudos de Casos e Controles , Biologia Computacional/métodos , Ilhas de CpG , Detecção Precoce de Câncer , Feminino , Loci Gênicos , Humanos , Imuno-Histoquímica , Gradação de Tumores , Estadiamento de Neoplasias , Curva ROCRESUMO
Purpose Epigenetic alterations measured in blood may help guide breast cancer treatment. The multisite prospective study TBCRC 005 was conducted to examine the ability of a novel panel of cell-free DNA methylation markers to predict survival outcomes in metastatic breast cancer (MBC) using a new quantitative multiplex assay (cMethDNA). Patients and Methods Ten genes were tested in duplicate serum samples from 141 women at baseline, at week 4, and at first restaging. A cumulative methylation index (CMI) was generated on the basis of six of the 10 genes tested. Methylation cut points were selected to maximize the log-rank statistic, and cross-validation was used to obtain unbiased point estimates. Logistic regression or Cox proportional hazard models were used to test associations between the CMI and progression-free survival (PFS), overall survival (OS), and disease status at first restaging. The added value of the CMI in predicting survival outcomes was evaluated and compared with circulating tumor cells (CellSearch). Results Median PFS and OS were significantly shorter in women with a high CMI (PFS, 2.1 months; OS, 12.3 months) versus a low CMI (PFS, 5.8 months; OS, 21.7 months). In multivariable models, among women with MBC, a high versus low CMI at week 4 was independently associated with worse PFS (hazard ratio, 1.79; 95% CI, 1.23 to 2.60; P = .002) and OS (hazard ratio, 1.75; 95% CI, 1.21 to 2.54; P = .003). An increase in the CMI from baseline to week 4 was associated with worse PFS ( P < .001) and progressive disease at first restaging ( P < .001). Week 4 CMI was a strong predictor of PFS, even in the presence of circulating tumor cells ( P = .004). Conclusion Methylation of this gene panel is a strong predictor of survival outcomes in MBC and may have clinical usefulness in risk stratification and disease monitoring.
Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Metilação de DNA , DNA de Neoplasias/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , DNA de Neoplasias/genética , Intervalo Livre de Doença , Feminino , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Metástase Neoplásica , Modelos de Riscos Proporcionais , Estudos Prospectivos , Taxa de SobrevidaRESUMO
High density oligonucleotide array technology is widely used in many areas of biomedical research for quantitative and highly parallel measurements of gene expression. Affymetrix GeneChip arrays are the most popular. In this technology each gene is typically represented by a set of 11-20 pairs of probes. In order to obtain expression measures it is necessary to summarize the probe level data. Using two extensive spike-in studies and a dilution study, we developed a set of tools for assessing the effectiveness of expression measures. We found that the performance of the current version of the default expression measure provided by Affymetrix Microarray Suite can be significantly improved by the use of probe level summaries derived from empirically motivated statistical models. In particular, improvements in the ability to detect differentially expressed genes are demonstrated.
Assuntos
Sondas de DNA/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sistema Nervoso Central/metabolismo , Sondas de DNA/normas , Perfilação da Expressão Gênica , Humanos , Fígado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/normas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , SoftwareRESUMO
We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Genoma Humano/genética , Genômica/métodos , Adolescente , Neoplasias do Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/terapia , Carcinoma Adrenocortical/patologia , Carcinoma Adrenocortical/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Metilação de DNA , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Avaliação de Resultados em Cuidados de Saúde , Prognóstico , Adulto JovemRESUMO
BACKGROUND: In this paper, we consider analytic methods for the integrated analysis of genomic DNA variation and mRNA expression (also named as eQTL data), to discover genetic networks that are associated with a complex trait of interest. Our focus is the systematic evaluation of the trade-off between network size and network search efficiency in the construction of these networks. RESULTS: We developed a modular approach to network construction, building from smaller networks to larger ones, thereby reducing the search space while including more variables in the analysis. The goal is achieving a lower computational cost while maintaining high confidence in the resulting networks. As demonstrated in our simulation results, networks built in this way have low node/edge false discovery rate (FDR) and high edge sensitivity comparing to greedy search. We further demonstrate our method in a data set of cellular responses to two chemotherapeutic agents: docetaxel and 5-fluorouracil (5-FU), and identify biologically plausible networks that might describe resistances to these drugs. CONCLUSION: In this study, we suggest that guided comprehensive searches for parsimonious networks should be considered as an alternative to greedy network searches.