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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.
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Metilación de ADN , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/genética , Neoplasias Ováricas/diagnóstico , Metilación de ADN/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisis , Persona de Mediana Edad , ADN/genética , ADN/análisis , Sensibilidad y Especificidad , Adulto , Prueba de Papanicolaou/métodos , Prueba de Papanicolaou/estadística & datos numéricosRESUMEN
Background: Cancer stem cells (CSCs) have emerged as pivotal players in tumorigenesis, disease progression, and resistance to therapies. Objective: This comprehensive review delves into the intricate relationship between CSCs and the cell-of-origin in diverse cancer types. Design: Comprehensive review of thematically-relevant literature. Methods: We explore the underlying molecular mechanisms that drive the conversion of normal cells into CSCs and the impact of the cell-of-origin on CSC properties, tumor initiation, and therapeutic responses. Moreover, we discuss potential therapeutic interventions targeting CSCs based on their distinct cell-of-origin characteristics. Results: Accruing evidence suggest that the cell-of-origin, the cell type from which the tumor originates, plays a crucial role in determining the properties of CSCs and their contribution to tumor heterogeneity. Conclusion: By providing critical insights into the complex interplay between CSCs and their cellular origins, this article aims to enhance our understanding of cancer biology and pave the way for more effective and personalized cancer treatments.
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There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Detección Precoz del Cáncer , Metilación de ADN/genética , Reacción en Cadena de la PolimerasaRESUMEN
With a 5-year survival rate of less than 50%, ovarian high-grade serous carcinoma (HGSC) is one of the most highly aggressive gynecological malignancies affecting women today. The high mortality rate of HGSC is largely attributable to delays in diagnosis, as most patients remain undiagnosed until the late stages of -disease. There are currently no recommended screening tests for ovarian cancer and there thus remains an urgent need for new diagnostic methods, particularly those that can detect the disease at early stages when clinical intervention remains effective. While diagnostics for ovarian cancer share many of the same technical hurdles as for other cancer types, the low prevalence of the disease in the general population, coupled with a notable lack of sensitive and specific biomarkers, have made the development of a clinically useful screening strategy particularly challenging. Here, we present a detailed review of the overall landscape of ovarian cancer diagnostics, with emphasis on emerging methods that employ novel protein, genetic, epigenetic and imaging-based biomarkers and/or advanced diagnostic technologies for the noninvasive detection of HGSC, particularly in women at high risk due to germline mutations such as BRCA1/2. Lastly, we discuss the translational potential of these approaches for achieving a clinically implementable solution for screening and diagnostics of early-stage ovarian cancer as a means of ultimately improving patient outcomes in both the general and high-risk populations.
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Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA methylation require sample DNA to be extracted, purified and chemically-processed through bisulfite conversion before downstream analysis. While some automated solutions exist for each of these individual process steps, a fully integrated solution for accomplishing the entire process in a high-throughput manner has yet to be demonstrated. Thus, sample processing methods still require numerous manual steps that may reduce sample throughput and precision, while increasing the risk of contamination and human error. In this work, we present an integrated, automated solution for performing the entire sample preparation process, including DNA extraction, purification, bisulfite conversion and PCR plate preparation within in an enclosed environment. The method employs silica-coated magnetic particles that eliminate the need for a centrifuge or vacuum manifold, thereby reducing the complexity and cost of the required automation platform. Toward this end, we also compare commercial DNA extraction and bisulfite conversion kits to identify a protocol suitable for automation to significantly improve genomic and bisulfite-treated DNA yields over manufacturer protocols. Overall, this research demonstrated development of an automated protocol that offers the ability to generate high-quality, bisulfite-treated DNA samples in a high-throughput and clean environment with minimal user intervention and comparable yields to manual processing.
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Metilación de ADN , ADN , ADN/análisis , Humanos , Reacción en Cadena de la Polimerasa , Manejo de EspecímenesRESUMEN
Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations. MSP requires multiple sample preparation steps including protein digestion, DNA isolation, and bisulfite conversion prior to detection. In this work, we present a streamlined assay platform and instrumentation for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples through the use of droplet magnetofluidic principles. In conjunction with this platform, we present a streamlined protocol for solid-phase DNA extraction from cells and bisulfite conversion of genomic DNA, minimizing the processing steps and reagent volume for implementation on a compact assay platform.
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Metilación de ADN , ADN , Bioensayo , ADN/análisis , ADN/genética , Procesamiento Proteico-Postraduccional , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity. Here, we describe the development of a rapid and sensitive magnetofluidic immuno-PCR platform that can address the current gap in point-of-care serological testing for COVID-19. Our magnetofluidic immuno-PCR platform automates a magnetic bead-based, single-binding, and one-wash immuno-PCR assay in a palm-sized magnetofluidic device and delivers results in â¼30 min. In the device, a programmable magnetic arm attracts and transports magnetically-captured antibodies through assay reagents pre-loaded in a companion plastic cartridge, and a miniaturized thermocycler and a fluorescence detector perform immuno-PCR to detect the antibodies. We evaluated our magnetofluidic immuno-PCR with 108 clinical serum/plasma samples and achieved 93.8% (45/48) sensitivity and 98.3% (59/60) specificity, demonstrating its potential as a rapid and sensitive point-of-care serological test for COVID-19.
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Técnicas Biosensibles , COVID-19 , Anticuerpos Antivirales , Prueba Serológica para COVID-19 , Prueba de COVID-19 , Humanos , Sistemas de Atención de Punto , Pruebas en el Punto de Atención , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA. METHODS: Hierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann-Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy. RESULTS: In the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue. CONCLUSIONS: We identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries.
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Biomarcadores de Tumor/análisis , Neoplasias del Colon/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Metilación de ADN , Femenino , Pruebas Genéticas/instrumentación , Pruebas Genéticas/métodos , Pruebas Genéticas/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana EdadRESUMEN
We previously reported CHFR methylation in a subset of colorectal cancer (CRC; â¼30%) with high concordance with microsatellite instability (MSI). We also showed that CHFR methylation predicted for sensitivity to docetaxel, whereas the MSI-high phenotypes were sensitive to gemcitabine. We hypothesized that this subset of patients with CRC would be selectively sensitive to gemcitabine and docetaxel. We enrolled a Phase 2 trial of gemcitabine and docetaxel in patients with MSI-high and/or CHFR methylated CRC. The primary objective was Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 response rate. Enrolled patients were treated with gemcitabine 800 mg/m2 on days 1 and 8 and docetaxel 70 mg/m2 on day 8 of each 21-day cycle. A total of 6 patients with CHFR-methylated, MSI-high CRC were enrolled from September 2012 to August 2016. The study was closed in September of 2017 due to poor accrual prior to reaching the first interim assessment of response rate, which would have occurred at 10 patients. No RECIST criteria tumor responses were observed, with 3 patients (50%) having stable disease as best response, 1 lasting more than 9 months. Median progression-free survival (PFS) was 1.79 months (95% confidence interval [CI] = 1.28, not available [NA]) and median overall survival (OS) was 15.67 months (95% CI = 4.24, NA). Common grade 3 toxicities were lymphopenia (67%), leukopenia (33%), and anemia (33%). Although negative, this study establishes a proof-of-concept for the implementation of epigenetic biomarkers (CHFR methylation/MSI) as inclusion criteria in a prospective clinical trial to optimize combinatorial strategies in the era of personalized medicine. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? CHFR silencing via DNA methylation has been suggested to be predictive of taxane sensitivity in diverse tumors. The frequent association of CHFR methylation with microsatellite instability (MSI) suggested a possible combination therapy with gemcitabine, because the MSI phenotype may result in sensitivity to nucleoside analogues. WHAT QUESTION DID THIS STUDY ADDRESS? We hypothesized that metastatic colorectal cancer (mCRC), which have CHFR methylation and MSI phenotype were sensitive to gemcitabine and docetaxel, and have designed this Phase 2 trial in biomarker-selected mCRC to test this prediction. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? The study enrolled a molecularly defined subgroup of patients with colorectal cancer (CRC) and showed that the combination is safe in this population. Nevertheless, due to poor enrollment and early termination, no conclusions on the primary and secondary end points could be made. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? This study supports the feasibility of implementing DNA methylation markers in a prospective clinical trial and further efforts toward their application as predictive biomarkers for therapeutic agents in defined subsets of patients are warranted.
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Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de Ciclo Celular/genética , Neoplasias Colorrectales/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Docetaxel/farmacología , Proteínas de Neoplasias/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Ubiquitina-Proteína Ligasas/genética , Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/secundario , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Toma de Decisiones Clínicas , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Metilación de ADN , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Docetaxel/uso terapéutico , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Femenino , Humanos , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Supervivencia sin Progresión , Regiones Promotoras Genéticas , Prueba de Estudio Conceptual , Criterios de Evaluación de Respuesta en Tumores Sólidos , GemcitabinaRESUMEN
A role for cancer cell epithelial-to-mesenchymal transition (EMT) in cancer is well established. Here, we show that, in addition to cancer cell EMT, ovarian cancer cell metastasis relies on an epigenomic mesenchymal-to-epithelial transition (MET) in host mesenchymal stem cells (MSCs). These reprogrammed MSCs, termed carcinoma-associated MSCs (CA-MSCs), acquire pro-tumorigenic functions and directly bind cancer cells to serve as a metastatic driver/chaperone. Cancer cells induce this epigenomic MET characterized by enhancer-enriched DNA hypermethylation, altered chromatin accessibility, and differential histone modifications. This phenomenon appears clinically relevant, as CA-MSC MET is highly correlated with patient survival. Mechanistically, mirroring MET observed in development, MET in CA-MSCs is mediated by WT1 and EZH2. Importantly, EZH2 inhibitors, which are clinically available, significantly inhibited CA-MSC-mediated metastasis in mouse models of ovarian cancer.
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Transición Epitelial-Mesenquimal/genética , Metástasis de la Neoplasia/genética , Neoplasias Ováricas/genética , Animales , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Epigenoma/genética , Epigenómica/métodos , Femenino , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos NOD , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Cultivo Primario de Células , Transducción de Señal/genética , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMEN
PURPOSE: The current paradigm in the development of high-grade serous ovarian carcinoma (HGSC) proposes that the majority of HGSCs arise from precursor serous tubal intraepithelial carcinoma (STIC) lesions of the fallopian tube. Here we survey genome-wide methylation in HGSC precursor lesions to identify genomic regions that exhibit high-specificity differential hypermethylation for potential use as biomarkers for detecting STIC and HGSC at stages when curative intervention likely remains feasible. EXPERIMENTAL DESIGN: We first identified quality control criteria for performing reliable methylomic analysis of DNA-limited tubal precursor lesions with the Illumina Infinium MethylationEPIC array. We then used this platform to compare genome-wide methylation among 12 STICs with paired adjacent-normal epithelia, one p53 signature lesion and two samples of concurrent HGSC. The resulting methylomic data were analyzed by unsupervised hierarchical clustering and multidimensional analysis. Regions of high-confidence STIC-specific differential hypermethylation were identified using selective bioinformatic criteria and compared with published MethylationEPIC data from 23 HGSC tumors and 11 healthy fallopian tube mucosae. RESULTS: Unsupervised analysis showed that STICs largely clustered with HGSCs, but were clearly distinct from adjacent-normal fallopian tube epithelia. Forty-two genomic regions exhibited high-confidence STIC-specific differential hypermethylation, of which 17 (40.5%) directly overlapped with HGSC-specific differentially methylated regions. Methylation at these shared loci was able to completely distinguish STIC and HGSC samples from normal and adjacent-normal specimens. CONCLUSIONS: Our results suggest that most STICs are epigenetically similar to HGSCs and share regions of differential hypermethylation that warrant further evaluation for potential use as biomarkers for early detection of ovarian HGSC.See related commentary by Ishak and De Carvalho, p. 6083.
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Biomarcadores de Tumor/genética , Cistadenocarcinoma Seroso/patología , Metilación de ADN , Neoplasias de las Trompas Uterinas/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/patología , Lesiones Precancerosas/patología , Adulto , Anciano , Estudios de Casos y Controles , Cistadenocarcinoma Seroso/genética , Neoplasias de las Trompas Uterinas/genética , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/genética , Lesiones Precancerosas/genética , Pronóstico , Estudios RetrospectivosRESUMEN
BACKGROUND: Despite improvements in cancer management, most pancreatic cancers are still diagnosed at an advanced stage. We have recently identified promoter DNA methylation of the genes ADAMTS1 and BNC1 as potential blood biomarkers of pancreas cancer. In this study, we validate this biomarker panel in peripheral cell-free tumor DNA of patients with pancreatic cancer. RESULTS: Sensitivity and specificity for each gene are as follows: ADAMTS1 87.2% and 95.8% (AUC = 0.91; 95% CI 0.71-0.86) and BNC1 64.1% and 93.7% (AUC = 0.79; 95% CI 0.63-0.78). When using methylation of either gene as a combination panel, sensitivity increases to 97.3% and specificity to 91.6% (AUC = 0.95; 95% CI 0.77-0.90). Adding pre-operative CA 19-9 values to the combined two-gene methylation panel did not improve sensitivity. Methylation of ADAMTS1 was found to be positive in 87.5% (7/8) of stage I, 77.8% (7/9) of stage IIA, and 90% (18/20) of stage IIB disease. Similarly, BNC1 was positive in 62.5% (5/8) of stage I patients, 55.6% (5/9) of stage IIA, and 65% (13/20) of patients with stage IIB disease. The two-gene panel (ADAMTS1 and/or BNC1) was positive in 100% (8/8) of stage I, 88.9% (8/9) of stage IIA, and 100% (20/20) of stage IIB disease. The sensitivity and specificity of the two-gene panel for localized pancreatic cancer (stages I and II), where the cancer is eligible for surgical resection with curative potential, was 94.8% and 91.6% respectively. Additionally, the two-gene panel exhibited an AUC of 0.95 (95% CI 0.90-0.98) compared to 57.1% for CA 19-9 alone. CONCLUSION: The methylation status of ADAMTS1 and BNC1 in cfDNA shows promise for detecting pancreatic cancer during the early stages when curative resection of the tumor is still possible. This minimally invasive blood-based biomarker panel could be used as a promising tool for diagnosis and screening in a select subset of high-risk populations.
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Proteína ADAMTS1/genética , Metilación de ADN , Proteínas de Unión al ADN/genética , Neoplasias Pancreáticas/diagnóstico , Factores de Transcripción/genética , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Ácidos Nucleicos Libres de Células/genética , Detección Precoz del Cáncer , Epigénesis Genética , Femenino , Humanos , Masculino , Estadificación de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas , Sensibilidad y EspecificidadRESUMEN
Liquid biopsies contain a treasure of genetic and epigenetic biomarkers that contain information for the detection and monitoring of human disease. DNA methylation is an epigenetic modification that is critical to determining cellular phenotype and often becomes altered in many disease states. In cancer, aberrant DNA methylation contributes to carcinogenesis and can profoundly affect tumor evolution, metastatic potential, and resistance to therapeutic intervention. However, current technologies are not well-suited for quantitative assessment of DNA methylation heterogeneity, especially in challenging samples such as liquid biopsies with low DNA input and high background. We present a multilayer microfluidic device for quantitative analysis of DNA methylation by digital PCR and high resolution melt (HRM). The multilayer design facilitates high-density array digitization aimed at maximizing sample loading efficiency. The platform achieves highly parallelized digital PCR-HRM-based discrimination of rare heterogeneous DNA methylation as low as 0.0001% methylated/unmethylated molecules of a classic tumor suppressor gene, CDKN2A (p14ARF).
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Metilación de ADN , Dispositivos Laboratorio en un Chip , Reacción en Cadena de la Polimerasa/instrumentación , Alelos , Biopsia , Epigénesis Genética , Genes p16 , Humanos , Desnaturalización de Ácido NucleicoRESUMEN
There is growing evidence that most high-grade serous ovarian carcinomas likely arise from local dissemination of precursor lesions of the fallopian tube. Evolution of these lesions from early p53 signatures to latter-stage, serous tubal intraepithelial carcinomas (STICs) is characterized by cytologic atypia, accumulation of somatic mutations, and genomic instability, the etiologies of which remain unclear. Long interspersed element 1 (LINE-1) retrotransposon is expressed in many carcinomas, including high-grade serous ovarian carcinoma, where it contributes to genomic instability; however, the timing of LINE-1 activation during this evolution has yet to be elucidated. In this study, we assessed LINE-1 open reading frame 1 protein expression in 12 p53 signature lesions, 32 STICs, and 112 various types of ovarian cancers via immunohistochemical staining and examined LINE-1 promoter methylation in representative cases. We found that 78% and 57% of STICs, with and without concurrent ovarian carcinomas, respectively, exhibited intense LINE-1 immunoreactivity compared with adjacent, normal-appearing fallopian tube epithelium. Hypomethylation of the LINE-1 promoter was found in all STICs exhibiting overexpression. None of the 12 p53 signatures demonstrated significant LINE-1 expression. In ovarian cancer, 84 (75%) of 112 ovarian carcinomas overexpressed LINE-1. Our results indicate that LINE-1 retrotransposons often become deregulated during progression of ovarian cancer precursor lesions from the p53 signature to STIC stages and remain highly expressed in carcinoma.
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Cistadenocarcinoma Seroso/metabolismo , Neoplasias de las Trompas Uterinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Elementos de Nucleótido Esparcido Largo , Neoplasias Ováricas/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Adulto , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Neoplasias de las Trompas Uterinas/genética , Neoplasias de las Trompas Uterinas/patología , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes. The capabilities of this platform are validated through serial dilutions of mixed epialleles, with demonstrated detection limits as low as 1 methylated variant in 2 million unmethylated templates (0.00005%) of a classic tumor suppressor gene, CDKN2A (p14ARF). The clinical potential of the platform is demonstrated using a digital assay for NDRG4, a tumor suppressor gene that is commonly methylated in colorectal cancer, in liquid biopsies of healthy and colorectal cancer patients. Overall, the platform provides the depth of information, simplicity of use, and single-molecule sensitivity necessary for rapid assessment of intermolecular variation contributing to genetic and epigenetic heterogeneity for challenging applications in embryogenesis, carcinogenesis, and rare biomarker detection.
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Neoplasias Colorrectales/genética , Metilación de ADN , Técnicas Genéticas/instrumentación , Microfluídica/instrumentación , Microfluídica/métodos , Neoplasias Colorrectales/patología , Diseño de Equipo , Humanos , Dispositivos Laboratorio en un Chip , Biopsia Líquida , Masculino , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Flujo de TrabajoRESUMEN
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.
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Biomarcadores de Tumor , Metilación de ADN , Perfilación de la Expresión Génica , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Transcriptoma , Biopsia , Estudios de Casos y Controles , Biología Computacional/métodos , Islas de CpG , Detección Precoz del Cáncer , Femenino , Sitios Genéticos , Humanos , Inmunohistoquímica , Clasificación del Tumor , Estadificación de Neoplasias , Curva ROCRESUMEN
DNA methylation is a fundamental means of epigenetic gene regulation that occurs in virtually all cell types. In many higher organisms, including humans, it plays vital roles in cell differentiation and homeostatic maintenance of cell phenotype. The control of DNA methylation has traditionally been attributed to a highly coordinated, linear process, whose dysregulation has been associated with numerous pathologies including cancer, where it occurs early in, and even prior to, the development of neoplastic tissues. Recent experimental evidence has demonstrated that, contrary to prevailing paradigms, methylation patterns are actually maintained through inexact, dynamic processes. These processes normally result in minor stochastic differences between cells that accumulate with age. However, various factors, including cancer itself, can lead to substantial differences in intercellular methylation patterns, viz. methylation heterogeneity. Advancements in molecular biology techniques are just now beginning to allow insight into how this heterogeneity contributes to clonal evolution and overall cancer heterogeneity. In the current review, we begin by presenting a didactic overview of how the basal bimodal methylome is established and maintained. We then provide a synopsis of some of the factors that lead to the accrual of heterogeneous methylation and how this heterogeneity may lead to gene silencing and impact the development of cancerous phenotypes. Lastly, we highlight currently available methylation assessment techniques and discuss their suitability to the study of heterogeneous methylation.
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Metilación de ADN/genética , Heterogeneidad Genética , Neoplasias/genética , Animales , Humanos , Modelos Genéticos , Procesos EstocásticosRESUMEN
Many cancers comprise heterogeneous populations of cells at primary and metastatic sites throughout the body. The presence or emergence of distinct subclones with drug-resistant genetic and epigenetic phenotypes within these populations can greatly complicate therapeutic intervention. Liquid biopsies of peripheral blood from cancer patients have been suggested as an ideal means of sampling intratumor genetic and epigenetic heterogeneity for diagnostics, monitoring and therapeutic guidance. However, current molecular diagnostic and sequencing methods are not well suited to the routine assessment of epigenetic heterogeneity in difficult samples such as liquid biopsies that contain intrinsically low fractional concentrations of circulating tumor DNA (ctDNA) and rare epigenetic subclonal populations. Here we report an alternative approach, deemed DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of epiallelic species at single-CpG-site resolution in fractions as low as 0.005%, providing facile and inexpensive ultrasensitive assessment of locus-specific epigenetic heterogeneity directly from liquid biopsies. The technique is demonstrated here for the evaluation of epigenetic heterogeneity at p14(ARF) and BRCA1 gene-promoter loci in liquid biopsies obtained from patients in association with non-small cell lung cancer (NSCLC) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), respectively.
Asunto(s)
Biopsia , Metilación de ADN , ADN de Neoplasias/sangre , Epigénesis Genética , Neoplasias/genética , Alelos , Carcinoma de Pulmón de Células no Pequeñas/genética , Islas de CpG , Cartilla de ADN , ADN de Neoplasias/química , Interpretación Estadística de Datos , Epigenómica/métodos , Variación Genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Síndromes Mielodisplásicos/genética , Neoplasias/patología , Desnaturalización de Ácido Nucleico , Análisis de Secuencia de ADN , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
The use of methylated tumor-specific circulating DNA has shown great promise as a potential cancer biomarker. Nonetheless, the relative scarcity of tumor-specific circulating DNA presents a challenge for traditional DNA extraction and processing techniques. Here we demonstrate a single tube extraction and processing technique dubbed "methylation on beads" that allows for DNA extraction and bisulfite conversion for up to 2 ml of plasma or serum. In comparison to traditional techniques including phenol chloroform and alcohol extraction, methylation on beads yields a 1.5- to 5-fold improvement in extraction efficiency. The technique results in far less carryover of PCR inhibitors yielding analytical sensitivity improvements of over 25-fold. The combination of improved recovery and sensitivity make possible the detection of rare epigenetic events and the development of high sensitivity epigenetic diagnostic assays.
Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , ADN/sangre , Neoplasias Pulmonares/sangre , Reacción en Cadena de la Polimerasa/métodos , Proteínas Supresoras de Tumor/sangre , Actinas/sangre , Actinas/genética , Anciano , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Metilación de ADN , Epigénesis Genética , Femenino , Expresión Génica , Células HeLa , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Masculino , Microesferas , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/normas , Sensibilidad y Especificidad , Sulfitos/química , Proteínas Supresoras de Tumor/genéticaRESUMEN
Iron oxide nanoparticles (IONPs) are promising neuroimaging agents and molecular cargo across neurovascular barriers. Development of intrinsically safe IONP chemistries requires a robust in vivo nanoneurotoxicity screening model. Herein, we engineered four IONPs of different surface and core chemistries: DMSA-Fe2O3, DMSA-Fe3O4, PEG-Fe3O4 and PEG-Au-Fe3O4. Capitalizing on the ability of the peripheral nervous system to recruit potent immune cells from circulation, we characterized a spatiotemporally controlled platform for the study of in vivo nanobiointerfaces with hematogenous immune cells, neuroglial and neurovascular units after intraneural IONP delivery into rat sciatic nerve. SQUID magnetometry and histological iron stain were used for IONP tracking. Among the IONPs, DMSA-Fe2O3 NPs were potent pro-apoptotic agents in nerve, with differential ability to regulate oxidative stress, inflammation and apoptotic signaling in neuroglia, macrophages, lymphocytes and endothelial cells. This platform aims to facilitate the development of predictive paradigms of nanoneurotoxicity based on mechanistic investigation of relevant in vivo bio-nanointerfaces. FROM THE CLINICAL EDITOR: This team of investigators report the development of a platform that enables screening of iron oxide nanoparticles from the standpoint of their potential neurotoxicity, utilizing rat sciatic nerves. Such screening tools are clearly needed with the potential advent of iron oxide nanoparticle-based diagnostic and therapeutic approaches.