RESUMEN
BACKGROUND: Copy number variation (CNV) is a key genetic characteristic for cancer diagnostics and can be used as a biomarker for the selection of therapeutic treatments. Using data sets established in our previous study, we benchmark the performance of cancer CNV calling by six most recent and commonly used software tools on their detection accuracy, sensitivity, and reproducibility. In comparison to other orthogonal methods, such as microarray and Bionano, we also explore the consistency of CNV calling across different technologies on a challenging genome. RESULTS: While consistent results are observed for copy gain, loss, and loss of heterozygosity (LOH) calls across sequencing centers, CNV callers, and different technologies, variation of CNV calls are mostly affected by the determination of genome ploidy. Using consensus results from six CNV callers and confirmation from three orthogonal methods, we establish a high confident CNV call set for the reference cancer cell line (HCC1395). CONCLUSIONS: NGS technologies and current bioinformatics tools can offer reliable results for detection of copy gain, loss, and LOH. However, when working with a hyper-diploid genome, some software tools can call excessive copy gain or loss due to inaccurate assessment of genome ploidy. With performance matrices on various experimental conditions, this study raises awareness within the cancer research community for the selection of sequencing platforms, sample preparation, sequencing coverage, and the choice of CNV detection tools.
Asunto(s)
Biología Computacional , Variaciones en el Número de Copia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Pérdida de Heterocigocidad , Neoplasias , Programas Informáticos , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Biología Computacional/métodos , Diploidia , Genoma Humano , Línea Celular Tumoral , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodosRESUMEN
BACKGROUND: The cancer genome is commonly altered with thousands of structural rearrangements including insertions, deletions, translocation, inversions, duplications, and copy number variations. Thus, structural variant (SV) characterization plays a paramount role in cancer target identification, oncology diagnostics, and personalized medicine. As part of the SEQC2 Consortium effort, the present study established and evaluated a consensus SV call set using a breast cancer reference cell line and matched normal control derived from the same donor, which were used in our companion benchmarking studies as reference samples. RESULTS: We systematically investigated somatic SVs in the reference cancer cell line by comparing to a matched normal cell line using multiple NGS platforms including Illumina short-read, 10X Genomics linked reads, PacBio long reads, Oxford Nanopore long reads, and high-throughput chromosome conformation capture (Hi-C). We established a consensus SV call set of a total of 1788 SVs including 717 deletions, 230 duplications, 551 insertions, 133 inversions, 146 translocations, and 11 breakends for the reference cancer cell line. To independently evaluate and cross-validate the accuracy of our consensus SV call set, we used orthogonal methods including PCR-based validation, Affymetrix arrays, Bionano optical mapping, and identification of fusion genes detected from RNA-seq. We evaluated the strengths and weaknesses of each NGS technology for SV determination, and our findings provide an actionable guide to improve cancer genome SV detection sensitivity and accuracy. CONCLUSIONS: A high-confidence consensus SV call set was established for the reference cancer cell line. A large subset of the variants identified was validated by multiple orthogonal methods.
Asunto(s)
Variaciones en el Número de Copia de ADN , Neoplasias , Humanos , Análisis de Secuencia de ADN/métodos , Variación Estructural del Genoma , Tecnología , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Genoma Humano , Neoplasias/genéticaRESUMEN
The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has benefitted society1,2. However, it still has many gaps and errors, and does not represent a biological genome as it is a blend of multiple individuals3,4. Recently, a high-quality telomere-to-telomere reference, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a nearly homozygous genome5. To address these limitations, the Human Pangenome Reference Consortium formed with the goal of creating high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and assembly approaches yield the most complete and accurate diploid genome assembly with minimal manual curation. Approaches that used highly accurate long reads and parent-child data with graph-based haplotype phasing during assembly outperformed those that did not. Developing a combination of the top-performing methods, we generated our first high-quality diploid reference assembly, containing only approximately four gaps per chromosome on average, with most chromosomes within ±1% of the length of CHM13. Nearly 48% of protein-coding genes have non-synonymous amino acid changes between haplotypes, and centromeric regions showed the highest diversity. Our findings serve as a foundation for assembling near-complete diploid human genomes at scale for a pangenome reference to capture global genetic variation from single nucleotides to structural rearrangements.
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Mapeo Cromosómico , Diploidia , Genoma Humano , Genómica , Humanos , Mapeo Cromosómico/normas , Genoma Humano/genética , Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas , Estándares de Referencia , Genómica/métodos , Genómica/normas , Cromosomas Humanos/genética , Variación Genética/genéticaRESUMEN
As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB, and ZFPM2) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
With the rapid advancement of sequencing technologies, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples and generated whole-genome (WGS) and whole-exome sequencing (WES) data using sixteen library protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.
Asunto(s)
Secuenciación del Exoma , Genoma Humano , Neoplasias/genética , Secuenciación Completa del Genoma , Benchmarking , Línea Celular Tumoral , Biología Computacional , Genómica , Humanos , Medicina de PrecisiónRESUMEN
Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.
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Benchmarking , Secuenciación del Exoma/normas , Neoplasias/genética , Análisis de Secuencia de ADN/normas , Secuenciación Completa del Genoma/normas , Línea Celular , Línea Celular Tumoral , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación , Neoplasias/patología , Reproducibilidad de los ResultadosRESUMEN
The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.
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Benchmarking , Neoplasias de la Mama/genética , Análisis Mutacional de ADN/normas , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Secuenciación Completa del Genoma/normas , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Células Germinativas , Humanos , Mutación , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
High-quality genomes are essential to resolve challenges in breeding, comparative biology, medicine, and conservation planning. New library preparation techniques along with better assembly algorithms result in continued improvements in assemblies for non-model organisms, moving them toward reference-quality genomes. We report on the latest genome assembly of the Atlantic bottlenose dolphin, leveraging Illumina sequencing data coupled with a combination of several library preparation techniques. These include Linked-Reads (Chromium, 10x Genomics), mate pairs (MP), long insert paired ends, and standard paired end. Data were assembled with the commercial DeNovoMAGIC assembly software, resulting in two assemblies, a traditional "haploid" assembly (Tur_tru_Illumina_hap_v1) that is a mosaic of the two parental haplotypes and a phased assembly (Tur_tru_Illumina_phased_v1) where each scaffold has sequence from a single homologous chromosome. We show that Tur_tru_Illumina_hap_v1 is more complete and more accurate compared to the current best reference based on the amount and composition of sequence, the consistency of the MP alignments to the assembled scaffolds, and on the analysis of conserved single-copy mammalian orthologs. The phased de novo assembly Tur_tru_Illumina_phased_v1 is the first publicly available for this species and provides the community with novel and accurate ways to explore the heterozygous nature of the dolphin genome.
Asunto(s)
Delfín Mular/genética , Genoma , Haplotipos , Secuenciación Completa del Genoma , Animales , Femenino , GenómicaRESUMEN
Haplotype-resolved genome sequencing promises to unlock a wealth of information in population and medical genetics. However, for the vast majority of genomes sequenced to date, haplotypes have not been determined because of cumbersome haplotyping workflows that require fractions of the genome to be sequenced in a large number of compartments. Here we demonstrate barcode partitioning of long DNA molecules in a single compartment using "on-bead" barcoded tagmentation. The key to the method that we call "contiguity preserving transposition" sequencing on beads (CPTv2-seq) is transposon-mediated transfer of homogenous populations of barcodes from beads to individual long DNA molecules that get fragmented at the same time (tagmentation). These are then processed to sequencing libraries wherein all sequencing reads originating from each long DNA molecule share a common barcode. Single-tube, bulk processing of long DNA molecules with â¼150,000 different barcoded bead types provides a barcode-linked read structure that reveals long-range molecular contiguity. This technology provides a simple, rapid, plate-scalable and automatable route to accurate, haplotype-resolved sequencing, and phasing of structural variants of the genome.
Asunto(s)
Código de Barras del ADN Taxonómico/métodos , Genoma Humano/genética , Genómica/métodos , Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , HumanosRESUMEN
The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.
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Benchmarking , Genoma Humano , Exoma , Genómica , Humanos , Mutación INDELRESUMEN
Alternative splicing shapes mammalian transcriptomes, with many RNA molecules undergoing multiple distant alternative splicing events. Comprehensive transcriptome analysis, including analysis of exon co-association in the same molecule, requires deep, long-read sequencing. Here we introduce an RNA sequencing method, synthetic long-read RNA sequencing (SLR-RNA-seq), in which small pools (≤1,000 molecules/pool, ≤1 molecule/gene for most genes) of full-length cDNAs are amplified, fragmented and short-read-sequenced. We demonstrate that these RNA sequences reconstructed from the short reads from each of the pools are mostly close to full length and contain few insertion and deletion errors. We report many previously undescribed isoforms (human brain: â¼13,800 affected genes, 14.5% of molecules; mouse brain â¼8,600 genes, 18% of molecules) and up to 165 human distant molecularly associated exon pairs (dMAPs) and distant molecularly and mutually exclusive pairs (dMEPs). Of 16 associated pairs detected in the mouse brain, 9 are conserved in human. Our results indicate conserved mechanisms that can produce distant but phased features on transcript and proteome isoforms.
Asunto(s)
Empalme Alternativo/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Animales , Química Encefálica , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Isoformas de ARN/genéticaRESUMEN
BACKGROUND: A single platform designed for the synchronous screening of multiple mutations can potentially enable molecular profiling in samples of limited tumor tissue. This approach is ideal for the assessment of advanced non-small-cell lung cancer (NSCLC) diagnostic specimens, which often comprise small biopsies. Therefore, we aimed in this study to validate the mass spectrometry-based Sequenom LungCarta panel and MassARRAY platform using DNA extracted from a single 5 µM formalin-fixed paraffin-embedded tissue section. METHODS: Mutations, including those with an equivocal spectrum, detected in 90 cases of NSCLC (72 lung biopsies, 13 metastatic tissue biopsies, three resections, and two cytology samples) were validated by a combination of standard sequencing techniques, immunohistochemical staining for p53 protein, and next-generation sequencing with the TruSight Tumor panel. RESULTS: Fifty-five mutations were diagnosed in 47 cases (52%) in the following genes: TP53 (22), KRAS (15), EGFR (5), MET (3), PIK3CA (3), STK11 (2), NRF-2 (2), EPHA5 (1), EPHA3 (1), and MAP2K1 (1). Of the 90 samples, one failed testing due to poor quality DNA. An additional 7 TP53 mutations were detected by next-generation sequencing, which facilitated the interpretation of p53 immunohistochemistry but required 5 × 10 µM tumor sections per sample tested. CONCLUSIONS: The LungCarta panel is a sensitive method of screening for multiple alterations (214 mutations across 26 genes) and which optimizes the use of limited amounts of tumor DNA isolated from small specimens.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/secundario , Análisis Mutacional de ADN/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Pulmón/patología , Quinasas de la Proteína-Quinasa Activada por el AMP , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Carcinoma de Pulmón de Células no Pequeñas/química , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Genes erbB-1/genética , Genes ras/genética , Pruebas Genéticas/métodos , Humanos , Neoplasias Pulmonares/química , MAP Quinasa Quinasa 1/genética , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Mutación , Factor 2 Relacionado con NF-E2/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptor EphA3 , Receptor EphA5/genética , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Renal tumor heterogeneity studies have utilized the von Hippel-Lindau VHL gene to classify disease into molecularly defined subtypes to examine associations with etiologic risk factors and prognosis. The aim of this study was to provide a comprehensive analysis of VHL inactivation in clear cell renal tumors (ccRCC) and to evaluate relationships between VHL inactivation subgroups with renal cancer risk factors and VHL germline single nucleotide polymorphisms (SNPs). VHL genetic and epigenetic inactivation was examined among 507 sporadic RCC/470 ccRCC cases using endonuclease scanning and using bisulfite treatment and Sanger sequencing across 11 CpG sites within the VHL promoter. Case-only multivariate analyses were conducted to identify associations between alteration subtypes and risk factors. VHL inactivation, either through sequence alterations or promoter methylation in tumor DNA, was observed among 86.6% of ccRCC cases. Germline VHL SNPs and a haplotype were associated with promoter hypermethylation in tumor tissue (ORâ=â6.10; 95% CI: 2.28-16.35, pâ=â3.76E-4, p-globalâ=â8E-5). Risk of having genetic VHL inactivation was inversely associated with smoking due to a higher proportion of wild-type ccRCC tumors [former: ORâ=â0.70 (0.20-1.31) and current: ORâ=â0.56 (0.32-0.99); P-trendâ=â0.04]. Alteration prevalence did not differ by histopathologic characteristics or occupational exposure to trichloroethylene. ccRCC cases with particular VHL germline polymorphisms were more likely to have VHL inactivation through promoter hypermethylation than through sequence alterations in tumor DNA, suggesting that the presence of these SNPs may represent an example of facilitated epigenetic variation (an inherited propensity towards epigenetic variation) in renal tissue. A proportion of tumors from current smokers lacked VHL alterations and may represent a biologically distinct clinical entity from inactivated cases.
Asunto(s)
Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Adulto , Anciano , Carcinoma de Células Renales/patología , Estudios de Casos y Controles , Islas de CpG/genética , Metilación de ADN , Epigénesis Genética , Femenino , Silenciador del Gen , Estudios de Asociación Genética , Mutación de Línea Germinal , Haplotipos , Humanos , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Factores de Riesgo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
We broadly profiled DNA methylation in breast cancers (n = 351) and benign parenchyma (n = 47) for correspondence with disease phenotype, using FFPE diagnostic surgical pathology specimens. Exploratory analysis revealed a distinctive primary invasive carcinoma subclass featuring extreme global methylation deviation. Subsequently, we tested the correlation between methylation remodeling pervasiveness and malignant biological features. A methyl deviation index (MDI) was calculated for each lesion relative to terminal ductal-lobular unit baseline, and group comparisons revealed that high-grade and short-survival estrogen receptor-positive (ER(+)) cancers manifest a significantly higher MDI than low-grade and long-survival ER(+) cancers. In contrast, ER(-) cancers display a significantly lower MDI, revealing a striking epigenomic distinction between cancer hormone receptor subtypes. Kaplan-Meier survival curves of MDI-based risk classes showed significant divergence between low- and high-risk groups. MDI showed superior prognostic performance to crude methylation levels, and MDI retained prognostic significance (P < 0.01) in Cox multivariate analysis, including clinical stage and pathological grade. Most MDI targets individually are significant markers of ER(+) cancer survival. Lymphoid and mesenchymal indexes were not substantially different between ER(+) and ER(-) groups and do not explain MDI dichotomy. However, the mesenchymal index was associated with ER(+) cancer survival, and a high lymphoid index was associated with medullary carcinoma. Finally, a comparison between metastases and primary tumors suggests methylation patterns are established early and maintained through disease progression for both ER(+) and ER(-) tumors.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Carcinoma Medular/genética , Metilación de ADN , Epigenómica , Receptores de Estrógenos/metabolismo , Anciano , Biomarcadores de Tumor , Mama/patología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/mortalidad , Carcinoma Ductal de Mama/secundario , Carcinoma Lobular/mortalidad , Carcinoma Lobular/secundario , Carcinoma Medular/mortalidad , Carcinoma Medular/secundario , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunofenotipificación , Estudios Longitudinales , Linfocitos/patología , Mesodermo/patología , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Tasa de SupervivenciaRESUMEN
Epigenetic remodeling is a hallmark of cancer, with the frequent acquisition of de novo DNA methylation in CpG islands. However, the functional relevance of de novo DNA methylation in cancer is less well defined. To begin to address this issue in B-cells, we used BeadArray assays to survey the methylation status of over 1,500 cancer-related CpG loci in two molecular subtypes of diffuse large B-cell lymphoma (ABC-DLBCL and GCB-DLBCL) and cognate normal B-cell populations. We identified 81 loci that showed frequent de novo DNA methylation in GCB-DLBCL and 67 loci that showed frequent de novo DNA methylation in ABC-DLBCL. These de novo methylated CpG loci included reported targets of polycomb repressive complexes (PRC) in stem cells. All candidate loci in GCB-DLBCL are proximal to genes that are poorly expressed or silent in purified normal germinal center (GC) B-cells. This is consistent with the hypothesis that de novo DNA methylation in cancer is more frequently involved in the maintenance rather than the initiation of gene silencing (de novo repression). This suggests that epigenetic switching occurs during tumorigenesis with de novo DNA methylation locking in gene silencing normally mediated by transcriptional repressors. Furthermore, we propose that similar to de novo genetic mutations, the majority of de novo DNA methylation events observed in tumors are passengers not causally involved in tumorigenesis.
Asunto(s)
Linfocitos B/patología , Metilación de ADN/fisiología , Linfoma de Células B Grandes Difuso/genética , Línea Celular Tumoral , Islas de CpG , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Linfoma de Células B Grandes Difuso/patología , Células Madre Neoplásicas , Proteínas del Grupo Polycomb , Proteínas Represoras/genéticaRESUMEN
Emerging technologies allow broad profiling of the cancer genome for differential DNA methylation relative to benign cells. Herein, bisulfite-modified DNA from lymph nodes with either reactive hyperplasia or follicular lymphoma (FL) were analyzed using a commercial C/UpG genotyping assay. Two hundred fifty-nine differentially methylated targets (DMT) distributed among 183 unique genes were identified in FL. Comparison of matched formalin-fixed, paraffin-embedded and frozen surgical pathology replicates showed the complete preservation of the cancer methylome among differently archived tissue specimens. Analysis of the DMT profile is consistent with a pervasive epigenomic remodeling process in FL that affects predominantly nonlymphoid genes.
Asunto(s)
Ganglios Linfáticos/patología , Linfoma Folicular/genética , Linfoma Folicular/patología , Islas de CpG , Metilación de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Formaldehído , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Adhesión en Parafina , Reproducibilidad de los Resultados , Fijación del TejidoRESUMEN
PURPOSE: To provide a comprehensive, thorough analysis of somatic mutation and promoter hypermethylation of the von Hippel-Lindau (VHL) gene in the cancer genome, unique to clear cell renal cancer (ccRCC). Identify relationships between the prevalence of VHL gene alterations and alteration subtypes with patient and tumor characteristics. EXPERIMENTAL DESIGN: As part of a large kidney cancer case-control study conducted in Central Europe, we analyzed VHL mutations and promoter methylation in 205 well-characterized, histologically confirmed patient tumor biopsies using a combination of sensitive, high-throughput methods (endonuclease scanning and Sanger sequencing) and analysis of 11 CpG sites in the VHL promoter. RESULTS: We identified mutations in 82.4% of cases, the highest VHL gene mutation prevalence reported to date. Analysis of 11 VHL promoter CpG sites revealed that 8.3% of tumors were hypermethylated and all were mutation negative. In total, 91% of ccRCCs exhibited alteration of the gene through genetic or epigenetic mechanisms. Analysis of patient and tumor characteristics revealed that certain mutation subtypes were significantly associated with Fuhrman nuclear grade, metastasis, node positivity, and self-reported family history of RCC. CONCLUSION: Detection of VHL gene alterations using these accurate, sensitive, and practical methods provides evidence that the vast majority of histologically confirmed ccRCC tumors possess genetic or epigenetic alteration of the VHL gene and support the hypothesis that VHL alteration is an early event in ccRCC carcinogenesis. These findings also indicate that VHL molecular subtypes can provide a sensitive marker of tumor heterogeneity among histologically similar ccRCC cases for etiologic, prognostic, and translational studies.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Adenocarcinoma de Células Claras/metabolismo , Carcinoma de Células Renales/metabolismo , Estudios de Casos y Controles , Islas de CpG , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Mutación , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
PURPOSE: The von Hippel-Lindau (VHL) gene is often inactivated (by mutation or promoter hypermethylation) in renal cell carcinoma but the relation to therapeutic outcome is unclear. MATERIALS AND METHODS: Patients with metastatic clear cell renal cell carcinoma with available baseline tumor samples who received vascular endothelial growth factor targeted therapy were included in analysis. Patient characteristics, VHL gene status and clinical outcome were documented. Our primary end point was to test for response rate in relation to VHL inactivation. Progression-free survival and overall survival in relation to VHL status were investigated as secondary end points. RESULTS: A total of 123 patients were evaluable. Response rate, median progression-free survival and median overall survival were 37% (95% CI 28-46), 10.8 (95% CI 7.7-14.8) and 29.8 (CI not estimable) months, respectively. Patients with VHL inactivation had a response rate of 41% vs 31% for those with wild-type VHL (p = 0.34). Patients with loss of function mutations (frameshift, nonsense, splice and in-frame deletions/insertions) had a 52% response rate vs 31% with wild-type VHL (p = 0.04). On multivariate analysis the presence of a loss of function mutation remained an independent prognostic factor associated with improved response. Progression-free survival and overall survival were not significantly different based on VHL status. CONCLUSIONS: To our knowledge this is the largest analysis investigating the impact of VHL inactivation on the outcome of vascular endothelial growth factor targeted agents in metastatic renal cell carcinoma. We did not find a statistically significant increase in response to vascular endothelial growth factor targeted agents in patients with VHL inactivation. Loss of function mutations identified a population of patients with a greater response. Investigation of downstream markers is under way.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Enfermedad de von Hippel-Lindau/genética , Anciano , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Axitinib , Bencenosulfonatos/uso terapéutico , Bevacizumab , Carcinoma de Células Renales/patología , Distribución de Chi-Cuadrado , Cartilla de ADN , Femenino , Humanos , Imidazoles/uso terapéutico , Indazoles/uso terapéutico , Indoles/uso terapéutico , Neoplasias Renales/patología , Modelos Logísticos , Masculino , Persona de Mediana Edad , Mutación/genética , Metástasis de la Neoplasia , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Pronóstico , Piridinas/uso terapéutico , Pirroles/uso terapéutico , Sorafenib , Sunitinib , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
OBJECTIVE: To describe the relationship among patient characteristics, Von Hippel-Lindau (VHL) gene status and clinical outcome in metastatic renal cell carcinoma (RCC) in patients receiving vascular endothelial growth factor (VEGF)-targeted therapy. PATIENTS AND METHODS: All patients with metastatic RCC who received therapy with interferon-alpha plus bevacizumab, SU11248 or AG013736 at the authors' institution were considered. Clinical features were collected and activation status of the VHL gene (VHL) was determined from baseline paraffin-embedded tumour samples. Tumour response, time to tumour progression (TTP) and overall survival were recorded. RESULTS: Forty-three patients were evaluable for determination of VHL status and clinical response. There was an objective response in 18 patients (43%; 95% confidence interval 28-59%). The median TTP for the entire cohort was 8.1 months. There was an improved clinical outcome in patients with the following clinical features: male gender, lack of hepatic metastases, no previous radiation therapy and higher baseline haemoglobin level. Twenty-six patients (60%) had evidence of VHL mutation or promoter methylation; such patients had an objective response rate of 48%, vs 35% in patients with no VHL mutation or methylation. Patients with VHL methylation or a mutation predicted to truncate or shift the VHL reading frame had a median TTP of 13.3 months, vs 7.4 months in patients with none of these features (P = 0.06). CONCLUSION: VEGF-targeted therapy is active in metastatic RCC and the response can be associated with certain clinical features. The TTP with VEGF-targeted therapy might be prolonged in patients with VHL methylation or mutations that truncate or shift the VHL reading frame. Further investigation of VHL pathway components is needed to understand the biology of the response to VEGF-targeted agents in metastatic RCC.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Enfermedad de von Hippel-Lindau/tratamiento farmacológico , Adulto , Anciano , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Axitinib , Bevacizumab , Carcinoma de Células Renales/complicaciones , Carcinoma de Células Renales/genética , Femenino , Humanos , Imidazoles/administración & dosificación , Indazoles/administración & dosificación , Indoles/administración & dosificación , Neoplasias Renales/complicaciones , Neoplasias Renales/genética , Masculino , Persona de Mediana Edad , Mutación/genética , Pirroles/administración & dosificación , Sunitinib , Resultado del Tratamiento , Enfermedad de von Hippel-Lindau/complicaciones , Enfermedad de von Hippel-Lindau/genéticaRESUMEN
Our previous studies demonstrate that introduction of a approximately 70 cM region (now estimated at 63.75 Mb by the Human Genome Project) of human chromosome 12 into the highly metastatic Dunning rat prostate cancer cell line AT6.1 results in >30-fold (>/=90%) reduction in the number of overt metastases in spontaneous metastasis assays. We report the further localization and biological characterization of the metastasis-suppressor activity encoded by a reduced region of chromosome 12. To localize this metastasis-suppressor activity, a panel of AT6.1 microcell hybrids that retain varying portions of human chromosome 12 was constructed and subjected to sequence-tagged site (STS)-based PCR analysis and assessment of in vivo metastatic ability. Data from these complementary approaches localized the metastasis-suppressor activity to a approximately 4.2 Mb portion of human chromosome 12q24.3 comprised of 3 separate regions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting were used for differential expression analyses to identify which characterized genes, predicted gene sequences and expressed sequence tags (ESTs) within this region could be responsible for the observed metastasis suppression. Comprehensive in vivo studies showed that suppressed AT6.1-12 hybrids that retain the metastasis-suppressor region on 12q24.3 are capable of arriving at the secondary site, but are not able to persist there. Thus, unlike other metastasis-suppressor genes characterized to date, the metastasis-suppressor gene encoded by this region appears to utilize a different biologic mechanism to suppress the growth of overt metastases at the secondary site.