RESUMEN

Chromosomal translocations resulting in fusion genes represent important oncogenic drivers and potential therapeutic targets in rare leukemia subtypes. Formalin-fixed, paraffin-embedded trephines are frequently used in hematologic diagnostic tests and provide relevant access to leukemic cells for further studies, for example, phenotyping in bone marrow fibrosis. However, high-throughput molecular analysis of nucleic acids obtained from this material is challenging, especially the reliable detection of RNA transcripts. Sixty-three formalin-fixed, paraffin-embedded bone marrow trephines of patients with chronic eosinophilic leukemia, chronic myeloid leukemia, acute myeloid leukemia, and myeloproliferative neoplasms were analyzed for gene mutations and the presence of fusion transcripts with a commercial amplicon-based next-generation sequencing approach. Fusion transcripts relevant for diagnosis and therapy could be detected and validated (by RT-PCR) in 25 patients (39.7%). Retrospectively selected material, up to 10 years old, was used for this purpose, and only one sample failed in the RNA analysis (1.6%). This study concludes that amplicon-based fusion transcript detection in bone marrow trephines is feasible and that bone marrow trephines taken for histologic assessment can also be applied for high-throughput molecular analysis.

Asunto(s)

Biomarcadores de Tumor , Células de la Médula Ósea/metabolismo , Médula Ósea/patología , Leucemia/diagnóstico , Leucemia/genética , Mutación , Proteínas de Fusión Oncogénica/genética , Biopsia con Aguja , Células de la Médula Ósea/patología , Biología Computacional/métodos , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN

RESUMEN

Besides histopathological findings there are no indicators of increased risk for fibrotic progression in myeloproliferative neoplasms (MPN). Age-related clonal hematopoiesis (ARCH/CHIP) is a frequent finding in the elderly and combinations with MPN driver mutations (JAK2, MPL, and CALR) have been described. To determine the impact of ARCH/CHIP-related mutations for development of fibrosis in primary myelofibrosis (PMF), the mutational status of cases with fibrotic progression from grade 0 to grade 2/3 (n = 77) as evidenced by follow-up bone marrow biopsies (median 6.2 years) was compared with prefibrotic PMF samples without development of fibrosis (n = 27; median follow-up 7.3 years). Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, and DNMT3A) demonstrable at presentation were not connected with fibrotic progression. However, mutations which are rarely found in ARCH/CHIP (SRSF2, U2AF1, SF3B1, IDH1/2, and EZH2) were present in 24.7% of cases with later development of fibrosis and not detectable in cases staying free from fibrosis (P = 0.0028). Determination of the tumor mutational burden (TMB) in a subgroup of cases (n = 32) did not show significant differences (7.68 mutations/MB vs. 6.85 mutations/MB). We conclude that mutations rarely found in ARCH/CHIP provide an independent risk factor for rapid fibrotic progression (median 2.0 years) when manifest already at first presentation.

Asunto(s)

Biomarcadores de Tumor/genética , Médula Ósea/patología , Evolución Clonal , Fibrosis/patología , Mutación , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/patología , Factores de Edad , Médula Ósea/metabolismo , Progresión de la Enfermedad , Femenino , Fibrosis/genética , Estudios de Seguimiento , Hematopoyesis , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos

RESUMEN

Reactivation of interspersed repetitive sequences due to loss of methylation is associated with genomic instability, one of the hallmarks of cancer cells. LINE-1 hypomethylation is a surrogate marker for global methylation loss and is potentially a new diagnostic and prognostic biomarker in tumors. However, the correlation of LINE-1 hypomethylation with clinicopathological parameters and the CpG island methylator phenotype (CIMP) in patients with liver tumors is not yet well defined, particularly in Caucasians who show quite low rates of HCV/HBV infection and a higher incidence of liver steatosis. Therefore, quantitative DNA methylation analysis of LINE-1, RASSF1A, and CCND2 using pyrosequencing was performed in human hepatocellular carcinomas (HCC, n = 40), hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5), and corresponding peritumoral liver tissues as well as healthy liver tissues (n = 5) from Caucasian patients. Methylation results were correlated with histopathological findings and clinical data. We found loss of LINE-1 DNA methylation only in HCC. It correlated significantly with poor survival (log rank test, p = 0.007). An inverse correlation was found for LINE-1 and RASSF1A DNA methylation levels (r2 = -0.47, p = 0.002). LINE-1 hypomethylation correlated with concurrent RASSF1/CCND2 hypermethylation (Fisher's exact test, p = 0.02). Both LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation were not found in benign hepatocellular tumors (HCA and FNH). Our results show that LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation are epigenetic aberrations specific for the process of malignant liver transformation. In addition, LINE-1 hypomethylation might serve as a future predictive biomarker to identify HCC patients with unfavorable overall survival.

Asunto(s)

Carcinoma Hepatocelular , Metilación de ADN , ADN de Neoplasias/metabolismo , Neoplasias Hepáticas , Elementos de Nucleótido Esparcido Largo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Ciclina D2/metabolismo , Supervivencia sin Enfermedad , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Tasa de Supervivencia , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

The detection of hotspot mutations in key cancer genes is now an essential part of the diagnostic work-up in molecular pathology. Nearly all assays for mutation detection involve an amplification step. A second single nucleotide variant (SNV) on the same allele adjacent to a mutational hotspot can interfere with primer binding, leading to unnoticed allele-specific amplification of the wild type allele and thereby false-negative mutation testing. We present two diagnostic cases with false negative sequence results for JAK2 and SRSF2. In both cases mutations would have escaped detection if only one strand of DNA had been analysed. Because many commercially available diagnostic kits rely on the analysis of only one DNA strand they are prone to fail in cases like these. Detailed protocols and quality control measures to prevent corresponding pitfalls are presented.

Asunto(s)

Pruebas Genéticas/normas , Mutación , Policitemia Vera/genética , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/normas , Reacciones Falso Negativas , Pruebas Genéticas/métodos , Humanos , Janus Quinasa 2/genética , Análisis de Secuencia de ADN/métodos , Factores de Empalme Serina-Arginina/genética

RESUMEN

Circulating cell-free DNA (cfDNA), which is isolated from blood plasma, represents a noninvasive source for the detection of mutations conferring resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small-cell lung cancer patients. In advanced disease stages, performing regular biopsies is often not possible because of the general health condition of the patients. Furthermore, a biopsy of a single tumor lesion or metastasis may not reflect the heterogeneous genotype of the tumor and its metastases. Plasma cfDNA represents an alternative material for molecular monitoring of patients under therapy. Herein, we present a cross-platform comparison of three different molecular methods [digital PCR, next-generation sequencing (NGS), and quantitative PCR] to detect clinically relevant mutations in cfDNA. We validated our workflow with commercially available cfDNA reference material (5.0%, 1.0%, and 0.1% mutation frequency, respectively). Digital PCR and NGS detect reliably 0.1% allele frequency of the EGFR p.T790M mutation. Furthermore, we analyzed 55 cfDNA preparations from patients with lung cancer to compare reliability and sensitivity of the three methods under routine conditions and achieved 96.0% concordance of p.T790M results. A limit of detection for mutation calling using digital PCR (>0.1%) and NGS (>0.2%) was established. In total, 62.5% of known primary EGFR mutations were successfully detected in cfDNA. In 56.0% of the patients with detectable EGFR primary mutations, we identified a resistance conferring the EGFR p.T790M mutation.

Asunto(s)

Biomarcadores de Tumor , ADN Tumoral Circulante , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Análisis Mutacional de ADN/métodos , Análisis Mutacional de ADN/normas , Pruebas Diagnósticas de Rutina/métodos , Pruebas Diagnósticas de Rutina/normas , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mutación , Reacción en Cadena de la Polimerasa , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

AIM: To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC). METHODS: Knockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables. RESULTS: miRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes. CONCLUSION: Our data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.

Asunto(s)

Carcinoma Hepatocelular/genética , Metilación de ADN , Neoplasias Hepáticas/genética , MicroARNs/genética , Anciano , Biomarcadores de Tumor , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/mortalidad , Línea Celular Tumoral , Islas de CpG , Epigénesis Genética , Femenino , Células Hep G2 , Hepatocitos/citología , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/mortalidad , Masculino , MicroARNs/química , Persona de Mediana Edad , Pronóstico , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sulfitos/química , Resultado del Tratamiento

RESUMEN

Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions.To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192). A customized amplicon-based gene panel including 23 genes frequently mutated in myeloid neoplasms was established and implemented. Thereby, 629,691 reads per sample (range 179,847-1,460,412) and a mean coverage of 2,702 (range 707-6,327) could be obtained, which are sufficient for comprehensive mutational profiling. Seven samples failed in sequencing (3.6%). In 185 samples we found in total 269 pathogenic variants (mean 1.4 variants per patient, range 0-5), 125 Patients exhibit at least one pathogenic mutation (67.6%). Variants show allele frequencies ranging from 6.7% up to 95.7%. Most frequently mutated genes were TET2 (28.7%), SRSF2 (19.5%), ASXL1 (8.6%) and U2AF1 (8.1%). The mutation profiling increases the diagnostic precision and adds prognostic information.

Asunto(s)

Neoplasias Hematológicas/diagnóstico , Síndromes Mielodisplásicos/diagnóstico , Enfermedades Mielodisplásicas-Mieloproliferativas/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biopsia , Médula Ósea/patología , Análisis Mutacional de ADN/métodos , Proteínas de Unión al ADN/genética , Dioxigenasas , Frecuencia de los Genes , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Persona de Mediana Edad , Mutación , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Enfermedades Mielodisplásicas-Mieloproliferativas/genética , Enfermedades Mielodisplásicas-Mieloproliferativas/patología , Pronóstico , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Análisis de Secuencia de ADN , Factores de Empalme Serina-Arginina/genética , Factor de Empalme U2AF/genética , Adulto Joven

RESUMEN

BACKGROUND: Aberrant DNA methylation at imprinted loci is an important molecular mechanism contributing to several developmental and pathological disorders including cancer. However, knowledge about imprinting defects due to DNA methylation changes is relatively limited in hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide. Therefore, comprehensive quantitative DNA methylation analysis at imprinted loci showing ~50 % methylation in healthy liver tissues was performed in primary HCC specimens and the peritumoural liver tissues. RESULTS: We found frequent and extensive DNA methylation aberrations at many imprinted loci in HCC. Unsupervised cluster analysis of DNA methylation patterns at imprinted loci revealed subgroups of HCCs with moderate and severe loss of methylation. Hypomethylation at imprinted loci correlated significantly with poor overall survival (log-rank test, p = 0.02). Demethylation at imprinted loci was accompanied by loss of methylation at LINE-1, a commonly used marker for global DNA methylation levels (p < 0.001). In addition, we found that loss of methylation at imprinted loci correlated with the presence of a CTNNB1 mutation (Fisher's exact test p = 0.03). Re-analysis of publically available genome-wide methylation data sets confirmed our findings. The analysis of benign liver tumours (hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH)), the corresponding adjacent liver tissues, and healthy liver tissues showed that aberrant DNA methylation at imprinted loci is specific for HCC. CONCLUSIONS: Our analyses demonstrate frequent and widespread DNA methylation aberrations at imprinted loci in human HCC and identified a hypomethylated subgroup of patients with shorter overall survival.

RESUMEN

BACKGROUND: Aberrations in DNA methylation patterns are well-described in human malignancies. However, the existence of the 'CpG island methylator phenotype' (CIMP) in human breast cancer is still controversial. MATERIALS & METHODS: Illumina's HumanMethylation 450K BeadChip was used to analyze genome-wide DNA methylation patterns. Chromosomal abnormalities were determined by array-based CGH. RESULTS: Invasive lobular breast carcinomas exhibit the highest number of differentially methylated CpG sites and a strong inverse correlation of aberrant DNA hypermethylation and copy number alterations. Nine differentially methylated regions within seven genes discriminating the investigated subgroups were identified and validated in an independent validation cohort and correlated to a better relapse-free survival. CONCLUSION: These results depict a clear difference between genetically and epigenetically unstable breast carcinomas indicating different ways of tumor progression and/or initiation, which strongly supports the association of CIMP with the lobular subtype and provide new options for detection and therapy.

Asunto(s)

Neoplasias de la Mama/genética , Islas de CpG , Metilación de ADN , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Epigénesis Genética , Femenino , Expresión Génica , Humanos , Fenotipo

RESUMEN

The tumour suppressor gene RB1 is frequently silenced in many different types of human cancer, including hepatocellular carcinoma (HCC). However, mutations of the RB1 gene are relatively rare in HCC. A systematic screen for the identification of imprinted genes deregulated in human HCC revealed that RB1 shows imprint abnormalities in a high proportion of primary patient samples. Altogether, 40% of the HCC specimens (16/40) showed hyper- or hypomethylation at the CpG island in intron 2 of the RB1 gene. Re-analysis of publicly available genome-wide DNA methylation data confirmed these findings in two independent HCC cohorts. Loss of correct DNA methylation patterns at the RB1 locus leads to the aberrant expression of an alternative RB1-E2B transcript, as measured by quantitative real-time PCR. Demethylation at the intron 2 CpG island by DNMT1 knock-down or aza-deoxycytidine (DAC) treatment stimulated expression of the RB1-E2B transcript, accompanied by diminished RB1 main transcript expression. No aberrant DNA methylation was found at the RB1 locus in hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5) and their corresponding adjacent liver tissue specimens. Deregulated RB1 expression due to hyper- or hypomethylation in intron 2 of the RB1 gene is found in tumours without loss of heterozygosity and is associated with a decrease in overall survival (p = 0.032) if caused by hypermethylation of CpG85. This unequivocally demonstrates that loss of imprinting represents an important additional mechanism for RB1 pathway inactivation in human HCC, complementing well-described molecular defects.

Asunto(s)

Carcinoma Hepatocelular/genética , Regulación hacia Abajo/genética , Genes Supresores de Tumor , Impresión Genómica/genética , Neoplasias Hepáticas/genética , Proteína de Retinoblastoma/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Islas de CpG/genética , Metilación de ADN/genética , ADN de Neoplasias/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Proteína de Retinoblastoma/metabolismo

Asunto(s)

Leucemia Linfocítica Crónica de Células B/genética , Humanos

Asunto(s)

Proteínas Nucleares/genética , Mielofibrosis Primaria/genética , Ribonucleoproteínas/genética , Anciano , Anciano de 80 o más Años , Enfermedades Asintomáticas , Recuento de Células , Femenino , Fibrosis , Humanos , Masculino , Persona de Mediana Edad , Mutación/fisiología , Mielofibrosis Primaria/patología , Factores de Empalme Serina-Arginina

RESUMEN

Epigenetic inactivation by aberrant DNA methylation has been reported for many microRNA genes in various human malignancies. However, relatively little is known about microRNA gene methylation in hepatocellular carcinoma (HCC). Therefore, a systematic screen for identification of aberrantly hypermethylated microRNA genes in HCC was initiated. The methylation status of 39 intergenic CpG island associated microRNA genes was analyzed in HCC cell lines (n = 7), immortalized hepatocytes (n = 2) and normal liver samples (n = 5). Subsequently, 13 differentially methylated microRNA genes were analyzed in primary human HCC samples (n = 40), benign liver tumors (n = 15) and the adjacent liver tissues employing pyrosequencing. Expression of microRNA genes was measured using quantitative real-time polymerase chain reaction (RT-PCR). In addition, DNA methylation and expression of microRNA genes were measured after DNMT1 knockdown or DNMT inhibition. Aberrant hypermethylation and concomitant reduction in expression of intergenic microRNA genes is a frequent event in human HCC: hsa-mir-9-2 (23%), hsa-mir-9-3 (50 %), hsa-mir-124-1 (20%), hsa-mir-124-2 (13%), hsa-mir-124-3 (43%), hsa-mir-129-2 (58%), hsa-mir-596 (28%) and hsa-mir-1247 (38%). Altogether, it affects 90% of the HCC specimens under study. MicroRNA gene methylation is not found in hepatocellular adenoma (n = 10) and focal nodular hyperplasia (n = 5). DNMT1 knockdown or DNMT inhibition reduced microRNA gene methylation and stimulated expression. In primary human HCC specimens hypermethylation and expression of microRNA genes showed an inverse correlation. Concordant hypermethylation of three or more microRNA genes is a highly specific marker for the detection of HCC and for poor prognosis.

Asunto(s)

Carcinoma Hepatocelular/genética , Metilación de ADN , Neoplasias Hepáticas/genética , MicroARNs/genética , Secuencia de Bases , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/diagnóstico , Línea Celular Tumoral , Islas de CpG/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Hepatocitos/citología , Humanos , Neoplasias Hepáticas/diagnóstico , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño , Análisis de Secuencia de ADN

RESUMEN

Deregulation of imprinted genes is an important molecular mechanism contributing to the development of cancer in humans. However, knowledge about imprinting defects in human hepatocellular carcinoma (HCC), the third leading cause of cancer mortality worldwide, is still limited. Therefore, a systematic meta-analysis of the expression of 223 imprinted loci in human HCC was initiated. This screen revealed that the DLK1-MEG3 locus is frequently deregulated in HCC. Deregulation of DLK1 and MEG3 expression accompanied by extensive aberrations in DNA methylation could be confirmed experimentally in an independent series of human HCC (n = 40) in more than 80% of cases. Loss of methylation at the DLK1-MEG3 locus correlates linearly with global loss of DNA methylation in HCC (r(2) = 0.63, p<0.0001). Inhibition of DNMT1 in HCC cells using siRNA led to a reduction in MEG3-DMR methylation and concomitant increase in MEG3 RNA expression. Allele-specific expression analysis identified loss of imprinting in 10 out of 31 informative samples (32%), rendering it one of the most frequent molecular defects in human HCC. In 2 cases unequivocal gain of bi-allelic expression accompanied by substantial loss of methylation at the IG-DMR could be demonstrated. In 8 cases the tumour cells displayed allelic switching by mono-allelic expression of the normally imprinted allele. Allelic switching was accompanied by gains or losses of DNA methylation primarily at IG-DMR1. Analysis of 10 hepatocellular adenomas (HCA) and 5 cases of focal nodular hyperplasia (FNH) confirmed that this epigenetic instability is specifically associated with the process of malignant transformation and not linked to increased proliferation per se. This widespread imprint instability in human HCC has to be considered in order to minimize unwanted side-effects of therapeutic approaches targeting the DNA methylation machinery. It might also serve in the future as predictive biomarker and for monitoring response to epigenetic therapy.

Asunto(s)

Alelos , Carcinoma Hepatocelular/genética , Impresión Genómica , Péptidos y Proteínas de Señalización Intercelular/genética , Neoplasias Hepáticas/genética , Proteínas de la Membrana/genética , ARN Largo no Codificante/genética , Proteínas de Unión al Calcio , Carcinoma Hepatocelular/patología , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/patología

RESUMEN

BACKGROUND: The newly released 450k DNA methylation array from Illumina, Inc. offers the possibility to analyze more than 480,000 individual CpG sites in a user friendly standardized format. In this study the relationship between the ß-values provided by the Illumina, Inc. array for each individual CpG dinucleotide and the quantitative methylation levels obtained by pyrosequencing were analyzed. In addition, the representation of microRNA genes and imprinted loci on the Illumina, Inc. array was assessed in detail. Genomic DNA from 4 human breast cancer cell lines (IPH-926, HCC1937, MDA-MB-134, PMC42) and 18 human breast cancer specimens as well as 4 normal mammary epithelial fractions was analyzed on 450k DNA methylation arrays. The ß-values for 692 individual CpG sites from 62 different genes were cross-validated using conventional quantitative pyrosequencing. FINDINGS: The newly released 450k methylation array from Illumina, Inc. shows a high concordance with quantitative pyrosequencing if identical CpG sites are analyzed in cell lines (Spearman r = 0.88, p ≪ 0.0001), which is somewhat reduced in primary tumor specimens (Spearman r = 0.86, p ≪ 0.0001). 80.7% of the CpG sites show an absolute difference in methylation level of less than 15 percentage points. If different CpG sites in the same CpG islands are targeted the concordance is lower (r = 0.83 in cell lines and r = 0.7 in primary tumors). The number of CpG sites representing microRNA genes and imprinted loci is very heterogeneous (range: 1 - 70 CpG sites for microRNAs and 1 - 288 for imprinted loci). CONCLUSIONS: The newly released 450k methylation array from Illumina, Inc. provides a genome-wide quantitative representation of DNA methylation aberrations in a convenient format. Overall, the congruence with pyrosequencing data is very good. However, for individual loci one should be careful to translate the ß-values directly into percent methylation levels.

Asunto(s)

Emparejamiento Base/genética , Metilación de ADN/genética , Análisis de Secuencia de ADN/instrumentación , Análisis de Secuencia de ADN/métodos , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Sitios Genéticos/genética , Impresión Genómica/genética , Humanos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/normas , Proteínas Nucleares snRNP/genética

RESUMEN

BACKGROUND: New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed. RESULTS: To this end the methylation patterns of 12 loci (GSTπ1, p16INK4a, RASSF1A, SOCS1, MAL, hsa-mir-1-1, hsa-mir-9-3, hsa-mir-34a, hsa-mir-596, hsa-mir-663, MINT31, and LINE-1) were analyzed in ten primary hepatocellular carcinoma specimens. After applying stringent quality control criteria, 35749 sequences entered further analysis. The methylation level of individual CpG dinucleotides obtained by 454 sequencing was systematically compared with the corresponding values obtained by conventional pyrosequencing. Statistical analyses revealed an excellent concordance of methylation levels for all individual CpG dinucleotides under study (r2 = 0.927). CONCLUSIONS: Our results confirm that 454 sequencing of bisulfite treated genomic DNA provides reliable high quality quantitative methylation data and identify MAL, hsa-mir-9-3, hsa-mir-596, and hsa-mir-663 as new targets of aberrant DNA methylation in human hepatocellular carcinoma. In addition, the single molecule resolution of 454 sequencing provides unprecedented information about the details of DNA methylation pattern heterogeneity in clinical samples.

Asunto(s)

Metilación de ADN , ADN/química , Análisis de Secuencia de ADN/métodos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Análisis de Regresión , Reproducibilidad de los Resultados

RESUMEN

BACKGROUND: The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases. METHODS: The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer. RESULTS: Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer. CONCLUSIONS: Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

Asunto(s)

Neoplasias de la Mama Masculina/metabolismo , Neoplasias de la Mama Masculina/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Neoplasias de la Mama Masculina/genética , Análisis por Conglomerados , Epigénesis Genética , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , MicroARNs/metabolismo , Microscopía Fluorescente/métodos , Persona de Mediana Edad

RESUMEN

The identification of aberrantly hypermethylated genes may lead to the development of new diagnostic markers and the identification of novel targets of epigenetic therapy in myelodysplastic syndromes (MDS). We therefore investigated the methylation status of transcription factor genes KLF5, KLF11, and MAFB, shown to be aberrantly methylated in myelogeneous leukaemia cells, in a series of 115 MDS patient as well as in 25 control subjects. Using quantitative high-resolution pyrosequencing methodology, KLF11, MAFB, and KLF5 were shown for the first time to be hypermethylated in 17 (15%), 8 (7%), and 2 (1.7%) cases, respectively, but not in any of the patients with an isolated 5q-deletion. Patient samples harbouring KLF11 methylation displayed reduced KLF11 mRNA expression and KLF11 hypermethylation correlated with a high International Prognostic Scoring System score (P < 0.05). In conclusion, epigenetic inactivation and subsequent transcriptional repression of the KLF11 gene is quite frequent in MDS. Patients with an isolated 5q-deletion seem to harbour a distinct epigenetic profile.

Asunto(s)

Proteínas de Ciclo Celular/biosíntesis , Metilación de ADN , Epigénesis Genética , Silenciador del Gen , Genes Supresores de Tumor , Síndromes Mielodisplásicos/metabolismo , Proteínas Represoras/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis , Proteínas de Ciclo Celular/genética , Femenino , Humanos , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Factor de Transcripción MafB/biosíntesis , Factor de Transcripción MafB/genética , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Proteínas Represoras/genética

RESUMEN

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E-cadherin. ILBCs also lack beta-catenin expression and show aberrant cytoplasmic localization of the E-cadherin binding protein p120-catenin. The lack of a well-characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH-926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH-926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH-926 expressed various epithelial cell markers but lacked expression of E-cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH-926 from this particular tumour. IPH-926 also lacked beta-catenin expression and showed aberrant cytoplasmic localization of p120-catenin. Array-CGH analysis of IPH-926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH-926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH-926 cells were anti-cancer drug-resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH-926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH-926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo.

Asunto(s)

Neoplasias de la Mama/genética , Cadherinas/genética , Carcinoma Lobular/genética , Anciano , Desequilibrio Alélico , Animales , Antígenos CD , Secuencia de Bases , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/deficiencia , Cadherinas/metabolismo , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patología , Carcinoma Lobular/secundario , Línea Celular Tumoral , Femenino , Humanos , Cariotipificación , Ratones , Ratones Endogámicos NOD , Mutación , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Trasplante Heterólogo

Asunto(s)

Azacitidina/análogos & derivados , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Citogenética , Metilación de ADN/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Azacitidina/farmacología , Azacitidina/uso terapéutico , Citogenética/tendencias , Metilación de ADN/genética , Decitabina , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Resultado del Tratamiento