RESUMEN
The causes of early genomic events underlying the development of prostate cancer (CaP) remain unclear. The onset of chromosomal instability is likely to facilitate the formation of crucial genomic aberrations both in the precursor lesion high-grade prostatic intraepithelial neoplasia (HPIN) and in CaP. Instability generated by telomere attrition is one potential mechanism that could initiate chromosomal rearrangements. In this study, normalized telomere length variation was examined in a cohort of 68 men without CaP who had HPIN only on prostatic biopsies. Multiple significant associations between telomere attrition and eventual diagnosis of CaP in the HPIN and in the surrounding stroma were found. Kaplan-Meier analysis of telomere length demonstrated a significantly increased risk for the development of cancer with short telomeres in the surrounding stroma [P = .035; hazard ratio (HR) = 2.12; 95% confidence interval (95% CI) = 0.231-0.956], and a trend for HPIN itself (P = .126; HR = 1.72; 95% CI = 0.287-1.168). Cox regression analysis also demonstrated significance between the time from the original biopsy to the diagnosis of cancer and telomere length in HPIN and in the surrounding stroma. These analyses showed significance, both alone and in combination with baseline prostate-specific antigen, and lend support to the hypothesis that telomere attrition in prostatic preneoplasia may be fundamental to the generation of chromosomal instability and to the emergence of CaP.
Asunto(s)
Inestabilidad Cromosómica , Neoplasia Intraepitelial Prostática/genética , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Telómero , Anciano , Anciano de 80 o más Años , Daño del ADN , Fluorescencia , Humanos , Masculino , Persona de Mediana Edad , Análisis de RegresiónRESUMEN
The progression to cancer is often associated with instability and the acquisition of genomic heterogeneity, generating both clonal and non-clonal populations. Chromosomal instability (CIN) describes the excessive rate of numerical and structural genomic change in tumors. Mitotic segregation errors strongly influences copy number, while structural aberrations can occur at unstable genomic regions, or through aberrant DNA repair or methylation. Combined molecular cytogenetic analyses can evaluate cell-to-cell variation, and define the complexity of numerical and structural alterations. Because structural change may occur independently of numerical alteration, we propose the term structural chromosomal instability [(S)-CIN] to distinguish numerical from structural CIN.
Asunto(s)
Genoma Humano , Neoplasias/genética , Elementos Alu , Animales , Núcleo Celular/metabolismo , Inestabilidad Cromosómica , Aberraciones Cromosómicas , Cromosomas/ultraestructura , Citogenética , Metilación de ADN , Reparación del ADN , Humanos , Hibridación Fluorescente in Situ , Mitosis , Modelos Genéticos , Neoplasias/metabolismoRESUMEN
BACKGROUND: Advances made in the area of microarray comparative genomic hybridization (aCGH) have enabled the interrogation of the entire genome at a previously unattainable resolution. This has lead to the discovery of a novel class of alternative entities called large-scale copy number variations (CNVs). These CNVs are often found in regions of closely linked sequence homology called duplicons that are thought to facilitate genomic rearrangements in some classes of neoplasia. Recently, it was proposed that duplicons located near the recurrent translocation break points on chromosomes 9 and 22 in chronic myeloid leukemia (CML) may facilitate this tumor-specific translocation. Furthermore, approximately 15-20% of CML patients also carry a microdeletion on the derivative 9 chromosome (der(9)) and these patients have a poor prognosis. It has been hypothesised that der(9) deletion patients have increased levels of chromosomal instability. RESULTS: In this study aCGH was performed and identified a CNV (RP11-125A5, hereafter called CNV14q12) that was present as a genomic gain or loss in 10% of control DNA samples derived from cytogenetically normal individuals. CNV14q12 was the same clone identified by Iafrate et al. as a CNV. Real-time polymerase chain reaction (Q-PCR) was used to determine the relative frequency of this CNV in DNA from a series of 16 CML patients (both with and without a der(9) deletion) together with DNA derived from 36 paediatric solid tumors in comparison to the incidence of CNV in control DNA. CNV14q12 was present in approximately 50% of both tumor and CML DNA, but was found in 72% of CML bearing a der(9) microdeletion. Chi square analysis found a statistically significant difference (p Asunto(s)
Aberraciones Cromosómicas
, Cromosomas Humanos Par 14
, Dosificación de Gen
, Variación Genética
, Neoplasias/genética
, Niño
, Mapeo Cromosómico
, Estudios de Cohortes
, Femenino
, Duplicación de Gen
, Genoma Humano
, Humanos
, Hibridación Fluorescente in Situ
, Leucemia Mielógena Crónica BCR-ABL Positiva/genética
, Masculino
, Análisis por Micromatrices/métodos
, Reacción en Cadena de la Polimerasa/métodos
RESUMEN
Spontaneous tumor regression of pediatric low-grade gliomas (PLGG). We speculated that lack of telomere maintenance is responsible for this behavior. We first looked for evidence of telomerase activity and alternative lengthening of telomeres (ALT) in 56 PLGG. Telomerase activity was observed in 0 of 11 PLGG, in contrast to 10 of 13 high-grade pediatric brain tumors. There was no ALT in 45 of 45 samples. We then applied Q-FISH to eight patients whose indolent PLGG underwent two metachronous biopsies over a lag of several years. Telomere shortening was observed in the second biopsy in all tumors, but not in normal brain control (P < .0001), indicating that lack of telomere maintenance is associated with continuous telomere erosion. Based on these observations, we found that younger PLGG patients, who exhibit more aggressive and frequently recurrent tumors, had significantly longer telomeres than older ones (P = .00014). Tumors with a terminal restriction fragment length <7.5 did not recur, whereas the presence of longer telomeres (>8.0) conferred a high likelihood of late recurrences in PLGG. Our findings provide a plausible biologic mechanism to explain the tendency of PLGG to exhibit growth arrest and spontaneous regression. Telomere maintenance may therefore represent the first known biologic prognostic marker in PLGG.
Asunto(s)
Neoplasias Encefálicas/patología , Glioma/patología , Regresión Neoplásica Espontánea , Telómero/fisiología , Astrocitoma/genética , Astrocitoma/patología , Biopsia , División Celular , Niño , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Ependimoma/genética , Ependimoma/patología , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Telómero/patologíaRESUMEN
The role of BRCA1 in breast and ovarian tumor suppression has been primarily ascribed to the maintenance of genome integrity. BRCA1 interacts with components of the non-homologous end-joining pathway previously shown to play a role in telomere maintenance in yeast. Here, we provide evidence that links Brca1 with telomere integrity. Brca1(-/-) T-cells display telomere dysfunction in both loss of telomere repeats as well as defective telomere capping. Loss of Brca1 synergizes with p53 deficiency in the onset and frequency of tumorigenesis. Karyotyping of tBrca1(-/-)p53(-/-) thymic lymphomas revealed the presence of telomere dysfunction accompanied by clonal chromosomal translocations. The telomere dysfunction phenotype in Brca1-deficient cells suggests that loss of telomere integrity might contribute to chromosome end dysfunction and permit the formation of potentially oncogenic translocations.
Asunto(s)
Proteína BRCA1/fisiología , Telómero/metabolismo , Animales , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Hibridación Fluorescente in Situ , Ratones , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Linfocitos T/metabolismo , Linfocitos T/patología , Telómero/genética , Telómero/patología , Timoma/genética , Timoma/patología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Osteosarcoma (OS) is characterized by chromosomal instability and high-copy-number gene amplification. The breakage-fusion-bridge (BFB) cycle is a well-established mechanism of genomic instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. However, until now, there have been no high-resolution cytogenetic or genomic array studies of BFB events in OS. In the present study, multicolor banding (mBAND) FISH and submegabase resolution tiling set (SMRT) array comparative genomic hybridization (CGH) were used to identify and map genomic signatures of BFB events in four OS cell lines and one patient tumor. The expected intermediates associated with BFB-dicentric chromosomes, inverted duplications, and intra- and interchromosomal amplifications-were identified. mBAND analysis provided detailed mapping of rearrangements in 1p, 6p, and 8q and showed that translocation junctions were often in close proximity to fragile sites. More detailed mBAND studies of OS cell line MG-63 revealed ladderlike FISH signals of equally spaced interchromosomal coamplifications of 6p21, 8q24, and 9p21-p22 in a homogeneously staining region (hsr). Focal amplifications that concordantly mapped to the hsr were localized to discrete genomic intervals by SMRT array CGH. The complex amplicon structure in this hsr suggests focal amplifications immediately adjacent to microdeletions. Moreover, the genomic regions in which there was deletion/amplification had a preponderance of fragile sites. In summary, this study has provided further support for the role of the BFB mechanism and fragile sites in facilitating gene amplification and chromosomal rearrangement in OS.
Asunto(s)
Amplificación de Genes , Eliminación de Gen , Hibridación de Ácido Nucleico , Osteosarcoma/genética , Línea Celular Tumoral , Cromosomas Humanos Par 6 , Cromosomas Humanos Par 8 , Humanos , Sondas Moleculares , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteosarcoma/patología , Ácidos Nucleicos de Péptidos , Translocación GenéticaRESUMEN
The advancement of fluorescence in situ hybridization-based assays has permitted more refined delineation of chromosomal loci involved in complex chromosomal rearrangements (CCRs) and gene amplification. In this detailed molecular cytogenetic analysis, spectral karyotyping (SKY), multicolor banding (mBAND) analysis, and microarray comparative genomic hybridization (CGH) were used to refine the analysis of chromosomes with amplifications and small intrachromosomal rearrangements such as inverted duplications and interstitial deletions present in the osteosarcoma cell line MG-63. SKY analysis has limited resolving power to delineate cryptic chromosomal rearrangements, so mBAND assays were performed for a subset of chromosomes (i.e., 6, 8, 17, and 20). Of the 10 clonal CCRs analyzed in detail with mBAND, 5 were found to have rearrangements between 8q24 and either 6p23 approximately pter or 6p21, with multiple copies of this translocation inserted at various sites in the different chromosomes. In two CCRs, 6p21 and 8q24 generated an alternating pattern of mBAND probe hybridization, indicating the presence of a large coamplified repeat unit within homogeneously staining regions. Microarray CGH analysis demonstrated focal high-level amplification of 8q23 approximately q24, 6p22 approximately pter, and 6p21, in agreement with the pattern of chromosome subband gains identified with mBAND. Thus, sequential SKY, mBAND, and microarray CGH provided a comprehensive description of some of the intricate chromosomal aberrations present in the complex MG-63 karyotype and permitted reconstruction of the fine structure of the genomic rearrangements, thus providing some important mechanistic clues concerning the details of the amplification process in tumors.
Asunto(s)
Neoplasias Óseas/genética , Bandeo Cromosómico , Amplificación de Genes/genética , Reordenamiento Génico/genética , Cariotipificación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Osteosarcoma/genética , Línea Celular Tumoral , Mapeo Cromosómico , Humanos , Hibridación de Ácido Nucleico/métodosRESUMEN
Mechanisms underlying prostate cancer (CaP) initiation and progression are poorly understood. A chromosomal instability mechanism leading to the generation of numerical and structural chromosomal changes has been implicated in the preneoplastic and neoplastic stages of CaP. Telomere dysfunction is one potential mechanism associated with the onset of such instability. To determine whether there was alteration in telomere length and chromosome number, 15 paraffin-embedded prostatectomy specimens were investigated using quantitative peptide nucleic acid (PNA) FISH analysis of representative foci of carcinoma, putative precancerous lesions (high-grade prostatic intraepithelial neoplasia, HPIN) and nondysplastic prostate epithelium. A significant decrease in telomere length was shown in both HPIN and CaP in comparison with normal epithelium. In addition, elevated rates of aneusomy suggested that increased levels of chromosomal aberrations were associated with decreased telomere length. Moreover, multiple foci of HPIN were shown to have a heterogeneous overall reduction of telomere length. This reduction was more evident in the histologic regions of the prostate containing CaP. Such observations lend support to the hypothesis that telomere erosion may be a consistent feature of CaP oncogenesis and may also be associated with the generation of chromosomal instability that characterizes this malignancy.
Asunto(s)
Adenocarcinoma/genética , Neoplasia Intraepitelial Prostática/genética , Neoplasias de la Próstata/genética , Telómero/ultraestructura , Adenocarcinoma/química , Adenocarcinoma/ultraestructura , Anciano , Envejecimiento/genética , Aneuploidia , Transformación Celular Neoplásica/genética , Aberraciones Cromosómicas , Progresión de la Enfermedad , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Hibridación Fluorescente in Situ , Interfase , Antígeno Ki-67/análisis , Masculino , Persona de Mediana Edad , Adhesión en Parafina , Neoplasia Intraepitelial Prostática/química , Neoplasia Intraepitelial Prostática/ultraestructura , Neoplasias de la Próstata/química , Neoplasias de la Próstata/ultraestructura , Proteína p53 Supresora de Tumor/análisisRESUMEN
Prostate cancer (CaP) is a multifocal heterogenous disease. A major challenge in CaP research is to identify genetic biomarkers that herald aggressive transformation. To investigate the effect of tumor heterogeneity on the analysis of genomic aberration, we compared the results of comparative genomic hybridization (CGH) analysis of DNA extracted from tumor bulk against that of DNA amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from homogeneous cell population obtained by laser capture microdissection of discrete tumor foci. Sampling by microdissection, aberrations were observed in three of three foci of carcinoma involved with prostatic capsule, and in two of three prostatic intraepithelial neoplasia (PIN) foci examined. Carcinoma foci consistently exhibited more extensive aberrations than the PIN samples obtained from the same tumor. Within these samples, the different tumor foci exhibited gain of 8q, whereas PIN showed no consistent aberration. Using bulk extracted DNA, CGH detected aberrations in only 3 of 21 samples investigated, despite the known trisomy 8 status, as revealed by fluorescence in situ hybridization. The results of this study demonstrate that CGH analysis using bulk dissected fresh tissue is insufficiently sensitive to fully detect the chromosomal numerical aberrations in CaP. Given the considerable intratumor genomic heterogeneity, CGH with microdissection and DOP-PCR amplification provides a more complete repertoire of aberrations as well as a better phenotype-genotype correlation in prostate tumors.