RESUMEN
Clinical management of ductal carcinoma in situ (DCIS) remains a challenge because significant proportions of patients experience recurrence after conservative surgical treatment. Unfortunately, it is difficult to prospectively identify, using objective criteria, patients who are at high risk of recurrence and might benefit from additional treatment. We conducted a multi-institutional, collaborative case-control study to identify nuclear morphometric features that would be useful for identifying women with DCIS at the highest risk of recurrence. Tissue sections of archival breast tissue of 29 women with recurrent and 73 matched women with nonrecurrent DCIS were stained for DNA, and nuclei in the DCIS lesions were evaluated by image analysis. A clear correlation between mean fractal2_area (FA2) and nuclear grade was observed (P < 0.001), allowing an objective determination of nuclear grade. Several nuclear morphometric features, including mean and variance of variation of radius, mean area, mean and variance of frequency of high boundary harmonics (FQH), and variance in sphericity, were found to be useful in discriminating recurrent from nonrecurrent DCIS subjects. However, the nuclear features associated with recurrence differed between high- and low-grade lesions. For lesions with high FA2 (nuclear grade 3), mean variation of radius, mean FQH, and mean area alone yielded recurrence odds ratios of 4.55 [95% confidence interval (CI) 0.45-45.96], 3.86 (95% CI, 0.88-16.98), 2.90 (95% CI, 0.31-27.2), respectively. Using a summed feature model, high-FA2 lesions showing three poor prognostic features had an odds ratio of 15.63 (95% CI, 1.22-200), compared with those with zero or one poor prognostic feature. Lesions with low mean FA2 (nuclear grade 1 or 2) showing high variances in sphericity and FQH had an odds ratio of 7.71 (95% CI, 1.77-33.60). Addition of other features did not enhance the odds ratio or its significance. These results suggest that nuclear image analysis of DCIS lesions may provide an adjunctive tool to conventional pathological analysis, both for the objective assessment of nuclear grade and for the identification of features that predict patient outcome.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/patología , ADN de Neoplasias/análisis , Procesamiento de Imagen Asistido por Computador , Recurrencia Local de Neoplasia/epidemiología , Recurrencia Local de Neoplasia/patología , Matriz Nuclear/patología , Adulto , Anciano , Anciano de 80 o más Años , Biopsia con Aguja , Neoplasias de la Mama/epidemiología , Carcinoma Intraductal no Infiltrante/epidemiología , Estudios de Casos y Controles , Estudios de Cohortes , Intervalos de Confianza , Femenino , Humanos , Incidencia , Persona de Mediana Edad , Oportunidad Relativa , Valor Predictivo de las Pruebas , Probabilidad , Valores de Referencia , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
Several studies have shown an association between high nuclear grade or necrosis of ductal carcinoma in situ (DCIS) lesions and the risk of local disease recurrence in patients with DCIS treated surgically with less than mastectomy. Although criteria for separating low from high nuclear grade lesions have been published, no information exists regarding interobserver reproducibility (IR). To assess IR in the classification of DCIS, six surgical pathologists from four institutions used the Lagios grading system to grade 125 DCIS lesions. Before meeting to evaluate the cases, a training set of 12 glass slides, including cases chosen to present conflicting cues for classification, was mailed to the participants with a written criteria summary. This was followed by a working session in which criteria were reviewed and agreed on. The pathologists then graded the lesions independently. The area of interest was marked on each slide before grading. After initial grading, the pathologists met again to resolve discrepant lesion classifications. A complete agreement among raters was achieved in 43 (35%) cases, with five of six raters agreeing in another 45 (36%) cases. In no case did two raters differ by more than one grade. The pairwise kappa agreement values ranged from fair to substantial (0.30 to 0.61). Generalized kappa value indicated moderate agreement (0.46, standard error = 0.02). Kappa statistics for the distinction between grades 1 and 2 and 2 and 3 were 0.29 and 0.48, respectively, (standard error = 0.02). Only one of the six raters differed significantly in scoring. With adherence to specific criteria, IR in the classification of DCIS cases can be obtained in most cases. Although these pathologists made a few grading system modifications, further refinements are needed, especially if grading will influence future therapy.
Asunto(s)
Neoplasias de la Mama/clasificación , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/clasificación , Carcinoma Intraductal no Infiltrante/patología , Núcleo Celular/patología , Femenino , Humanos , Variaciones Dependientes del Observador , Reproducibilidad de los ResultadosRESUMEN
Genetic predisposition to lung cancer was determined by observing nonrandom chromosomal alterations in peripheral blood lymphocytes (PBLs) of lung cancer patients. The histological distribution of the cases showed that chromosomes 7 and 9 were frequently altered in squamous cell lung carcinoma (SCLC) patients. We analyzed PBLs of 26 SCLC patients and 5 controls using fluorescent in situ hybridization (FISH) with whole chromosome painting probes of chromosomes 7 and 9 to further investigate the frequency of rearrangements in these chromosomes. Our results suggested that seeking nonrandom aberrations in larger numbers of cells using FISH strengthened our previous observation of mosaicism and involvement of specific chromosomes in lung cancer patients. On combining our previous data, aberrations in chromosome 7 (16 of 26 patients), chromosome 9 (14 of 26), and the present study, we could actually pinpoint more individuals with abnormalities of chromosome 7 (23 of 26) and chromosome 9 (21 of 26). Thus, analyzing more cells in PBLs and adding FISH analysis serve as useful adjuncts to our studies of nonrandom chromosomal aberrations and genetic mosaicicm.
Asunto(s)
Carcinoma de Células Pequeñas/sangre , Aberraciones Cromosómicas , Neoplasias Pulmonares/sangre , Linfocitos/ultraestructura , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Pequeñas/genética , Cromosomas Humanos Par 7 , Cromosomas Humanos Par 9 , Femenino , Humanos , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana EdadRESUMEN
The fusion of mouse and human melanoma cells that were tumorigenic but had different metastatic capabilities resulted in hybrids that were metastatic when injected intravenously or subcutaneously into nude mice, regardless of whether it was the mouse or the human melanoma clone that was metastatic. The H7 hybrid line, formed by fusing murine nonmetastatic K1735 C19 cells with human metastatic A375 C15 cells retained high metastatic potential over more than 50 sub-culture passages, suggesting that the dominant metastatic phenotype in these hybrid cells was stable. Using fluorescent in situ hybridization (FISH), human chromosome 17 was consistently identified as the predominant human chromosome in the majority of H7 cells tested between passages 20 and 60. Western blot analysis showed that the hybrid cells expressed human nm23 protein, indicating that at least one gene on the human chromosome 17 was functional. Immunocytochemistry and immunoprecipitation showed that the metastatic A375 C15 and H7 cells expressed p53 protein, but that the nonmetastatic K1735 C19 melanoma cells did not. Sequencing the human p53 gene in A375 C15N and H7 showed mutations in exon 7. Using a bioassay technique, we showed that K1735 C19 cells can spread from subcutaneous tumors to the lungs of nude mice yet fail to form metastases. With the addition of human chromosome 17 from A375 C15 cells, which carries a mutant p53 gene, the cells readily formed lung metastases. In this melanoma hybrid, a mutant p53 gene appears to confer a survival advantage on cells arrested in the lungs of nude mice and thus contributes to the growth of metastatic cells.
Asunto(s)
Melanoma Experimental/patología , Melanoma/patología , Proteínas de Unión al GTP Monoméricas , Metástasis de la Neoplasia , Nucleósido-Difosfato Quinasa , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 17 , Cartilla de ADN/química , Genes Dominantes , Genes p53 , Humanos , Células Híbridas , Melanoma Experimental/genética , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Nucleósido Difosfato Quinasas NM23 , Metástasis de la Neoplasia/genética , Fenotipo , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
We have previously shown that in interspecific mouse-human melanoma cell hybrids obtained by fusion of nonmetastatic mouse with metastatic human melanoma cells, the metastatic phenotype predominates. The purpose of this study was to identify human chromosome(s) which could be responsible for conferring metastatic potential upon nonmetastatic mouse melanoma cells and therefore harbor the genes important for the metastatic properties of human melanoma cells. Seven mouse-human melanoma hybrids were examined; five were derived from the fusion of nonmetastatic (C19) and metastatic (C3) mouse K-1735 melanoma clones with highly metastatic clone (C15) of human melanoma A375 and the two others had as a human partner a nonmetastatic clone (Cls) of the A375 melanoma. The hybrids were examined during segregation of human chromosomes in vitro and in vivo for metastatic properties in nude mice and for the retaining human chromosomes. In the hybrid H7, which demonstrated the highest metastatic potential, the presence of human chromosomes was studied by GTG-banding and by fluorescence in situ hybridization (FISH) analysis. In the other hybrids, only FISH detection of human chromosomes was applied. All hybrids derived from nonmetastatic mouse and metastatic human melanoma cells demonstrated metastatic properties from early passages, when they contained the majority of the human chromosomes. Their metastatic phenotype remained stable during further segregation of most of the human chromosomes except for 17. Chromosome 17 was retained most consistently in all examined hybrids. However, the metastatic phenotype of the hybrids was associated only with the presence of chromosome 17 from the metastatic human donor cells. This chromosome was also found in almost 100% of cells recovered from lung metastases derived from the hybrid cells. In one lung metastasis developed from the H7 hybrid, chromosome 17 was detected as the sole human chromosome and these cells preserved the acquired high metastatic properties. Based on these results we conclude that human chromosome 17 from metastatic melanoma cells (A375 C15), when functional in the mouse genetic background, can be sufficient to render the recipient nonmetastatic mouse cells to a fully malignant phenotype. Additional data suggest that this ability might be related to the expression of the mutated human p53 gene.
RESUMEN
The purpose of this study was to determine whether production of liver metastasis by human colon carcinoma (HCC) cells depends on the response of tumor cells to organ-derived growth factors. HCC cells were isolated from several surgical specimens whose malignant potential differed (Dukes' stage B or D tumors), adapted to grow in culture, and assessed for expression of the epidermal growth factor receptor (EGF-R). Northern blot analyses revealed that highly metastatic HCC cells expressed >5-fold the number of EGF-R mRNA transcripts as low metastatic cells. The level of mRNA correlated with the amount of EGF-R protein as detected by Western blotting, immunohistochemistry, and Scatchard analyses. HCC growth response in vitro to picograms of transforming growth factor alpha was associated with functional cell surface EGF-Rs as determined by receptor tyrosine kinase activity assays. The EGF-R gene was not amplified or rearranged in highly metastatic cells. However, fluorescence in situ hybridization analysis showed that the copy number of chromosome 7 was higher in the highly metastatic cells. HCC cells were selected in vitro for low or high expression of EGF-R. Subsequent to injection into nude mice, only cells with high expression of EGF-R produced a high incidence of liver metastasis. These data demonstrate that expression of EGF-R by HCC cells directly correlates with their ability to produce hepatic metastasis.
Asunto(s)
Neoplasias del Colon/patología , Receptores ErbB/genética , Neoplasias Hepáticas/secundario , Metástasis de la Neoplasia , Transcripción Genética , Animales , Carcinoma de Células Escamosas , División Celular/efectos de los fármacos , Mapeo Cromosómico , Cromosomas Humanos Par 7 , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Receptores ErbB/análisis , Receptores ErbB/metabolismo , Humanos , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , ARN Mensajero/análisis , Factor de Crecimiento Transformador alfa/farmacología , Factor de Crecimiento Transformador alfa/fisiología , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Programmed cell death is an inherent characteristic of all somatic cells, including some immortalized cell lines. Cell death by apoptosis is attributed to nuclear disintegration, chromatin condensation, DNA fragmentation and cytoplasmic boiling. The purpose of this communication is to provide another cytogenetic marker of extensive telomeric associations between chromosomes of cells that are undergoing apoptosis. This can occur in normal fibroblasts, EBV-transformed lymphoblastoid B-cell lines, and even in certain tumor cell lines (including cell lines derived from pancreatic and head and neck tumors). From our preliminary data, we conclude that acquisition of ability to form dicentric chromosomes is associated with programmed cell death that can occur not only in normal fibroblasts but even in some tumor cell lines.
RESUMEN
We have previously reported the isolation of the cytotoxic factors, tumor necrosis factor-alpha (TNF-alpha), from monocytes and lymphotoxin or TNF-beta, from an established human B lymphoblastoid cell line. In the current study, we examined the production of cytotoxic factors by primary lymphoid cells derived from patients with different malignancies and correlated it with the chromosomal abnormalities in producer cells. Lymphoid cell lines were established from 78 untreated patients with breast carcinoma, 2 with cervical carcinoma, 24 with colon carcinoma, 12 with prostate carcinoma, 3 with melanoma, and the remaining from asymptomatic family members. Lymphoid cells from all 119 patients and their asymptomatic family members constitutively produced as much as 240 units/ml of TNF-beta in vitro. In contrast, TNF-alpha was produced by lymphoid cells from only 6 patients; a related cytokine with similar biological properties to TNF-beta. Sixty-five of these 119 samples were also analyzed for chromosomal abnormalities by standard cytogenetic technique. The production of TNFs was accompanied with varying degrees of chromosomal abnormalities in the cells from all patients. These included both structural and numerical abnormalities. We also examined the direct effect of TNF-beta on the induction of chromosomal abnormalities in growing cultures of both T and B lymphocytes. Our preliminary results indicate that treatment of lymphocytes with this cytokine induced chromosomal rearrangements in a dose-dependent manner. Thus, we provide for the first time both indirect and direct evidence that a soluble mediator such as TNF-beta can induce chromosomal alterations commonly associated with different types of tumors.
Asunto(s)
Linfocitos B/inmunología , Aberraciones Cromosómicas , Neoplasias/genética , Neoplasias/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Femenino , Reordenamiento Génico de Linfocito B , Humanos , Linfotoxina-alfa/biosíntesis , Masculino , Células Tumorales Cultivadas/inmunologíaRESUMEN
Clonal expansion of cells with a certain abnormal chromosome constitution has been demonstrated in EBV-transformed lymphoblastoid cell lines, and a new monoclonal tumorigenic cell line (48,XX,+X,+12) has been established. Based on these data and our previous hypothesis that predisposition to tumor development could be associated with inherited susceptibility of certain chromosomes and chromosome regions to breakage, we emphasize that cytogenetic analysis of immortalized cell populations established from normal individuals and cancer patients might be of great importance for predicting development of different primary and secondary malignancies.
Asunto(s)
Línea Celular Transformada , Cromosomas Humanos Par 12 , Células Clonales , Trisomía , Cromosoma X , Animales , Neoplasias de la Mama/genética , Transformación Celular Neoplásica , Transformación Celular Viral , Bandeo Cromosómico , Herpesvirus Humano 4 , Humanos , Cariotipificación , Metafase , Ratones , Ratones Desnudos , Neoplasias Experimentales/etiologíaRESUMEN
Karyotypes of 36 lymphoblastoid cell lines established by Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) of eight normal individuals and 28 patients with various nonhematologic malignancies were analyzed. In seven lines (19.4%), cells with trisomy 12 were noted, with clonality in two of these lines. In two of 11 metaphases with such trisomy, chromosome 12 was involved in structural rearrangements [t(8;12)(q12;p12) and t(12;12)(q11;q24)]. No cells with trisomy 12 were observed in phytohemagglutinin (PHA)-stimulated PBL cultures of these individuals. In 250 individuals (normal and with nonhematologic malignancies) examined in our laboratory in the last 5 years, extra copies of chromosome 12 in PHA-stimulated PBL cultures were observed in only five of 23,216 cells (0.02%). There were no cases of clonality in these samples. The frequency of an extra chromosome 12 was comparable to that of the other chromosomes except 21 and X, whose frequency of occurrence was 0.08% and 0.09%, respectively. These findings should be considered random events in PHA-stimulated PBL. On the contrary, in lymphoblastoid cell lines established by EBV transformation, trisomy of chromosome 12 was the most frequent numerical abnormality. It was observed in 64.7% of all cases with chromosome gains and therefore could not be considered a random occurrence. The specificity of this phenomenon for EBV transformation is supported by the results of cytogenetic analysis of eight lymphoblastoid cell lines established by an alternative procedure in our laboratory [1]. In 400 cells analyzed not a single cell with trisomy 12 was observed. We suggest that EBV transformation might either randomly induce formation of such cells in immortalized B-cell populations or show potentially blastomogenic cells or proneness to their formation in certain individuals who could be predisposed to develop lymphoproliferative diseases, especially chronic lymphocytic leukemia (CLL) in which trisomy of chromosome 12 is the most common alteration.
Asunto(s)
Neoplasias de la Mama/genética , Cromosomas Humanos Par 12 , Neoplasias del Colon/genética , Neoplasias/genética , Trisomía , Línea Celular Transformada , Herpesvirus Humano 4/genética , Humanos , Leucemia/genéticaAsunto(s)
Corteza Suprarrenal/metabolismo , Corticosterona/metabolismo , Animales , Masculino , Perfusión , Ratas , Ratas EndogámicasRESUMEN
Total fraction of histones increased the permeability of isolated lysosomes as showed the elevation in nonsedimented and free enzymatic activity of the matrix. S-like shape of curves, observed in dose-dependent effect of the histones increased concentrations on the enzymatic activity, indicated the structure transformations of membranes occurring by the cooperative type. Membranotropic effect of histones exceeded the action on non-ionic detergent Triton X-100. Hydrophobic interactions of histones with lysosomal membranes appear to be responsible for the effects observed.
Asunto(s)
Histonas/metabolismo , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Bovinos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Embrión de Pollo , Histonas/farmacología , Técnicas In Vitro , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/enzimología , Hígado/análisis , Ratones , Ratones Endogámicos C3H , Ribonucleasas/metabolismo , Timo/análisisRESUMEN
Histones at concentration of 2-10 micrograms/ml activated the mitochondrial cytochrome oxidase and at concentration of 25 micrograms/ml and higher--inhibited the enzyme in vitro. Cytochrome oxidase was completely inactivated by histones at concentration of 100 mg/microliter and higher. After administration in vivo histones modified the reaction of liver mitochondria in response to the subsequent treatment with a non-ion detergent Triton X-100. Possible pathogenetic importance of the phenomena observed is discussed.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Histonas/farmacología , Mitocondrias Hepáticas/enzimología , Animales , Relación Dosis-Respuesta a Droga , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Activación Enzimática , Femenino , Técnicas In Vitro , Ratones , Ratones Endogámicos C3H , RatasRESUMEN
Total histones increased the permeability of lysosomal membranes for enzymes of matrix in the preparation of isolated lysosomes, where free and nonsedimented activity of acid phosphatase was estimated. The phenomenon depended on concentration of histones in a mixture. Histones apparently destructed the lysosomal membranes at concentration of 100 mg/ml and higher, since the level of free and nonsedimented activity of acid phosphatase approximated and even exceeded the enzymatic activity after treatment of the lysosomes with 0.2% Triton X-100. These properties of histones depended on their macromolecular structure. Histones did not affect the activity of the solubilized enzyme.