¿Es posible utilizar las muestras de colecistectomías con cáncer en investigación?: Calidad del ADN de muestras obtenidas del sistema público y privado de salud / Quality of DNA from archival pathological samples of gallbladder cancer

Background: The quality of the archival samples stored at pathology services could be a limiting factor for molecular biology studies. Aim: To determine the quality of DNA extracted from gallbladder cancer samples at different institutions. Material and Methods: One hundred ninety four samples coming from fve medical centers in Chile, were analyzed. DNA extraction was quantifed determining genomic DNA concentration. The integrity of DNA was determined by polymerase chain reaction amplification of different length fragments of a constitutive gene (β-globin products of 110, 268 and 501 base pairs). Results: The mean DNA concentration obtained in 194 gallbladder cancer samples was 48 ± 43.1 ng/µl. In 22% of samples, no amplification was achieved despite obtaining a mean DNA concentration of 58.3 ng/ul. In 81, 67 and 22% of samples, a DNA amplification of at least 110, 268 or 501 base pairs was obtained, respectively. No differences in DNA concentration according to the source of the samples were demonstrated. However, there were marked differences in DNA integrity among participating centers. Samples from public hospitals were of lower quality than those from private clinics. Conclusions: Despite some limitations, in 80% of cases, the integrity of DNA in archival samples from pathology services in our country would allow the use of molecular biology techniques.

Análisis de expresión global del cáncer cérvico uterino: rutas metabólicas y genes alterados / Global expression analysis in uterine cervical cancer: Metabolic pathways and altered genes

High risk human papillomavirus (HPV) infection is considered to be the most important etiological factor of Cervical Uterine Cancer. In order to determine the global expression pattern and to identify possible molecular markers of cervical cancer, cDNA arrays with probe sets complementary to 8,000 human genes were used to examine the expression profiles among 5 cell lines derived from human cervical cancer, three HPV16(+) tumor samples and three normal cervical tissues HPV(-). The levels of expression of different cellular processes were identified. Hierarchical clustering was performed and the gene expression using RT-PCR was confirmed. Two genes were found to be consistently overexpressed in invasive cervical cancer biopsies; one of them, IL-6 was previously reported to be overexpressed in cervical cancer and one novel gene, MMP10, previously not known to be related to cervical cancer. Hierarchical clustering of the expression data revealed that samples with common HPV type infection grouped together, maybe this could mean that differences between HPV types could be indirectly determined by expression profiles.

La infección por virus de papiloma de alto riesgo (VPH) es considerada como el factor etiológico más importante del cáncer cérvico uterino (CaCU). Con el fin de determinar el patrón de expresión global e identificar algunos posibles genes marcadores del CaCU, se utilizaron microhileras de DNA que contenían 8,000 secuencias que codificaban para transcritos diferentes, para estudiar los perfiles de expresión de cinco líneas celulares derivadas de CaCU, tres muestras tumorales conteniendo VPH 16 y tres muestras normales negativas para la presencia de VPH. Se identificaron los niveles de expresión de genes relacionados con diferentes rutas metabólicas. Se llevó a cabo el análisis de agrupamiento jerárquico y posteriormente se confirmó la sobrexpresión de dos genes mediante RT-PCR. Estos dos genes se encontraron sobrexpresados en biopsias tumorales cervicales. Uno de ellos, el gen de IL6, que ha sido previamente reportado en relación con CaCU, así como el gen de la matriz-metaloproteasa 10 (MMP10) por primera vez relacionado con esta neoplasia. El análisis de agrupamiento jerárquico, además, reveló que las muestras que contienen el mismo tipo viral están asociadas, sugiriendo posibles diferencias en expresión entre tipos virales.

Cáncer de mama: contenido de ADN como factor pronóstico / Breast cancer: DNA content as prognostic factor

El estudio y caracterización biológica en el cáncer de la mama permitir adoptar terapéuticas acordes con el grado de agresividad biológica. La utilidad de la determinación del contenido de ADN en el cáncer de mama ha demostrado resultados contradictorios. Se determinó el contenido de ADN de 100 carcinomas de la mama mediante citofotometría y su relación con: tipo histológico, atipia, grado tumoral, mitosis y presencia de metástasis. El promedio de edad del grupo fue de 57,1 años (26-86 años). En 77 casos (77 por ciento) se realizó mastectomía. El tamaño promedio de los tumores fue 3,5 cm (DSñ 2,0 cm). El carcinoma ductal infiltrante fue el más frecuente en 95 casos (95 por ciento). En 37 de los 84 casos (44 por ciento) con linfoadenectomía axilar se encontró metástasis. En el 60 por ciento de los casos se observó contenido aneuploide de ADN. El índice promedio de ADN en los tumores aneuploides fue de 1,2 (DSñ 0,29). No se observaron diferencias entre los tumores diploides y aneploides respecto de la edad, raza, procedimiento quirúrgico ni tamaño tumoral. La frecuencia de aneuploidia fue mayor en relación al grado de atipia (grado I 28,6 por ciento, grado III 68 por ciento), mitosis (I 55 por ciento, III 60 por ciento) y grado tumoral (grado I 65 por ciento, grado III 70 por ciento), sin embargo, estas diferencias no fueron significativas. En los tumores diploides de ADN se observó un 29 por ciento de metástasis linfática, en cambio, en los tumores aneuploides un 55 por ciento (p= 0,02). Nuestro estudio demuestra la relación existente entre contenido de ADN y la presencia de metástasis linfáticas en el cáncer de mama y su potencial uso como factor pronóstico

Loss of heterozygosity for chromosome 11 in Epstein-Barr-virus associated nasopharyngeal carcinoma.

In order to demonstrate and define possible tumor suppressor gene loci on chromosome 11 associated with NPC, we used 7 STR to test for LOH on 25 NPC samples. LOH was detected in 46 per cent of cases. Most LOH loci were clustered on the long arm. Further study demonstrated 22 per cent and 45.5 per cent of cases with LOH on 11q13 and 11q23 respectively.

Use of PCR-PIRA for screening of a point mutation at codon 12 in K-ras oncogene obtained from paraffin embedded tissue sections.

Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results.