Hypermethylation of CDKN2A exon 2 in tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients.

BACKGROUND: Breast carcinogenesis is a multistep process involving genetic and epigenetic changes. Tumor tissues are frequently characterized by gene-specific hypermethylation and global DNA hypomethylation. Aberrant DNA methylation levels have, however, not only been found in tumors, but also in tumor-surrounding tissue appearing histologically normal. This phenomenon is called field cancerization. Knowledge of the existence of a cancer field and its spread are of clinical relevance. If the tissue showing pre-neoplastic lesions is not removed by surgery, it may develop into invasive carcinoma. METHODS: We investigated the prevalence of gene-specific and global DNA methylation changes in tumor-adjacent and tumor-distant tissues in comparison to tumor tissues from the same breast cancer patients (n = 18) and normal breast tissues from healthy women (n = 4). Methylation-sensitive high resolution melting (MS-HRM) analysis was applied to determine methylation levels in the promoters of APC, BRCA1, CDKN2A (p16), ESR1, HER2/neu and PTEN, in CDKN2A exon 2 and in LINE-1, as indicator for the global DNA methylation extent. The methylation status of the ESR2 promoter was determined by pyrosequencing. RESULTS: Tumor-adjacent and tumor-distant tissues frequently showed pre-neoplastic gene-specific and global DNA methylation changes. The APC promoter (p = 0.003) and exon 2 of CDKN2A (p < 0.001) were significantly higher methylated in tumors than in normal breast tissues from healthy women. For both regions, significant differences were also found between tumor and tumor-adjacent tissues (p = 0.001 and p < 0.001, respectively) and tumor and tumor-distant tissues (p = 0.001 and p < 0.001, respectively) from breast cancer patients. In addition, tumor-adjacent (p = 0.002) and tumor-distant tissues (p = 0.005) showed significantly higher methylation levels of CDKN2A exon 2 than normal breast tissues serving as control. Significant correlations were found between the proliferative activity and the methylation status of CDKN2A exon 2 in tumor (r = -0.485, p = 0.041) and tumor-distant tissues (r = -0.498, p = 0.036). CONCLUSIONS: From our results we can conclude that methylation changes in CDKN2A exon 2 are associated with breast carcinogenesis. Further investigations are, however, necessary to confirm that hypermethylation of CDKN2A exon 2 is associated with tumor proliferative activity.

Comparison of protocols for DNA extraction from long-term preserved formalin fixed tissues.

The current study compared the applicability of protocols to extract DNA from formalin fixed heart tissues that have been preserved for more than 50 years. Ten methods were tested: a cetyltrimethylammonium bromide (CTAB) standard protocol, seven variants of this standard protocol, and two commercial kits. In the case of younger specimens (fixed in 1951, 1934, or 1914), extracts with DNA concentrations ≥ 10.0 ng/µl were obtained with the standard CTAB protocol, two variants of the standard protocol including prolonged tissue digestion (72 h instead of 1-2h), and a commercial kit particularly recommended for DNA extraction from formalin fixed paraffin embedded tissues (FFPE Kit). With the FFPE Kit, DNA could also be extracted from older tissues (fixed in 1893, 1850/1851, or before 1820). In general, the purity of the DNA extracts, assessed from the ratio of the absorbance at 260 and 280 nm, was not very high. In spite of their rather low purity, the DNA extracts could, however, be used to amplify a 122-bp sequence and, in most cases, also a 171-bp sequence of the gene coding for human albumin by the polymerase chain reaction (PCR).

Pinacol Coupling Strategy for the Construction of the Bicyclo[6.4.1]tridecane Framework of Schiglautone A.

The synthesis of the tricyclic carbon framework of schiglautone A (1) is reported herein. The generation of the bicyclo[6.4.1]tridecane 19 was accomplished via a SmI2-mediated pinacol coupling of dialdehyde 18. The side chain in 18 was introduced using a selective 1,4-addition. A further key step of the synthesis was the homologation of a Wieland-Miescher ketone derivative to establish the 7-membered ring.

Cervical cancer screening program of Paraná: cost-effective model in a developing country.

The purpose of this study is to report the organization of a cost-effective screening program for cervical cancer in a developing country such as Brazil. The Cervical Cancer Screening Program of Paraná (CCSPP) was launched in October 1997 and was the result of a joint collaboration between the government of Paraná (Secretary of Health of the State of Paraná), scientific societies (pathologists, gynecologists, and nurses), and a non governmental organization called the Women's Popular Forum of Paraná. The main goal of the program was to enhance the Papanicolaou (Pap) smear screening to coverage up to 85% of female adult population with a 3-yr interval between examinations, as well as to reduce the incidence and mortality from cervical cancer in the state of Paraná, a Southern state of Brazil. The cytological findings in all Pap smears recorded in a central computer-based register during 5 yr of the program (October 1997-October 2002) are discussed. During that period, 2,244,158 Pap smears were performed in women included in the program from the 398 cities of the state of Paraná. The cytological smears were analyzed according to the Bethesda System. The previous year, before the program was launched, a Pap smear was taken from 43% of women of Paraná. At the end of 5 yr, coverage was increased to around 86%. The great majority of examinations had a negative result (98%). Only 2% of examinations had cytological abnormalities (n = 44,621). Low-grade lesions predominated in women aged 15-30 yr, and the high-grade lesions were more common in women aged 25-45 yr. Patients older than 40 yr had the greatest incidence of invasive cancer. Although the program is only 5 yr old, a decrease in the mortality from cervical cancer in women from Paraná is clearly apparent: in 1998, 297 women died of cervical cancer, as compared with 188 as of September 2002.