T-blocker: a simple and robust probe-free quantitative PCR assay to detect somatic mutations down to 0.1% frequency.

We have developed a simple and robust probe-free quantitative PCR (qPCR) assay method that can detect minor mutant alleles with a frequency as low as 0.1% in a heterogeneous sample by introducing a novel T-blocker concept to the allele-specific PCR method. Four new KRAS and BRAF mutation detection assays were developed and their performance was demonstrated by testing a large number of replicates, utilizing a customized PCR protocol.

Aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokers.

An important method for silencing tumor suppressor genes in cancers is by aberrant methylation (referred to as methylation) of CpG islands in gene promoter regions. In lung cancer, methylation of the genes retinoic acid receptor beta-2 (RARbeta-2), CDH13 (H-cadherin), p16(INK4a) (p16), RASSF1A (RAS association domain family I) is frequent. Thus, we investigated methylation of these genes in 4 different types of specimens (oropharyngeal brushes, sputum samples, bronchial brushes and bronchioloalveolar lavage [BAL] samples) of the upper aerodigestive tract epithelium from heavy smokers without evidence of cancer but with morphometric evidence of sputum atypia and compared the frequencies of methylation in the different types of specimens.

Aberrant methylation of the HIC1 promoter is a frequent event in specific pediatric neoplasms.

Purpose: To determine the role of methylation of HIC1, a candidate tumor suppressor gene on 17p13.3, in various types of pediatric tumors.

Experimental Design: We examined the methylation status of the HIC1 promoter by methylation specific PCR in 157 pediatric tumors and 27 nonmalignant tissues.

Frequent hypermethylation of the 5' CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer.

Chfr, a mitotic stress checkpoint gene, regulates a prophase delay in cells exposed to agents that disrupt microtubules, such as nocodazole and taxol. In the present study, we report that Chfr is frequently methylated in cell lines derived from tumors of the colon (80%), brain (100%) and bone (100%). In addition, Chfr was methylated in 37% of primary colon adenocarcinomas and in 10% of primary non-small cell lung carcinomas.

Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer.

Aberrant methylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers. There are major smoke exposure, histology, geography and gender-related changes in non-small cell lung cancer (NSCLC). We investigated smoking-related, histologic, geographic and gender differences in the methylation profiles of resected NSCLCs.

Methylation profiles of sporadic ovarian tumors and nonmalignant ovaries from high-risk women.

Purpose: The purpose of this research was to examine the DNA methylation profiles of primary sporadic ovarian cancers and ovarian tissues from high-risk women.

Experimental Design: We analyzed the DNA methylation status of nine cancer-related genes in 49 primary ovarian tumors, 39 nonmalignant ovarian tissues obtained from 16 women with no known risk and from 23 high-risk women with a strong family history of breast and/or ovarian cancer or BRCA1 germ-line mutations, and 11 ovarian cancer cell lines, by methylation-specific PCR.

Results: Our findings are as follows: (a) methylation rates of four of nine genes, RASSF1A (41%), HIC1 (35%), E-cadherin (29%), and APC (18%) were significantly higher in tumors compared with controls.

Hierarchical clustering of lung cancer cell lines using DNA methylation markers.

Recent analyses of global and gene-specific methylation patterns in cancer cells have suggested that cancers from different organs demonstrate distinct patterns of CpG island hypermethylation. Although certain CpG islands are frequently methylated in many different kinds of cancer, others are methylated only in specific tumor types. Because distinct patterns of CpG island hypermethylation can be seen in tumors from different organs, it seems likely that histological subtypes of cancer within a given organ may exhibit distinct methylation patterns as well.

Aberrant promoter methylation profile of prostate cancers and its relationship to clinicopathological features.

Purpose: We investigated the aberrant methylation profile of prostate cancers and correlated the data with clinical findings.

Experimental Design: Gene promoter methylation was analyzed in 101 prostate cancer samples. In addition, we analyzed 32 nonmalignant prostate tissue samples, which included 25 with benign disease, benign prostatic hypertrophy, or prostatitis, and 7 normal tissues adjacent to cancer.