Tumor-associated DNA has been detected in plasma of colorectal cancer (CRC) patients using various techniques but with limited gene or mutation coverage. We report a highly sensitive scanning methodology for mutational assessment of the APC and TP53 genes, which typically pose an analytical challenge because of their significant genotypic heterogeneity as well as specific mutational scoring assays for K-RAS and BRAF. Plasma DNA isolated from 20 CRC patients were scanned for mutations in these targets without knowledge of the molecular or pathological analyses of the matched primary tumors. We chose mutation scanning technology and these molecular targets to provide a comprehensive screen for somatic mutations known to be associated with sporadic CRC. Mutations were identified with a novel denaturing high-performance liquid chromatography (DHPLC) platform that uses post-separation fluorescence technology to enable the detection of variants that represent <0.1% of the total analyzed DNA. Mutant allele specific amplification (MASA) followed by detection with the same platform was used to identify low-level target mutations (mutation scoring) in K-RAS codons 12, 13, and 61, and BRAF codon 599. Using this combined scanning and scoring approach, we were able to identify at least one mutational event in 20/20 (100%) CRC patients. The thoroughness of a mutation scanning and scoring panel may have important implications for CRC screening and disease monitoring during and following therapy.
The human tumor stem-cell assay was used to investigate the in vitro chemosensitivity of 27 evaluable samples to cisplatin and its analogues, iproplatin and carboplatin, as well as to BCNU, teniposide, vindesine, and dibromodulcitol. All agents exhibited some antitumor activity with the exception of dibromodulcitol (zero response out of 19 evaluable samples). Vindesine, BCNU, and carboplatin were the three most active compounds, with response rates of 29%, 23%, and 22%, respectively. There was a lack of complete cross-resistance between carboplatin and cisplatin as well as between carboplatin and BCNU. Our data suggest that clinical studies with carboplatin and combinations of vindesine plus cisplatin and its analogues may be worthwhile.
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