Quantitative Detection of p53 Mutations in Plasma DNA from Tobacco Smokers

Abstract

In lung tumors, the p53 tumor suppressor gene is commonly mutated with a characteristic mutation spectrum. The amount of and alterations in plasma DNA, such as mutations in p53, were associated with several cancers. Few studies used quantitative methods of high sensitivity. Previously, we observed p53 mutations in the noncancerous tissue that differed from those in lung tumors using the highly sensitive p53 mutation load assay. Based on our observation of an increased p53 mutation load in nontumorous lung tissue in smokers, we hypothesized that plasma DNA may contain mutant p53 indicative of tobacco smoke exposure and will be an effective biomarker of lung cancer or smoking exposure. We modified the p53 mutation load assay to detect mutations at p53 codons 248 and 249, common mutations in lung cancer, in plasma DNA samples with a sensitivity of 1:5,000. The assay was applied to a set of lung cancer cases (n = 39), hospital controls (n = 21), and population controls (n = 20) from a larger study. Controls were selected to consist of equal numbers of both ever and never smokers. The p53 mutation load (mutated p53 copies per total number of p53 copies) was associated with smoking (P = 0.06), but not with lung cancer (P = 0.59). Most of the individuals with p53 mutations observed in plasma DNA were ever smokers and the p53 mutation load was higher in those who smoked for longer durations (P = 0.04). In summary, we were able to detect p53 mutations in plasma DNA from healthy individuals and our data suggest that p53 mutations in plasma DNA may be a marker of carcinogen exposure from tobacco smoke.

Optimizing the yield and utility of circulating cell-free DNA from plasma and serum

“…Some authors use small volumes of elution buffer (50µl) to obtain a concentrated DNA solution for subsequent PCR analysis [24,26]. However, we found that using low volumes of elution buffer led to a significantly lower DNA yield.…”

Molecular Signatures of Environmental Mutagens in Hepatocellular Carcinoma

“…This is however not the only instance where mutations are detectable in blood samples of healthy individuals. In heavy smokers, mutations known to be induced by tobacco carcinogens are detected in CFDNA even in the absence of any deˆned lung pathology (51). In a prospective study on cancer risk in a cohort of neversmokers, we have shown that mutations in TP53 and in KRAS could be detected in CFDNA up to several years ahead of diagnosis of speciˆc cancers including bladder cancer (52).…”

Circulating tumour-derived nucleic acids in cancer patients: potential applications as tumour markers

“…Their findings provided direct evidence for the presence of tumour-derived DNA in the circulation of cancer patients. Recently, studies have also reported that tumour-associated mutations could be found in the plasma/serum of individuals exposed to carcinogens, and the presence of such mutations in the circulation may indicate increased risk of developing cancers (Gormally et al, 2006;Hagiwara et al, 2006). Through the detection of common p53 mutations at codons 273, 249 and 248, Hagiwara et al (2006) reported that p53 mutations were readily detectable in the plasma of some smokers without cancer but not in the plasma of non-smokers.…”

“…However, the design of allele-specific primers requires the sequence information of the mutation, which can only be obtained when the primary tumour tissue is available. Alternatively, hotspot-mutations, for example mutations at codons 248 and 249 of the p53 gene and mutations at the codon 12 of the KRAS gene, can be screened for in the plasma (Gormally et al, 2006;Hagiwara et al, 2006). However, this approach would miss the tumour-associated mutations occurring outside the mutation hot spots and, hence, might reduce the sensitivity of the test.…”