Detection plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis

Recent arguments have suggested that tumor DNA in cancer patients could be found in plasma, but different points remain unclear, Using a series of 117 head and neck squamous cell carcinoma tumors, our goals for this study wer e: (a) to quantify the amount of plasma DNA; (b) to evaluate the presence o f plasma tumor DNA; and (c) to analyze the clinical relevance of tests base d on plasma DNA analyses. Low levels of plasma DNA were found in most sampl es, but all were successfully amplified, Two different methods:were used to detect tumor-specific genetic alterations: (a) microsatellite instability at UT5085 with an established sensitivity of 1:500; and (b) p53 mutation sc reening. Of the 117 tumors typed at UT5085, 65 demonstrated bandshifts (55% ). Plasma and tumor DNA a showed similar alteration in only one case among these samples, and the prevalence of tumor DNA in plasma was estimated to b e <2% using microsatellite analysis, Tumor DNA was detected in plasma at a higher prevalence (2 of 11 cases) when using p53 mutant allele-specific amp lification. These results showed that in plasma, tumor DNA is largely dilut ed by normal DNA, By comparison with previously published studies, the prev alence of microsatellite alterations in plasma in this series of head and n eck squamous cell carcinomas is very low, despite the fact that a large ser ies of tumors was analyzed. To explain this discrepancy, we analyzed the po ssibility of PCR artifacts as suspected by the presence of loss of heterozy gosity in two plasma DNA samples without a similar tumor DNA alteration. Wh en DNA concentrations were under the threshold of detection (<100 ng/ml), w e demonstrated that PCR artifacts could occur at random, and, if misinterpr eted, these false genetic alterations could artificially enhance the freque ncy of plasma DNA alterations. This may have been suspected in previously p ublished series, but it has never been discussed before. :Microsatellite an alysis on plasma DNA is difficult to interpret and can frequently be mislea ding. Plasma DNA should be analyzed with very sensitive and specific method s such as mutant allele-specific amplification, which excludes artifacts bu t requires specific optimization that is probably not compatible with routi ne and clinical use.