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    • Finally we sequenced the DNA extracted from the

    2018-10-23

    Finally, we sequenced the DNA extracted from the white-blood glucokinase inhibitor (WBC) of 39 cfDNA TP53-positive patients, from which material was available (19 cases and 20 controls). Five cfDNA TP53 mutations (from one case and four controls) were detected in the WBC DNA, with similar AFs to those found in the cfDNA (Table 2). For one control (MLT-14), the AFs in both cfDNA and WBC DNA were around 50%, being consistent with a heterozygous germ-line variant. The other four mutations were detected at AFs consistent with a somatic origin (AFs below 11%) in both cfDNA and WBC DNA (Table S2). Taken altogether, cancer-like TP53 mutations were identified in 25 of the 225 non-cancer controls analyzed in this study (11.1%). We checked if the presence of TP53 mutations in the controls was correlated with age, smoking status, or alcohol (adjusting one for the other), but none of these factors was found to be associated.
    Discussion Inactivation of TP53 by mutation has been reported to occur in over 90% of SCLC cases (George et al., 2015). In this study we were able to detect TP53 mutations in the cfDNA of 49% SCLC patients and, when stratifying by stage, in the cfDNA of 35.7% early-stage cases. These proportions matched those reported for other cancer types (Bettegowda et al., 2014). Unfortunately, we did not have the correspondent tumors to confirm that the TP53 mutations detected in the cfDNA originate from the SCLC tumors. However, our method detected TP53 mutations in 60% of the cfDNA samples of an independent French series of 10 SCLC patients (all of them carrying TP53 somatic mutations in their tumors). Importantly, each of the TP53 mutations found in the cfDNA matched the one found in the SCLC tumor (data not shown). We also observed cfDNA TP53-mutated fragments in 11.4% of 123 matched non-cancer controls. Acknowledging the potential for bias in our selection of controls (such as differential performance in QC criteria or cfDNA amount, between cases and controls), we screened a second series of 102 non-cancer controls, and found a comparable proportion of TP53 mutated samples in vascular system independent group (13 TP53 mutations in 11 controls, 10.8%). Altogether, the detection of TP53 mutations in 11.1% of the 225 non-cancer controls, from two independent groups of samples, suggests that the presence of circulating-mutated fragments among individuals without any diagnosed cancer is a common occurrence, and poses serious challenges for the development of ctDNA screening tests for the early detection of cancer. Only two other studies have explored the potential presence of circulating-mutated fragments in non-cancer subjects. A study within the EPIC prospective cohort (GENAIR) that used blood samples from controls, found that KRAS and TP53 mutations were detectable in the cfDNA of 1% and 3.2% healthy subjects, respectively, without a cancer diagnoses five years subsequent to blood draw (Gormally et al., 2006). The higher percentage of TP53-positive controls in our analyses is likely to be explained by the fact that these analyses within EPIC were undertaken using DHPLC (denaturing high-pressure liquid chromatography) and Sanger sequencing, and these techniques are less sensitive glucokinase inhibitor and only allow for detection of mutations with allelic fractions of 3% or more. Further, only TP53 exons five to nine were analyzed. If we limited our analysis to mutations from exons five to nine and AFs greater that 3%, we would have found a comparable number of TP53-positive controls (2.7%, 6/225). More recently, Krimmel and colleagues have reported extremely low-frequency cancer-like TP53 mutations in the peritoneal fluid from both women with ovarian cancer and those with benign lesions, using duplex sequencing (Krimmel et al., 2016). They also showed that low frequency TP53 mutagenesis increases with age and cancer. Overall, these results support the need for further ctDNA studies to incorporate series of non-cancer controls in order to improve validation of detection and analysis techniques.