Mutations are believed to accumulate in normal tissues at extremely low levels as a result of exposure to various carcinogenic factors. The degree of accumulation, namely mutation burden, is likely to be associated with cancer risk. However, owing to the limits of current detection methods for such extremely low frequency mutations, the mutation burden present in normal human lung tissues has been unclear. To overcome this limitation, we established a novel method for the quantification of extremely low frequency mutations in DNA samples. Using this method, we aimed to reveal the presence of mutation burden in normal lung tissues and its association with cancer risk.
Somatic mutations were quantified in normal lung tissues without smoking history (n = 11) (“entirely normal lung tissues”:G1), normal lung tissues with smoking history (n = 11) (“smoking-exposed normal tissues”:G2), and non-cancerous lung tissues of patients with lung cancer and smoking history (n = 11) (“smoking-exposed non-cancerous tissues”:G3). A sequence library (15,724 bases of 291 regions of 55 cancer-related genes) was prepared by multiplex PCR using 100 DNA molecules. Libraries were sequenced using a next generation sequencer.
The mutation burden in G3 (2.7 ± 0.8 × 10−5 mutations/base) was significantly higher than that in G1 (1.8 ± 0.5 × 10−5 mutations/base) (p = 0.0189). Accumulation of somatic mutations tended to be associated with increased cancer risk (OR = 3.75; 95% CI = 0.54–26.046). C>T mutations were significantly more frequent in G2 and G3 than in G1, which is in accordance with reported mutation signatures in cancer tissues [Alexandrov et al., Science, 354:2016]. GCC>GTC and CCC>CTC mutations, signatures of exposure to the nitrosamines contained in tobacco smoke, were significantly enriched in G2 and G3.
To the best of our knowledge, this is the first study showing that mutations accumulate in high-risk lung tissues due to exposure to tobacco smoking. This will lead to a novel approach to precision cancer risk diagnosis.
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All authors have declared no conflicts of interest.