We have a new paper at Sci Rep “High degree of polyclonality hinders somatic mutation calling in lung brush samples of COPD cases and controls”.
It took a long time from my initial grant application at Sander Stiftung in Dec 2009 (where it was rejected), to the field work within the scope of the EvA study (where the PI Loems Ziegler-Heitbrock retired), followed by some first analysis together with Francesc at CNAG in Barcelona until the final publication. My gratulations to Gian-Andri and Ivo Gut and his lab for their hard work!
Chronic obstructive pulmonary disease (COPD) is induced by cigarette smoking and characterized by inflammation of airway tissue. Since smokers with COPD have a higher risk of developing lung cancer than those without, we hypothesized that they carry more mutations in affected tissue.
We called somatic mutations in airway brush samples from medium-coverage whole genome sequencing data from healthy never and ex-smokers (n=8), as well as from ex-smokers with variable degrees of COPD (n=4). Owing to the limited concordance of resulting calls between the applied tools we built a consensus, a strategy that was validated with high accuracy for cancer data.
However, consensus calls showed little promise of representing true positives due to low mappability of corresponding sequence reads and high overlap with positions harbouring known genetic polymorphisms. A targeted re-sequencing approach suggested that only few mutations would survive stringent verification testing and that our data did not allow the inference of any difference in the mutational load of bronchial brush samples between former smoking COPD cases and controls.
So we would have probably needed a higher genome coverage on our brush sample mix? Or should we have sequenced more single cells as discussed in the paper?
I am rather sure, however, that in this (pre-cancer) COPD epithelium there are less pre-malignant lesions than usually expected. In spite of higher susceptibility to lung cancer in COPD patients, sequencing to an intermediate read depth did not provide the resolution to detect somatic mutations in COPD airway brush samples.
What could be the reason? An answer may be in the recent Sanchez-Vega paper who analysed the Cancer Genome Atlas (TCGA) and found an accumulation of mutations usually in only one of ten signalling pathways. My hypothesis right now – there is no gradually accumulation of mutations (until the second hit) but there is a clonal expansion of a single bronchial cell, hit by a single smoke stream. With this hypothesis, smoking would not kill by accumulation of deleterious mutations, it would kill by repetition until the ultimate deleterious mutation occurs (eg some of the RTK_RAS hits described by Sanchez-Vega. This particular cell may even produce its own passenger support.
Many of these timeline studies will be necessary to explain why the tumor risk decreases after quitting smoking. At least this paper may be some good starting point.