High degree of polyclonality hinders somatic mutation calling in lung brush samples of COPD cases and controls

Abstract

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. High polyclonality in airway brush samples renders medium-depth sequencing insufficient to provide the resolution to detect somatic mutations. Deep sequencing data of airway biopsies are needed to tackle the question.

Figure 1

Numbers of somatic single-nucleotide mutation (SSM) calls in lower lobe bronchial brushings per subject. Numbers represent the intersected results of three somatic mutation calling tools. Shades of blue represent COPD cases (dark), ex-smokers without COPD (azure) and never smokers (light).

Figure 2

Characteristics of somatic single-nucleotide mutation calls in lung brushings of subjects from the EvA study (N = 12) as well as in tumour tissue of two cancer cases based on the same calling procedure. Coverages and VAFs were based on the Strelka results. MB = medulloblastoma, CLL = chronic lymphocytic leukemia, DHS = DNase I hypersensitive sites, IQR = interquartile range, VAF = variant allele frequency.

Figure 3

Representation of mutant clones in the bronchial epithelium. Coloured cylinders represent different cellular clones of recent origin in the airway epithelium, each containing cells that share the same somatic mutations. Such a clone (shown for the one in salmon at the bottom) may contain a mixture of cell types, such as differentiated secretory cells (shown with granules in the cytoplasm), ciliated cells (depicted with cilia at the outer surface) and unstratified basal cells (square shaped), all sitting on the basement membrane (line at the very bottom). Such a cellular arrangement is named pseudostratified columnar epithelium and is typical for the airways. Clone size may vary, but presumably lies in the sub-millimeter range, which is much smaller than the diameter and the length of a brush used in bronchoscopy (shown on the right). Consequently, a brush sample contains many different clones and the proportion of cells carrying the same somatic mutations is very small and falls below the detection limit. Successful somatic mutation calling requires sampling a smaller area (i.e. coming closer to the detection limit by harvesting a higher proportion of cells carrying the same somatic mutations) and/or sequencing to a higher read depth (i.e. lowering the detection limit).