Single-Cell Analysis of Somatic Mutations in Human Bronchial Epithelial Cells in Relation to Aging and Smoking

While lung cancer risk among smokers is dependent on smoking dose, it remains unknown if this increased risk reflects an increased rate of somatic mutation accumulation in normal lung cells. Here we applied single-cell whole genome sequencing of proximal bronchial basal cells from 33 subjects, aged between 11 and 86 years, with smoking histories varying from never smoking to 116 pack years. We found an increase in the frequency of single-nucleotide variants and small insertions and deletions with chronological age in never-smokers, with mutation frequencies significantly elevated among smokers. However, when plotted against smoking pack-years, mutations were found to follow the linear increase in cancer risk only until about 23 pack years, after which no further increase in mutation frequency was observed, pointing towards individual selection for mutation avoidance. Known lung cancer-defined global mutation signatures tracked with both age and smoking. Within study power limits, no significant enrichment for somatic mutations in lung cancer driver genes was observed.]]>
The 31 clinical subjects were recruited at Montefiore Medical Center among individuals undergoing bronchoscopy for clinical purposes. Bronchoscopy was typically performed for lung nodule evaluation (e.g., possibility of cancer), for concern over opportunistic/exotic infections, or for exploring structural anomalies, and other clinical indications. Normal PBBCs of two healthy teenage subjects were obtained commercially from Lonza Walkersville Inc.]]>
By written informed consent, under protocol approved by the Institutional Review Board/Ethics Committee at Einstein-Montefiore, consenting subjects permitted pre-procedure interview, four additional research-directed bronchial brushings, and electronic medical record verification of clinical, imaging, and pathologic data. For the patients with carcinomas (squamous cell carcinomas or carcinoma in situ, as well as those with adenocarcinomas), a brush biopsy of normal bronchial tissue was taken from a contralateral site, distant from the tumor of clinical concern. Additionally, a representative sampling of 14 subjects revealed 12/14 with normal cytopathology, and 2/14 remaining subjects with mild atypia, per highly experienced, donor phenotype-blinded cytopathologist. There were no overt dysplastic or malignant cells in any of these 14 samples.]]>