*Article* **Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients**

**Mouadh Barbirou <sup>1</sup> , Amanda Miller <sup>1</sup> , Yariswamy Manjunath 2,3, Arturo B. Ramirez <sup>4</sup> , Nolan G. Ericson <sup>4</sup> , Kevin F. Staveley-O'Carroll 2,3,5, Jonathan B. Mitchem 2,3,5 , Wesley C. Warren 5,6, Aadel A. Chaudhuri 5,7 , Yi Huang 5,7, Guangfu Li 2,3,5, Peter J. Tonellato <sup>1</sup> and Jussuf T. Kaifi 1,2,3,5,\* ,†**


**Abstract:** Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide. Liquid biopsy by a blood draw to detect circulating tumor cells (CTCs) is a tool for molecular profiling of cancer using single-cell and next-generation sequencing (NGS) technologies. The aim of the study was to identify somatic variants in single CTCs isolated from NSCLC patients by targeted NGS. Thirty-one subjects (20 NSCLC patients, 11 smokers without cancer) were enrolled for blood draws (7.5 mL). CTCs were identified by immunofluorescence, individually retrieved, and DNA-extracted. Targeted NGS was performed to detect somatic variants (single-nucleotide variants (SNVs) and insertions/deletions (Indels)) across 65 oncogenes and tumor suppressor genes. Cancer-associated variants were classified using OncoKB database. NSCLC patients had significantly higher CTC counts than control smokers (*p* = 0.0132; Mann–Whitney test). Analyzing 23 CTCs and 13 white blood cells across seven patients revealed a total of 644 somatic variants that occurred in all CTCs within the same subject, ranging from 1 to 137 per patient. The highest number of variants detected in ≥1 CTC within a patient was 441. A total of 18/65 (27.7%) genes were highly mutated. Mutations with oncogenic impact were identified in functional domains of seven oncogenes/tumor suppressor genes (*NF1, PTCH1, TP53, SMARCB1, SMAD4, KRAS,* and *ERBB2*). Single CTC-targeted NGS detects heterogeneous and shared mutational signatures within and between NSCLC patients. CTC single-cell genomics have potential for integration in NSCLC precision oncology.

**Keywords:** circulating tumor cells; non-small-cell lung cancer; single cell next generation sequencing
