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.

A cell-free DNA (cfDNA) assay would be a promising approach to early cancer diagnosis, especially for patients with dense tissues. Consistent cfDNA signatures have been observed for many carcinogens. Recently, investigations of cfDNA as a reliable early detection bioassay have presented a powerful opportunity for detecting dense tissue screening complications early. We performed a prospective study to evaluate the potential of characterizing cfDNA as a central element in the early detection of dense tissue breast cancer (BC). Plasma samples were collected from 32 consenting subjects with dense tissue and positive mammograms, 20 with positive biopsies and 12 with negative biopsies. After screening and before biopsy, cfDNA was extracted, and whole-genome next-generation sequencing (NGS) was performed on all samples. Copy number alteration (CNA) and single nucleotide polymorphism (SNP)/insertion/deletion (Indel) analyses were performed to characterize cfDNA. In the positive-positive subjects (cases), a total of 5 CNAs overlapped with 5 previously reported BC-related oncogenes (KSR2, MAP2K4, MSI2, CANT1 and MSI2). In addition, 1 SNP was detected in KMT2C, a BC oncogene, and 9 others were detected in or near 10 genes (SERAC1, DAGLB, MACF1, NVL, FBXW4, FANK1, KCTD4, CAVIN1; ATP6V0A1 and ZBTB20-AS1) previously associated with non-BC cancers. For the positive–negative subjects (screening), 3 CNAs were detected in BC genes (ACVR2A, CUL3 and PIK3R1), and 5 SNPs were identified in 6 non-BC cancer genes (SNIP1, TBC1D10B, PANK1, PRKCA and RUNX2; SUPT3H). This study presents evidence of the potential of using cfDNA somatic variants as dense tissue BC biomarkers from a noninvasive liquid bioassay for early cancer detection.

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