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

Mouadh Barbirou,Arturo B Ramirez,Wesley C Warren,Jussuf T Kaifi,Yi Huang,Nolan G Ericson,Guangfu Li,Amanda Miller,Aadel A Chaudhuri,Peter J Tonellato,Yariswamy Manjunath,Jonathan B Mitchem,Kevin F Staveley-O’Carroll

doi:10.3390/cimb44020052

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.

Highlights

Lung cancer is by far the leading cause of cancer-related deaths worldwide
20 patients were diagnosed with Non-small-cell lung cancer (NSCLC) and 11 subjects were risk-matched controls consisting of longterm smokers without evidence of lung cancer on screening low-dose computed tomography (LDCT)
In contrast to invasive lung cancer tissue biopsies, which are associated with significant morbidities and costs, minimally invasive liquid biopsies by simple blood draws hold great promise to improve clinical management of NSCLC

Summary

Introduction

Lung cancer is by far the leading cause of cancer-related deaths worldwide. Nonsmall-cell lung cancer (NSCLC) accounts for >80% of all lung cancer subtypes [1]. The eligibility of NSCLC patients with advanced disease to receive targeted therapy relies on profiling of driver oncogenes and tumor suppressor genes mutation analyses performed on invasive tumor tissue biopsies [4]. These tumor tissue biopsies are associated with significant morbidities and costs. Due to these limitations, invasive biopsies are typically only performed once and do not reflect tumor evolution over time and development of resistant clones during therapies [5,6]. Developing non-invasive and repeatable, real-time diagnostic tests for NSCLC patients appears critical to improve management [7]

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