Cell-Free DNA Variant Sequencing Using CTC-Depleted Blood for Comprehensive Liquid Biopsy Testing in Metastatic Breast Cancer

Abstract

Liquid biopsy analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for personalized treatment. Since cfDNA and CTCs are considered to give additive information and blood specimens are limited, isolation of cfDNA and CTC in an “all from one tube” format is desired. We investigated whether cfDNA variant sequencing from CTC-depleted blood (CTC-depl. B; obtained after positive immunomagnetic isolation of CTCs (AdnaTest EMT-2/Stem Cell Select, QIAGEN)) impacts the results compared to cfDNA variant sequencing from matched whole blood (WB). Cell-free DNA was isolated using matched WB and CTC-depl. B from 17 hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) metastatic breast cancer patients (QIAamp MinElute ccfDNA Kit, QIAGEN). Cell-free DNA libraries were constructed (customized QIAseq Targeted DNA Panel for Illumina, QIAGEN) with integrated unique molecular indices. Sequencing (on the NextSeq 550 platform, Illumina) and data analysis (Ingenuity Variant Analysis) were performed. RNA expression in CTCs was analyzed by multimarker quantitative PCR. Cell-free DNA concentration and size distribution in the matched plasma samples were not significantly different. Seventy percent of all variants were identical in matched WB and CTC-depl. B, but 115/125 variants were exclusively found in WB/CTC-depl. B. The number of detected variants per patient and the number of exclusively detected variants per patient in only one cfDNA source did not differ between the two matched cfDNA sources. Even the characteristics of the exclusively detected cfDNA variants in either WB or CTC-depl. B were comparable. Thus, cfDNA variants from matched WB and CTC-depl. B exhibited no relevant differences, and parallel isolation of cfDNA and CTCs from only 10 mL of blood in an “all from one tube” format was feasible. Matched cfDNA mutational and CTC transcriptional analyses might empower a comprehensive liquid biopsy analysis to enhance the identification of actionable targets for individual therapy strategies.

Targeted deep sequencing revealed variants in cell-free DNA of hormone receptor-positive metastatic breast cancer patients

“…Sufficient sequencing quality of all samples for clinical interpretation had been guaranteed by exclusion of libraries with less than 5 million read fragments, an UMI coverage lower than 400 and if less than 95% of the target region was covered with at least 5% of the mean UMI coverage, as described [16,18]. The QIAGEN Biomedical Genomics Workbench and the Ingenuity Variant Analysis plugin (IVA; QIAGEN) were used for further annotation, scoring, filtering (described previously [16,18]) and interpretation of variants detected in the UMI-based analysis. Called variants were separately annotated for the different transcript isoforms.…”

The combination of CTCs and CEA can help guide the management of patients with SPNs suspected of being lung cancer

“…Circulating tumour cells (CTCs) analysis has recently emerged as a liquid biopsy approach for the early diagnosis [1,2], biomarker [3], evaluation of curative efficacy [4], evaluation of relapse [5] and prognostic prediction [6,7] in several solid tumours, including breast cancer [8,9],gastric cancer [10], prostate cancer [11], head and neck cancer [12], bladder cancer [13], and lung cancer [14][15][16]. CTCs also showed the potential efficacy in metastasis cancer, such as metastatic breast cancer [17,18] and colorectal cancer [19].…”

The combination of CTCs and CEA can help guide the management of patients with SPNs suspected of being lung cancer

“…Circulating tumour cells (CTCs) analysis has recently emerged as a liquid biopsy approach for the early diagnosis [1,2] , biomarker [3] , evaluation of curative efficacy [4] , evaluation of relapse [5] and prognostic prediction [6,7] in several solid tumours, including breast cancer [8,9] ,gastric cancer [10] , prostate cancer [11] , head and neck cancer [12] , bladder cancer [13] , and lung cancer [14][15][16] . CTCs also showed the potential efficacy in metastasis cancer, such as metastatic breast cancer [17,18] and colorectal cancer [19] .…”