Quantitative Whole Genome Sequencing of Circulating Tumor Cells Enables Personalized Combination Therapy of Metastatic Cancer

Abstract

Much effort has been dedicated to developing circulating tumor cells (CTC) as a noninvasive cancer biopsy, but with limited success as yet. In this study, we combine a method for isolation of highly pure CTCs using immunomagnetic enrichment/fluorescence-activated cell sorting with advanced whole genome sequencing (WGS), based on long fragment read technology, to illustrate the utility of an accurate, comprehensive, phased, and quantitative genomic analysis platform for CTCs. Whole genomes of 34 CTCs from a patient with metastatic breast cancer were analyzed as 3,072 barcoded subgenomic compartments of long DNA. WGS resulted in a read coverage of 23× per cell and an ensemble call rate of >95%. These barcoded reads enabled accurate detection of somatic mutations present in as few as 12% of CTCs. We found in CTCs a total of 2,766 somatic single-nucleotide variants and 543 indels and multi-base substitutions, 23 of which altered amino acid sequences. Another 16,961 somatic single nucleotide variant and 8,408 indels and multi-base substitutions, 77 of which were nonsynonymous, were detected with varying degrees of prevalence across the 34 CTCs. On the basis of our whole genome data of mutations found in all CTCs, we identified driver mutations and the tissue of origin of these cells, suggesting personalized combination therapies beyond the scope of most gene panels. Taken together, our results show how advanced WGS of CTCs can lead to high-resolution analyses of cancers that can reliably guide personalized therapy. .

Genomic and expression profiling reveal molecular heterogeneity of disseminated tumor cells in bone marrow of early breast cancer

“…Previous studies have demonstrated the robustness of the IE/FACS method for detection and isolation of highly pure CTCs (>90%), 8,9 and downstream molecular analyses have confirmed the malignant nature of IE/FACS-isolated CTCs. 8–10 …”

“…1 Our group has used EPCAM-based immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) for detection and isolation of circulating tumor cells (CTC) from blood of cancer patients. 8,9 This method involves an initial IE step using magnetic beads coated with monoclonal antibody to EPCAM, followed by FACS to detect and purify CTCs away from blood cells. Previous studies have demonstrated the robustness of the IE/FACS method for detection and isolation of highly pure CTCs (>90%), 8,9 and downstream molecular analyses have confirmed the malignant nature of IE/FACS-isolated CTCs.…”

“…8,9 This method involves an initial IE step using magnetic beads coated with monoclonal antibody to EPCAM, followed by FACS to detect and purify CTCs away from blood cells. Previous studies have demonstrated the robustness of the IE/FACS method for detection and isolation of highly pure CTCs (>90%), 8,9 and downstream molecular analyses have confirmed the malignant nature of IE/FACS-isolated CTCs. 8–10 …”

Introductory Chapter: Current Studies in Transcriptional Control System; Toward the Establishment of Therapies against Human Diseases

“…Somatic mutations on driver genes in various cancers have been identified [43] and the statistical data will be applied on diagnosis or even on the prediction of cancer risks and incidences [44]. Very recently, it was proposed that analysis of WGS data from circulating tumor cells could be applied for personalized therapy for malignant cancer [45]. Importantly, mutations in 5′-upstream region of the human TERT gene are frequently identified in melanoma [46,47].…”

The integration of genomics testing and functional proteomics in the era of personalized medicine

“…Intra-tumor heterogeneity promotes therapeutic resistance through clonal replacement, whereas cancer cell plasticity promotes recurrence through the reactivation of dormant cancer stem cells [31]. A more comprehensive molecular view of cancer is necessary to enable the precise selection of combinatorial targeted therapies for tumor elimination with minimal side effects [32]. Therapeutic resistance to RTK inhibitors is classified into innate resistance, owing to the insufficient activation of the targeted RTK, the aberrant activation of other RTKs that bypass the targeted therapy, or canonical WNT ligand-dependent β-catenin signaling activation, and acquired resistance, owing to secondary mutations in the targeted RTK [23,[33][34][35][36].…”