Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer

Abstract

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. (J Mol Diagn 2016, 18: 688e696; http://dx

A novel multimarker assay for the phenotypic profiling of circulating tumor cells in hepatocellular carcinoma

“…Circulating tumor cells (CTCs) are thought to originate from the primary tumor or metastatic sites, can be detected in the peripheral blood, and are implicated as a potential cause of postsurgical recurrence and metastases . Although CTCs can serve as prognostic biomarkers in solid tumors, studies evaluating CTCs in HCC have found limited utility .…”

“…detected in the peripheral blood, and are implicated as a potential cause of postsurgical recurrence and metastases. (4,5) Although CTCs can serve as prognostic biomarkers in solid tumors, studies evaluating CTCs in HCC have found limited utility. (6,7) One reason is that most CTC enrichment assays, including the US Food and Drug Administration-approved CellSearch CTC Assay, rely on the use of antibodies against the epithelial cell surface marker called epithelial cell adhesion molecule (EpCAM) to "capture" CTCs by antigen-specific immunomagnetic separation from leukocytes.…”

Circulating Tumor Cells Predict Occult Metastatic Disease and Prognosis in Pancreatic Cancer

“…8 We have previously used the single-cell sequencing ability of the microfluidic NanoVelcro CTC assay to validate our immunocytochemical (ICC) CTC definition by confirming the tumor origin of PDAC CTCs through demonstration of mutational congruence between the primary tumor and the isolated CTCs. 9 We then used our PDAC CTC assay to show that CTCs are present in the majority of PDAC patients, but not patients with non-malignant pancreatic disease, and that CTCs may have utility as a diagnostic biomarker at the time of disease presentation. 10 …”

Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis

“…This is not an obstacle in settings such as fecal microbiome studies, where abundant organisms are present, but in other scenarios (e.g., in scenarios with low-biomass environmental samples and uncultured bacteria or in single cell analysis), the number of cells and the amount of genetic material can be limited (13)(14)(15)(16)(17). It is in these situations that whole-genome amplification (WGA) has the potential to play an important role in pathogen detection.…”