Circulating tumor cells in hepatocellular carcinoma: a pilot study of detection, enumeration, and next-generation sequencing in cases and controls

Abstract

BackgroundCirculating biomarkers are urgently needed in hepatocellular carcinoma (HCC). The aims of this study were to determine the feasibility of detecting and isolating circulating tumor cells (CTCs) in HCC patients using enrichment for epithelial cell adhesion molecule (EpCAM) expression, to examine their prognostic value, and to explore CTC-based DNA sequencing in metastatic HCC patients compared to a control cohort with non-malignant liver diseases (NMLD).MethodsWhole blood was obtained from patients with metastatic HCC or NMLD. CTCs were enumerated by CellSearch then purified by immunomagnetic EpCAM enrichment and fluorescence-activated cell sorting. Targeted ion semiconductor sequencing was performed on whole genome-amplified DNA from CTCs, tumor specimens, and peripheral blood mononuclear cells (PBMC) when available.ResultsTwenty HCC and 10 NMLD patients enrolled. CTCs ≥ 2/7.5 mL were detected in 7/20 (35%, 95% confidence interval: 12%, 60%) HCC and 0/9 eligible NMLD (p = 0.04). CTCs ≥ 1/7.5 mL was associated with alpha-fetoprotein ≥ 400 ng/mL (p = 0.008) and vascular invasion (p = 0.009). Sequencing of CTC DNA identified characteristic HCC mutations. The proportion with ≥ 100x coverage depth was lower in CTCs (43%) than tumor or PBMC (87%) (p < 0.025). Low frequency variants were higher in CTCs (p < 0.001).ConclusionsCTCs are detectable by EpCAM enrichment in metastatic HCC, without confounding false positive background from NMLD. CTC detection was associated with poor prognostic factors. Sequencing of CTC DNA identified known HCC mutations but more low-frequency variants and lower coverage depth than FFPE or PBMC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1195-z) contains supplementary material, which is available to authorized users.

Circulating Tumor Cells for Predicting the Prognostic of Patients with Hepatocellular Carcinoma: A Meta Analysis

“…4E). RRs for Serum AFP level were available in 8 studies [24,27,30,31,34,37,40,42]. The estimated pooled RRs indicated that CTC was associated with AFP≥400 ng/mL (RR 2.05, …”

Improving the CellSearch® system

“…The CellSearch test used for the FDA clearance has also been tested in many other cancers [15], and the presence of CTC is also associated with poor clinical outcome in gastric cancer [16,17], small-cell lung cancer [18][19][20], melanoma [21,22], endometrial cancer [23], esophageal squamous-cell carcinoma [24,25], cholangiocarcinoma [26], colon cancer, liver metastasis [27], hepatocellular carcinoma [28], pancreatic cancer [29], rectal cancer [30], bladder cancer [31], head and neck cancer [32], and ovarian cancer [33]. Publications referred to above are just examples.…”

“…An inevitable step therefore is to pick and isolate the single CTCs in individual tubes or as a pure CTC fraction for genomic analysis. Methods used to isolate single cell for individual analysis in combination with CellSearch-enriched CTCs: Micromanipulation [113], ALS Cellselector [114], the Silicon Biosystems DEPArray [115][116][117][118], FACS [28,119,120], and the Punch system using self-seeding Microwell Chips [121]. This step seems a minor intermediate part of the whole workflow but is crucial for the end result.…”

Relationship between hepatocellular carcinoma circulating tumor cells and tumor volume

“…CTCs have been enriched through a variety of technologies, with the majority utilizing positive CTC antigen capture or size based filtration methods (Yu et al, 2011b). The broadly used Veridex platform utilizing EpCAM positive immunomagnetic enrichment demonstrated 35% and 41% capture rate in two independent HCC studies (Kelley et al, 2015;Sun et al, 2013). This relatively low CTC detection rate is not unexpected given that HCC has the lowest expression level of EpCAM compared to all of the major epithelial cancers as represented in the Cancer Cell Line Encyclopedia (Barretina et al, 2012) (Additional file 1: Figure S1).…”