title: Circulating tumor cells in hepatocellular carcinoma: a pilot study of detection, enumeration, and next-generation sequencing in cases and controls.
abstract: BACKGROUND
  Circulating 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).
  
  
  METHODS
  Whole 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.
  
  
  RESULTS
  Twenty 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).
  
  
  CONCLUSIONS
  CTCs 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.
authors.affiliation: Helen Diller Family Comprehensive Cancer Center and The Liver Center, University of California San Francisco (UCSF), 550 16th St., Box 3211, San Francisco, CA, 94143, USA. Katie.kelley@ucsf.edu.
authors.forename: Robin K
authors.initials: RK
authors.lastname: Kelley
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA, 94143, USA. Mark.Magbanua@ucsf.edu.
authors.forename: Mark Jesus M
authors.initials: MJ
authors.lastname: Magbanua
authors.affiliation: Department of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Mail Code #L103, Portland, OR, 97239, USA. butlerti@ohsu.edu.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Butler
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA, 94143, USA. Eric.Collisson@ucsf.edu.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Collisson
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA, 94143, USA. jhwang@cc.ucsf.edu.
authors.forename: Jimmy
authors.initials: J
authors.lastname: Hwang
authors.affiliation: University of Vermont Medical Center, 89 Beaumont Ave., Burlington, VT, 05405, USA. Nikoletta.Sidiropoulos@vtmednet.org.
authors.forename: Nikoletta
authors.initials: N
authors.lastname: Sidiropoulos
authors.affiliation: Department of Pathology, UCSF, 513 Parnassus Ave., San Francisco, CA, 94143, USA. Kimberley.Evason@ucsfmedctr.org.
authors.forename: Kimberley
authors.initials: K
authors.lastname: Evason
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA, 94143, USA. McWhirterR@cc.ucsf.edu.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: McWhirter
authors.affiliation: Division of Hepatology and Liver Transplant, UCSF, 513 Parnassus Ave., S-357, San Francisco, CA, 94143, USA. Bilal.Hameed@ucsf.edu.
authors.forename: Bilal
authors.initials: B
authors.lastname: Hameed
authors.affiliation: Department of Transplantation-Abdominal, UCSF, 513 Parnassus Ave., S-357, San Francisco, CA, 94143, USA. Elizabeth.Wayne@ucsf.edu.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Wayne
authors.affiliation: Division of Hepatology and Liver Transplant and The Liver Center, UCSF, 513 Parnassus Ave., S-357, San Francisco, CA, 94143, USA. Francis.Yao@ucsf.edu.
authors.forename: Francis Y
authors.initials: FY
authors.lastname: Yao
authors.affiliation: Helen Diller Family Comprehensive Cancer Center and The Liver Center, University of California San Francisco (UCSF), 550 16th St., Box 3211, San Francisco, CA, 94143, USA. venook@cc.ucsf.edu.
authors.forename: Alan P
authors.initials: AP
authors.lastname: Venook
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA, 94143, USA. jpark@cc.ucsf.edu.
authors.forename: John W
authors.initials: JW
authors.lastname: Park
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000951:Antigens, Neoplasm / Q000097:blood / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000235:genetics* / Q000473:pathology; D015815:Cell Adhesion Molecules / Q000097:blood / Q000235:genetics*; D000071858:Epithelial Cell Adhesion Molecule; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053208:Kaplan-Meier Estimate; D008107:Liver Diseases / Q000097:blood / Q000235:genetics / Q000473:pathology; D008113:Liver Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating; D020641:Polymorphism, Single Nucleotide; D011379:Prognosis
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule