title: Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) in muscle invasive and metastatic bladder cancer patients.
abstract: BACKGROUND
  While programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes.
  
  
  METHODS
  Blood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)+and (CK)-CTCs (CD45-, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis.
  
  
  RESULTS
  CTCs were detected in 20/25 (80 %) patients, inclusive of CK+CTCs (13/25, 52 %), CK-CTCs (14/25, 56 %), CK+CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1+CTCs; 4 of these patients had exclusively CK-/CD45-/PD-L1+CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1+/CD45-CTC burden and low burden of apoptotic CTCs had worse overall survival.
  
  
  CONCLUSIONS
  CTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK+and CK-CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients.
authors.affiliation: Division of Hematology-Oncology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, 1825 4th Street, 6th Floor, San Francisco, CA, 94158, USA.
authors.forename: Archana
authors.initials: A
authors.lastname: Anantharaman
authors.affiliation: Division of Hematology-Oncology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, 1825 4th Street, 6th Floor, San Francisco, CA, 94158, USA. Terence.Friedlander@ucsf.edu.
authors.forename: Terence
authors.identifier: http://orcid.org/0000-0002-3630-4941
authors.initials: T
authors.lastname: Friedlander
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Lu
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Krupa
authors.affiliation: Department of Urology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, CA, USA.
authors.forename: Gayatri
authors.initials: G
authors.lastname: Premasekharan
authors.affiliation: Division of Hematology-Oncology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, 1825 4th Street, 6th Floor, San Francisco, CA, 94158, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hough
authors.affiliation: Division of Hematology-Oncology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, 1825 4th Street, 6th Floor, San Francisco, CA, 94158, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Edwards
authors.affiliation: Division of Hematology-Oncology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, 1825 4th Street, 6th Floor, San Francisco, CA, 94158, USA.
authors.forename: Rosa
authors.initials: R
authors.lastname: Paz
authors.affiliation: Department of Urology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, CA, USA.
authors.forename: Karla
authors.initials: K
authors.lastname: Lindquist
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: Ryon
authors.initials: R
authors.lastname: Graf
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Jendrisak
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Louw
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: Lyndsey
authors.initials: L
authors.lastname: Dugan
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Baird
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: Yipeng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Epic Sciences, San Diego, CA, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Dittamore
authors.affiliation: Division of Hematology-Oncology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, 1825 4th Street, 6th Floor, San Francisco, CA, 94158, USA.|Department of Urology, Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, CA, USA.
authors.forename: Pamela L
authors.initials: PL
authors.lastname: Paris
keywords: Biomarkers; Bladder cancer; Circulating tumor cells; Liquid biopsy; PD-L1