Citation: Shishido, S.N.; Ghoreifi, A.; Sayeed, S.; Courcoubetis, G.; Huang, A.; Ye, B.; Mrutyunjaya, S.; Gill, I.S.; Kuhn, P.; Mason, J.; et al. Liquid Biopsy Landscape in Patients with Primary Upper Tract Urothelial Carcinoma. Cancers 2022, 14, 3007. https://doi.org/10.3390/ cancers14123007 Academic Editor: Sonia Vallet Received: 11 May 2022 Accepted: 10 June 2022 Published: 18 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article Liquid Biopsy Landscape in Patients with Primary Upper Tract Urothelial Carcinoma Stephanie N. Shishido 1,† , Alireza Ghoreifi 2,† , Salmaan Sayeed 1 , George Courcoubetis 1 , Amy Huang 1 , Brandon Ye 1 , Sankalp Mrutyunjaya 1 , Inderbir S. Gill 2,3 , Peter Kuhn 1,2,3,4, * , Jeremy Mason 1,2,3, * ,‡ and Hooman Djaladat 2,3,‡ 1 Convergent Science Institute in Cancer, Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA; sshishid@usc.edu (S.N.S.); salmaans@usc.edu (S.S.); courcoub@usc.edu (G.C.); awhuang@usc.edu (A.H.); branye@usc.edu (B.Y.); mrutyunj@usc.edu (S.M.) 2 Catherine and Joseph Aresty Department of Urology, Institute of Urology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; alireza.ghoreifi@med.usc.edu (A.G.); igill@med.usc.edu (I.S.G.); djaladat@med.usc.edu (H.D.) 3 Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 4 Department of Biological Sciences, Dornsife College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, CA 90089, USA * Correspondence: pkuhn@usc.edu (P.K.); masonj@usc.edu (J.M.); Tel.: +1-213–821–3980 (P.K.) † These authors contributed equally to this work. ‡ These authors contributed equally to this work. Simple Summary: The standard of care for patients diagnosed with upper tract urothelial carcinoma (UTUC) is surgery. However, the current standard tools for the diagnosis and preoperative risk stratification of primary UTUC leave much to be desired. Using a simple blood sample, we can detect rare events, such as circulating tumor cells (CTCs) and large extracellular vesicles (LEVs), which may reveal biomarkers indicative of disease and provide insight into progression. This study demonstrates a wide variety of CTCs and LEVs that are identifiable in the blood of patients with UTUC at higher counts compared to normal donors. The data presented indicate that a blood-based liquid biopsy can detect UTUC and help guide clinical decisions. Abstract: Urothelial carcinomas (UCs) are a broad and heterogeneous group of malignancies, with the prevalence of upper tract urothelial carcinoma (UTUC) being rare, accounting for only 5–10% of total malignancies. There is a need for additional toolsets to assist the current clinical paradigm of care for patients with UTUC. As a non-invasive tool for the discovery of cancer-related biomarkers, the liquid biopsy has the potential to represent the complex process of tumorigenesis and metastasis. Herein, we show the efficacy of the liquid biopsy as a source of biomarkers for detecting UTUC. Using the third-generation high-definition single-cell assay (HDSCA3.0) workflow, we investigate liquid biopsy samples collected from patients with UTUC and normal donors (NDs) to provide critical information regarding the molecular and morphological characteristics of circulating rare events. We document several important findings from the liquid biopsy analysis of patients diagnosed with UTUC prior to surgery: (1) Large extracellular vesicles (LEVs) and circulating tumor cells (CTCs) are detectable in the peripheral blood. (2) The rare-event profile is highly heterogeneous. (3) Clinical data elements correlate with liquid biopsy analytes. Overall, this study provides evidence for the efficacy of the liquid biopsy in understanding the biology of UTUC with the future intent of informing clinical decision making, ultimately improving patient outcomes. Keywords: upper tract urothelial carcinoma; liquid biopsy; HDSCA; circulating tumor cell; large extracellular vesicle; peripheral blood Cancers 2022, 14, 3007. https://doi.org/10.3390/cancers14123007 https://www.mdpi.com/journal/cancers