Platelet-Coated Circulating Tumor Cells Are a Predictive Biomarker in Patients with Metastatic Castrate-Resistant Prostate Cancer

Abstract

Metastatic castration-resistant prostate cancer (mCRPC) includes a subset of patients with particularly unfavorable prognosis characterized by combined defects in at least two of three tumor suppressor genes: PTEN, RB1, and TP53 as aggressive variant prostate cancer molecular signature (AVPC-MS). We aimed to identify circulating tumor cells (CTC) signatures that could inform treatment decisions of patients with mCRPC with cabazitaxel–carboplatin combination therapy versus cabazitaxel alone. Liquid biopsy samples were collected prospectively from 79 patients for retrospective analysis. CTCs were detected, classified, enumerated through a computational pipeline followed by manual curation, and subjected to single-cell genome-wide copy-number profiling for AVPC-MS detection. On the basis of immunofluorescence intensities, detected rare cells were classified into 8 rare-cell groups. Further morphologic characterization categorized CTC subtypes from 4 cytokeratin-positive rare-cell groups, utilizing presence of mesenchymal features and platelet attachment. Of 79 cases, 77 (97.5%) had CTCs, 24 (30.4%) were positive for platelet-coated CTCs (pc.CTCs) and 25 (38.5%) of 65 sequenced patients exhibited AVPC-MS in CTCs. Survival analysis indicated that the presence of pc.CTCs identified the subset of patients who were AVPC-MS–positive with the worst prognosis and minimal benefit from combination therapy. In AVPC-MS–negative patients, its presence showed significant survival improvement from combination therapy. Our findings suggest the presence of pc.CTCs as a predictive biomarker to further stratify AVPC subsets with the worst prognosis and the most significant benefit of additional platinum therapy. Implications: HDSCA3.0 can be performed with rare cell detection, categorization, and genomic characterization for pc.CTC identification and AVPC-MS detection as a potential predictive biomarker of mCRPC.

Characterization of Cellular and Acellular Analytes from Pre-Cystectomy Liquid Biopsies in Patients Newly Diagnosed with Primary Bladder Cancer

“…For HDSCA analysis, each test consisted of two slides generated from the PB sample for an average of 0.74 mL blood analyzed. Slides were processed at room temperature using the IntelliPATH FLX™ autostainer (Biocare Medical LLC, Irvine, CA, USA) as previously described [12]. Briefly, samples were stained with 2.5 ug/mL of a mouse IgG1 antihuman CD31:Alexa Fluor ® 647 mAb (clone: WM59, MCA1738A647, BioRad, Hercules, CA, USA) and 100 ug/mL of a goat anti-mouse IgG monoclonal Fab fragments (115-007-003, Jackson ImmunoResearch, West Grove, PA, USA), permeabilized using 100% cold methanol, followed by an antibody cocktail consisting of mouse IgG1/Ig2a anti-human cytokeratins (CKs) 1, 4, 5, 6, 8, 10, 13, 18, and 19 (clones: C-11, PCK-26, CY-90, KS-1A3, M20, A53-B/A2, C2562, Sigma, St. Louis, MO, USA), mouse IgG1 anti-human CK 19 (clone: RCK108, GA61561-2, Dako, Carpinteria, CA, USA), mouse anti-human CD45:Alexa Fluor ® 647 (clone: F10-89-4, MCA87A647, AbD Serotec, Raleigh, NC, USA), and rabbit IgG antihuman vimentin (Vim) (clone: D21H3, 9854BC, Cell Signaling, Danvers, MA, USA).…”

“…As previously reported [12], rare cell candidates were detected using a custom computational methodology termed OCULAR (Outlier Clustering Unsupervised Learning Automated Report). Fluorescent images were used to segment each cell using the "EBImage" R package (EBImage_4.12.2) and extract 761 quantitative morphometric parameters based on the nuclear and cytoplasmic morphology and biomarker expression (CK, Vim, CD45/CD31) in a 4-channel immunofluorescence assay (DAPI, AlexaFluor ® 488, AlexaFluor ® 555, AlexaFluor ® 647).…”

“…Rare events were classified into 12 categories (8 cellular, 4 LEV) based on the combination of immunofluorescent marker expression in the previously reported 4 channels. Epitheliallike CTCs (epi.CTCs) were classified as cells that were CK-positive, Vim-negative, and CD45/CD31-negative, with distinct appearing nucleus by DAPI morphology as previously described [12,18]. Epi.CTCs expressing Vim were classified as mesenchymal-like CTCs (mes.CTCs).…”

“…These studies indicate that the presence of CTCs in the PB is an independent predictive indicator of poor outcomes for BCa patients. The work presented here is based on a third-generation comprehensive liquid biopsy [12]. This nonenrichment based, high-content direct imaging methodology is capable of providing both the visualization and characterization of a broad range of CTCs that are present in circulation, along with molecular parameters (DNA and protein) at both the cellular and acellular (large extracellular vesicles (LEVs) and cell-free DNA (cfDNA)) levels.…”

“…This nonenrichment based, high-content direct imaging methodology is capable of providing both the visualization and characterization of a broad range of CTCs that are present in circulation, along with molecular parameters (DNA and protein) at both the cellular and acellular (large extracellular vesicles (LEVs) and cell-free DNA (cfDNA)) levels. We have previously reported the value of single-cell genomic analysis conducted on this platform showing compatibility with clinical practice [12][13][14].…”