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.

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.

S1. Platelet confirmation with CD61 staining in the SKBR3-spiked NBD sample S2. Enumeration of CTC subtypes S3. Survival analysis using AVPC-MS-CTC status S4. Survival analysis in patients without pc.CTC S5. Treatment decision tree Table S1. Correlation between CTC and clinical characteristics

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.

S1. Platelet confirmation with CD61 staining in the SKBR3-spiked NBD sample S2. Enumeration of CTC subtypes S3. Survival analysis using AVPC-MS-CTC status S4. Survival analysis in patients without pc.CTC S5. Treatment decision tree Table S1. Correlation between CTC and clinical characteristics

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.