High-definition Single Cell Analysis Research Articles

Single cell correlation analysis of liquid and solid biopsies in metastatic colorectal cancer.

As cancer care is transitioning to personalized therapies with necessary complementary or companion biomarkers there is significant interest in determining to what extent non-invasive liquid biopsies reflect the gold standard solid biopsy. We have established an approach for measuring patient-specific circulating and solid cell concordance by introducing tumor touch preparations to the High-Definition Single Cell Analysis workflow for high-resolution cytomorphometric characterization of metastatic colorectal cancer (mCRC). Subgroups of cells based on size, shape and protein expression were identified in both liquid and solid biopsies, which overall displayed high inter- and intra- patient pleomorphism at the single-cell level of analysis. Concordance of liquid and solid biopsies was patient-dependent and between 0.1-0.9. Morphometric variables displayed particularly high correlation, suggesting that circulating cells do not represent distinct subpopulations from the solid tumor. This was further substantiated by significant decrease in concentration of circulating cells after mCRC resection. Combined with the association of circulating cells with tumor burden and necrosis of hepatic lesions, our overall findings demonstrate that liquid biopsy cells can be informative biomarkers in the mCRC setting. Patient-specific level of concordance can readily be measured to establish the utility of circulating cells as biomarkers and define biosignatures for liquid biopsy assays.

Circulating Tumor Cells: High-Throughput Imaging of CTCs and Bioinformatic Analysis.

Circulating tumor cells (CTCs) represent novel biomarkers, since they are obtainable through a simple and noninvasive blood draw or liquid biopsy. Here, we review the high-definition single-cell analysis (HD-SCA) workflow, which brings together modern methods of immunofluorescence with more sophisticated image processing to rapidly and accurately detect rare tumor cells among the milieu of platelets, erythrocytes, and leukocytes in the peripheral blood. In particular, we discuss progress in methods to measure CTC morphology and subcellular protein expression, and we highlight some initial applications that lead to fundamental new insights about the hematogenous phase of cancer, as well as its performance in early-stage diagnosis and treatment monitoring. We end with an outlook on how to further probe CTCs and the unique advantages of the HD-SCA workflow for improving the precision of cancer care.

Abstract 4459: Morphologic and genomic characterization of circulating tumor cells in patients with ERBB2 mutant HER2 non-amplified metastatic breast cancer treated with neratinib

Abstract Metastatic breast cancer patients have a high risk of progression and face poor prognosis overall, with about one third (34%) surviving 5 years or more. In rare instances (2-4% of cases) patients with metastatic breast cancer have ERBB2 (HER2) activating mutations, but are HER2 negative (non-amplified. Neratinib is a potent, irreversible inhibitor that binds HER2 and inhibits downstream signaling. We used the previously validated high-definition single cell analysis (HD-SCA) workflow to investigate the clinical significance of circulating tumor cells (HD-CTCs) in HER2-mutant, HER2 non-amplified, post-menopausal metastatic breast cancer patients starting neratinib and fulvestrant combinational therapy. Genomic analysis of the cell-free DNA (cfDNA) and single cell HD-CTCs was conducted to monitor tumor evolution and identify potential mutational variants unique to the patient’s clinical response. A subset of 5 patients presented here were from the MutHER (NCT01670877) and/or SUMMIT (NCT01953926) trials. Patients had an average of 5.4 lines of therapy before enrollment, variable hormone receptor status, and ERBB2 mutations at diagnosis and during treatment. HD-CTC enumeration alone was not sufficient to predict clinical response. Interestingly, treatment pressure was shown to lead to an observable change in HD-CTC morphology and genomic instability, suggesting these parameters may inform prognosis. Single cell copy number variation (CNV) analysis indicated that the persistence or development of a clonal population of HD-CTCs during treatment was associated with a worse response. Hierarchical clustering analysis of the single cells across all patients and timepoints identified distinct aberrant regions shared among patients, comprised of 26 genes that are similarly affected and may be related to drug resistance. Additionally at the cfDNA level, we identified new mutations in ERBB2, PIK3CA, and TP53 that arose due to treatment pressure in a patient with poor response, further informing us on the dynamics of the cancer genome over the course of therapy. The data presented in this small cohort study demonstrates the feasibility of real-time molecular profiling of the cellular and acellular fractions of the liquid biopsy using the HD-SCA platform. Additional insight is warranted to determine the potential use of morphometric and genomic analysis as a prognostic tool to advance personalized oncology. Citation Format: Stephanie Nicole Shishido, Rahul Masson, Lisa Welter, Liya Xu, Anishka D'Souza, Darcy Spicer, James Hicks, Janice Lu, Peter Kuhn. Morphologic and genomic characterization of circulating tumor cells in patients with ERBB2 mutant HER2 non-amplified metastatic breast cancer treated with neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4459.

Single-Cell Analysis of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are rare cells that can be found in the peripheral blood of cancer patients. They have been demonstrated to be useful prognostic markers in many cancer types. Within the last decade various methods have been developed to detect rare cells within a liquid biopsy from a cancer patient. These methods have revealed the phenotypic diversity of CTCs and how they can represent the complement of cells that are found in a tumor. Single-cell proteogenomics has emerged as an all-encompassing next-generation technological approach for CTC research. This allows for the deconstruction of cellular heterogeneity, dynamics of metastatic initiation and progression, and response or resistance to therapeutics in the clinical settings. We take advantage of this opportunity to investigate CTC heterogeneity and understand their full potential in precision medicine.The high-definition single-cell analysis (HD-SCA) workflow combines detection of the entire population of CTCs and rare cancer related cells with single-cell genomic analysis and may therefore provide insight into their subpopulations based on molecular as well as morphological data. In this chapter we describe in detail the protocols from isolation of a candidate cell from a microscopy slide, through whole-genome amplification and library preparation, to CNV analysis of identified cells from the HD-SCA workflow. This process may also be applicable to any platform starting with a standard microscopy slide or isolated cell of interest.

Abstract 5656: Developing metal-labeled antibody panels for multiplex proteomic characterization of rare circulating tumor cells by imaging mass cytometry

Abstract Imaging mass cytometry (IMC) is a novel technology with the potential of multiplex proteomic profiling of rare circulating and disseminated tumor cells (CTCs and DTCs) and immune cells from liquid biopsies. The high-definition single-cell analysis (HD-SCA) identifies CTCs and DTCs by the virtue of morphometric and fluorescent staining with exact locations of all the cells, which could be further characterized using downstream genomics and proteomics platform. This study aims to develop and employ metal-labeled antibody panels for multiplex proteomic characterization of CTCs, DTCs and immune cells obtained from longitudinal liquid biopsies from cancer patients on the basis of their epithelial-to-mesenchymal transition (EMT) status, cancer stem cell (CSC), dormancy, proliferation and drug resistance markers. For every antibody panel, 6-10 biomarkers were chosen, and metal-labeled antibodies were processed in a three-stage validation program using known positive and negative control cell lines. In the first stage, basal level marker expression was evaluated in positive control cells seeded on a glass slide. In the second stage, cell lines were spiked in blood and slides were prepared as per the HD-SCA protocol, stored at -800 C, and expression of biomarkers was evaluated in IMC staining. In the third stage, cell line-spiked slides were first stained in three colors immunofluorescent staining in HD-SCA, mimicking the clinical setup followed by IMC staining with metal-labeled antibodies. Antibodies showing expressions in all three stages were included to build a panel targeting EMT, CSC, vasculature mimicry, dormancy, proliferation and drug resistance biomarkers. Out of 69 metal-labeled antibodies, 24 antibodies targeting cancer and immune cell-specific biomarkers have been validated for multiplex proteomic profiling in HD-SCA platform. This includes epithelial markers EpCAM and E-cadherin; mesenchymal markers vimentin and fibronectin; CSC markers CD44, CD24, ALDH and beta-catenin; prostate-specific markers PSA, PSMA, AR-C terminal, AR-N terminal; and immune cell markers CD45, CD66a, CD3, HLA-DR, CD45RA, CD8a, CD4, CD38, CD14 and CD31. Building and validating metal-labeled antibody panels for EMT, CSC, dormancy and drug resistance biomarkers could achieve multiplex proteomic characterization of the rare cell IMC. It would be further used in monitoring treatment efficacy, mutational switchovers, and immune surveillance from longitudinal liquid biopsies collected from cancer patients to better understand the biology of a disease. Citation Format: Milind Pore, Erik Gerdtsson, Christopher Boldt, Sophia Wix, Preeya Katti, Mohan Singh, Akil Merchant, James Hicks, Peter Kuhn. Developing metal-labeled antibody panels for multiplex proteomic characterization of rare circulating tumor cells by imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5656.

Abstract 4591: Integration of genomic analysis and assessment of pre-analytic variables in the HD-SCA workflow: A technical validation study

Abstract The liquid biopsy allows assessment of multiple biological analytes over time to provide dynamic temporal information with the potential for improving clinical management and guiding treatment decisions. While the promise of liquid biopsies for prediction and response monitoring are intensely investigated, the pre-analytic variables are of primary concern for its implementation in diagnostic clinical medicine, including such categories as collection method, shipping conditions, and sample storage. Here we utilize an integrated high-definition single cell analysis (HD-SCA) workflow for genomic analysis of rare cells and cfDNA from the liquid biopsy to characterize the effects of pre-analytical variation and reproducibility of data analysis from the same cohort of patients. The results presented here confirm consistent rare cell enumeration and morphometric characterization between 24h and 48h time to assay (TTA), with a high efficiency and capacity for both copy number variation (CNV) and single nucleotide variation (SNV) analysis at the single cell level. Additionally, the freezing process neither diminishes or increases the DNA quantity, nor does it affect the DNA quality for CNV or SNV analysis. The integration of genomic information is imperative to reveal essential disease-implicated mutational profiles, but effective molecular diagnostic tests require reproducible coverage over a broad dynamic range. The performance of the HD-SCA platform quantified here may be utilized as a guide for implementation into patient care and/or research biorepository processes. Citation Format: Stephanie Nicole Shishido, Mariam Rodriguez_Lee, Anand Kolatkar, Liya Xu, Sara Restrepo-Vassalli, Lisa Welter, Anders Carlsson, Emily Greenspan, Shelley Hwang, Kathryn Waitman, Jorge Nieva, Kelley Bethel, James Hicks, Peter Kuhn. Integration of genomic analysis and assessment of pre-analytic variables in the HD-SCA workflow: A technical validation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4591.

Abstract 1576: Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis

Abstract Tumor heterogeneity is prevalent in both treatment-naïve and end-stage metastatic castration-resistant prostate cancer (PCa), and may contribute to the broad range of clinical presentation, treatment response, and disease progression. To characterize molecular heterogeneity associated with de novo metastatic PCa, multiplatform single cell profiling was performed using High Definition Single Cell Analysis (HD-SCA). HD-SCA enabled morphoproteomic and morphogenomic profiling of single cells from touch preparations of tissue cores (prostate and bone marrow biopsies) as well as liquid samples (peripheral blood and bone marrow aspirate). Morphology, nuclear features, copy number alterations, and protein expression were analyzed. Tumor cells isolated from prostate tissue touch preparation (PTTP) and bone marrow touch preparation (BMTP) as well as metastatic tumor cells (MTCs) isolated from bone marrow aspirate were characterized by morphology and cytokeratin expression. Although peripheral blood was examined, circulating tumor cells were not definitively observed. Targeted proteomics of PTTP, BMTP, and MTCs revealed cell lineage and luminal prostate epithelial differentiation associated with PCa, including co-expression of EpCAM, PSA, and PSMA. Androgen receptor expression was highest in MTCs. Hallmark PCa copy number alterations, including PTEN and ETV6 deletions and NCOA2 amplification, were observed in cells within the primary tumor and bone marrow biopsy samples. Genomic landscape of MTCs revealed to be a mix of both primary and bone metastatic tissue. This multiplatform analysis of single cells reveals several clonal origins of metastatic PCa in a newly diagnosed, untreated patient with polymetastatic disease. This case demonstrates that real-time molecular profiling of cells collected through prostate and bone marrow biopsies is feasible and has the potential to elucidate the origin and evolution of metastatic tumor cells. Altogether, biological and genomic data obtained through longitudinal biopsies can be used to reveal the properties of PCa and can impact clinical management. Citation Format: Paymaneh D. Malihi, Michael Morikado, Lisa Welter, Anders Carlsson, Carmen Ruiz Velasco, Anand Kolatkar, Mariam Rodriguez-Lee, James Hicks, Peter Kuhn, Sandy T. Liu, Eric T. Miller, Radu M. Cadaneanu, Beatrice S. Knudsen, Michael S. Lewis, Isla P. Garraway. Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1576.

Abstract 4584: High-definition single-cell analysis of liquid and solid biopsies reveals heterogeneity of circulating tumor cells in metastatic colorectal cancer

Abstract Treatment of cancer patients is transitioning to personalized strategies using targeted drugs with the required companion biomarkers. Many current biomarkers are tissue-based, requiring a solid biopsy and limiting sampling frequency. Given this, there is significant interest in determining whether noninvasive, blood-based liquid biopsies, utilizing measurements of factors that exist in the blood such as circulating tumor cells (CTCs), reflect solid tumors and can serve as substitutes for solid tumor biopsies. Colorectal cancer with hepatic metastases (mCRC) is one of very few scenarios where metastatic patients routinely undergo surgical resection, providing an opportunity for accessible tissue and simultaneous blood sampling. We collected blood and tumor touch preparations from patients undergoing resection of CRC with the purpose to determine the correlation of solid biopsies and the liquid phase of solid tumors in the mCRC setting. Samples were processed using the previously described HD-SCA (High-Definition Single-Cell Analysis) workflow for high-resolution imaging and characterization of tumor cells at a single-cell level. Briefly, tumor cells were identified by presence of nuclear stain, pan-cytokeratin, and absence of CD45. The colon-specific marker CDX2 was included as a fourth color for further characterization. Subsets of cells were subjected to copy number variation (CNV) profiling. The comparison of pre- and post-surgery blood samples showed that the concentration of CTCs decreased significantly after surgery. The concentration was markedly higher in patients with synchronous disease undergoing simultaneous resection of the primary tumor, indicating that the presence of CTCs is related to tumor burden. Principal component analysis demonstrated that the CTCs were more similar to the metastatic cells than to the primary colon cells. While protein levels varied between the liquid and solid biopsy cell populations, morphologic parameters including, e.g., cell and nuclear area and roundness, were similar in the two. The intrapatient analysis showed heterogeneous CTC populations, in general with a small subset of cells bearing characteristics more similar to the solid tumor cells than the bulk of CTCs. The presence of subpopulations was further indicated by the CNV analysis, which showed that only a minority of CTCs had genomic rearrangements and that, in contrast to the solid biopsy cells, the rearranged cells were largely nonclonal. Based on the high-content HD-SCA data, the CTCs were further grouped into different subtypes. In conclusion, we introduce an approach for high-content comparison of liquid and solid biopsies at a single-cell level. We show that the concentration of CTCs is related to tumor burden, and present a subclassification of rare circulating cells in mCRC. Citation Format: Anna S. Gerdtsson, Jana-Aletta Thiele, Randolph Schaffer, Kelly Bethel, Steven Curley, Heinz-Josef Lenz, Diana L. Hanna, Jorge Nieva, Anand Kolatkar, Mariam Rodriguez Lee, James Hicks, Peter Kuhn. High-definition single-cell analysis of liquid and solid biopsies reveals heterogeneity of circulating tumor cells in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4584.

Multiplex protein detection on circulating tumor cells from liquid biopsies using imaging mass cytometry.

Molecular analysis of circulating and disseminated tumor cells (CTCs/DTCs) has great potential as a means for continuous evaluation of prognosis and treatment efficacy in near-real time through minimally invasive liquid biopsies. To realize this potential, however, methods for molecular analysis of these rare cells must be developed and validated. Here, we describe the integration of imaging mass cytometry (IMC) using metal-labeled antibodies as implemented on the Fluidigm Hyperion Imaging System into the workflow of the previously established High Definition Single Cell Analysis (HD-SCA) assay for liquid biopsies, along with methods for image analysis and signal normalization. Using liquid biopsies from a metastatic prostate cancer case, we demonstrate that IMC can extend the reach of CTC characterization to include dozens of protein biomarkers, with the potential to understand a range of biological properties that could affect therapeutic response, metastasis and immune surveillance when coupled with simultaneous phenotyping of thousands of leukocytes.

Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis.

Tumor heterogeneity is prevalent in both treatment-naïve and end-stage metastatic castration-resistant prostate cancer (PCa), and may contribute to the broad range of clinical presentation, treatment response, and disease progression. To characterize molecular heterogeneity associated with de novo metastatic PCa, multiplatform single cell profiling was performed using high definition single cell analysis (HD-SCA). HD-SCA enabled morphoproteomic and morphogenomic profiling of single cells from touch preparations of tissue cores (prostate and bone marrow biopsies) as well as liquid samples (peripheral blood and bone marrow aspirate). Morphology, nuclear features, copy number alterations, and protein expression were analyzed. Tumor cells isolated from prostate tissue touch preparation (PTTP) and bone marrow touch preparation (BMTP) as well as metastatic tumor cells (MTCs) isolated from bone marrow aspirate were characterized by morphology and cytokeratin expression. Although peripheral blood was examined, circulating tumor cells were not definitively observed. Targeted proteomics of PTTP, BMTP, and MTCs revealed cell lineage and luminal prostate epithelial differentiation associated with PCa, including co-expression of EpCAM, PSA, and PSMA. Androgen receptor expression was highest in MTCs. Hallmark PCa copy number alterations, including PTEN and ETV6 deletions and NCOA2 amplification, were observed in cells within the primary tumor and bone marrow biopsy samples. Genomic landscape of MTCs revealed to be a mix of both primary and bone metastatic tissue. This multiplatform analysis of single cells reveals several clonal origins of metastatic PCa in a newly diagnosed, untreated patient with polymetastatic disease. This case demonstrates that real-time molecular profiling of cells collected through prostate and bone marrow biopsies is feasible and has the potential to elucidate the origin and evolution of metastatic tumor cells. Altogether, biological and genomic data obtained through longitudinal biopsies can be used to reveal the properties of PCa and can impact clinical management.

Abstract 4966: High-definition single cell analysis (HD-SCA) reveals enrichment in androgen receptor (AR) expression in tumor cell clusters in bone marrow and blood of metastatic castration-resistant prostate cancer (mCRPC) patients

Abstract Introduction. The analysis of tumor cells in the circulation (CTC) provides an opportunity for non-invasive characterization of mCRPC and monitoring of therapy response, but it is the bone where prostate cancer cells almost consistently migrate to, expand and adapt to therapy. We adapted the HD-CTC assay to bone marrow aspirates (BMA) to discern features most significant to the tumorigenicity and the dynamics of metastatic progression in this disease, and to allow for serial comparison of tumor cells in the circulatory and bone compartments. Since available experimental data suggests that tumor cells organized in clusters are more important contributors to metastasis than single CTC, we evaluated their presence and characteristics in the 2 compartments. Methods. Peripheral blood and BMA obtained through the posterior iliac crest were synchronously collected from individual mCRPC patients (n = 89) while progressing on therapy, and tumor cells in them were detected and characterized using the HD-SCA high-content platform. This flexible non-enrichment-based approach allows for quantification of cell morphometric and protein expression parameters and provides easy access to individual cells and cell clusters for single cell genomics and proteomics analysis. Candidate tumor cells based on cell size and morphology were presented and manually classified as DAPI and cytokeratin-positive and CD45-negative. Clusters were defined as two or more tumor cells in direct contact. AR protein expression and sub-cellular localization were also examined for each tumor cell and tumor cell cluster. Results. Metastatic tumor cells were detected in 24/61 (39%) available BMAs. A greater number of tumor cells were organized in clusters, and clusters were generally larger, in BMAs than in blood (50% vs. 33%, and 20% vs. 5% with 6 or more cells, respectively). In 14 patient-matched and synchronously collected blood and BMA specimens with at least 1 tumor cell, we found 10 (71%) with clusters in the BMA (13-357 clusters/case, with the exception of 1 case that had 1 cluster), while only 3 (21%) had CTC clusters (2-4 clusters/case) (p = 0.02). The 4 cases that had no clusters in the marrow did not have any clusters in the blood. Patients with larger cluster size did significantly worse both in terms of progression-free (p = 0.002) and overall survival (p = 0.02). AR expression in individual cells was significantly higher in those forming part of cell clusters than in single cells, and was positively correlated with cluster size, both in blood and BMAs. Conclusions. Tumor cell clusters are frequent in the circulation and especially in the bone marrow of mCRPC patients whose disease has progressed to therapy. In this setting, the increasing AR expression observed in tumor cell clusters is of probable biological relevance. Citation Format: Amado J. Zurita, Anders Carlsson, Patricia Troncoso, James Hicks, Christopher J. Logothetis, Peter Kuhn. High-definition single cell analysis (HD-SCA) reveals enrichment in androgen receptor (AR) expression in tumor cell clusters in bone marrow and blood of metastatic castration-resistant prostate cancer (mCRPC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4966.

A clinically applicable strategy to concurrently profile prostate cancer (PCa) in circulation and bone using high-definition single cell analysis (HD-SCA).

5041Background: The bone is the most significant organ site to characterize advanced PCa, but repeated bone biopsies are not feasible. Clinically relevant differences may exist between PCa clones i...

777P - High-Definition Single Cell Analysis (Hd-Sca) of Prostate Cancer (Pca) Cells in Matched Bone Marrow and Blood from Patients

ABSTRACT Aim: The approval of multiple therapies with diverse mechanisms of action for advanced PCa highlights a need to refine methods to select patients, monitor therapy effects and develop more efficacious sequences or combinations. The bone marrow is typically enriched for tumor cells in PCa. We applied this knowledge to test the hypothesis that analysis of tumor cells in bone marrow aspirates (BMA) will complement and overcome some of the challenges associated to circulating tumor cell (CTC) evaluation (serial identification, recognition of driver among heterogeneous tumor cell populations). To establish robust single PCa cell characterization for both phenotype and genotype, we developed a novel HD-SCA assay that is applied to blood and BMA specimens. Methods: Matched peripheral blood and BMA (obtained through the posterior iliac crest) were collected synchronously from 9 individual PCa patients. Nucleated cells were stained for cytokeratins (epithelial cell marker), CD45 (leukocyte marker), and androgen receptor (AR), allowing for detailed characterization of morphology, AR expression and its subcellular distribution. For single cell sequencing, candidate tumor cells were relocated and isolated. Using whole genome amplification, high-resolution whole genome copy number variation profiles were obtained from individual cells and tumor microemboli. Results: Eight patients had progressive bone metastatic castration resistant disease and 1 patient had been newly diagnosed with high-risk non-metastatic PCa (therapy naive). The number of cancer cells per mL in the marrow (median 536, range 2-4381) exceeded that in the blood (10, 1-30) in all but the non-metastatic patient, who had 13 CTC/mL and 2 disseminated tumor cells per mL. PCa cells in BMAs showed heterogeneous expression of AR, which we found positive in 2 cases with AR-negative CTC. In the 2 cases with single cell genomic analysis available, we identified cancer sub-clones with different representation in blood versus BMA for both patients. Conclusions: Our results suggest that HD-SCA provides complementary characterization of tumor cells in the fluid phase of blood and bone marrow. Morphologic and genomic profiling of single cells in simultaneously collected marrow and blood may help identify the most relevant drivers of disease progression in the individual PCa patient. Disclosure: All authors have declared no conflicts of interest.