Abstract 631: Utilizing Vortex Chip for enumeration and determination of single-cell heterogeneity of circulating tumor cells in prostate cancer

148 Background: Molecular characterization of metastatic castration resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTC) offers an attractive noninvasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in mCRPC patients. Methods: Blood samples were drawn from 11 enzalutamide or abiraterone pre-treated mCRPC patients enrolled in the clinical program MOSCATO (NCT02613962). CTC enrichment, immunofluorescent detection and single cell isolation were performed using three methods (ISET filtration, CellSearch and the VyCap puncher system and RosetteSep enrichment) to obtain pools of 1-10 CTCs with distinct epithelial or mesenchymal phenotypes. After Whole Genome Amplification (WGA), WES was performed on the Illumina HiSeq 2000 platform. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively. Results: 189 WGA of CTC pools were performed. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5-10 cells. 17/34 (50%) CTC samples had shared sSNV with the paired tumor sample (range 0.35%-68%) Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large non epithelial CTC pointing out a high level of genetic heterogeneity between CTC. Overall, 89 deleterious protein-coding mutations were found only in pools of CTC, including mutations affecting oncogenic drivers such as MAPK1, HSP90AB1 or KDM5B. Conclusions: We present single cell WES of CTCs harboring distinct phenotypes. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity.

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Prevalence and tissue concordance of BRCA2 copy number loss evaluated by single-cell, shallow whole genome sequencing of circulating tumor cells (CTCs) in castration-resistant prostate cancer (CRPC).

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.5531 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 5531-5531

Author(s):

Ethan Barnett ◽

Joseph Schonhoft ◽

Nikolaus D. Schultz ◽

Jerry Lee ◽

Samir Zaidi ◽

...

Keyword(s):

Prostate Cancer ◽

Whole Genome Sequencing ◽

Single Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Genome Sequencing ◽

Copy Number ◽

Cellular Heterogeneity ◽

Cancer Tissue ◽

Whole Genome

5531 Background: Genomic studies have shown that up to 25% of prostate cancer tissue specimens harbor alterations in DNA Damage Repair (DDR) genes, which may sensitize the tumor to poly ADP-ribose polymerase inhibitors (PARPi). Trials evaluating PARPi in patients with DDR deficiencies have shown varied response rates and differences regarding which genomic alterations predict for sensitivity to these agents, with the majority of objective responses seen in BRCA2-altered tumors. These results highlight the need to develop biomarker assays which can predict benefit from PARPi therapy. Tissue and cell-free DNA (cfDNA) have been the most utilized sources of tumor material for analysis in this setting, but success rates of obtaining sufficient tumor for analysis from bone are low and detecting tumor-derived copy number variants (CNVs) in cfDNA is challenging. Circulating tumor cells (CTCs) represent an alternate source of genetic information, for which assays are available to isolate and sequence individual cells in a manner that eliminates background noise from stroma and healthy cells, while capturing inter-cellular heterogeneity. Methods: Blood samples, collected from 138 progressing metastatic CRPC patients within 30 days of a pre-treatment biopsy intended for sequencing using MSK-IMPACT, were sent to EPIC Sciences for CTC analysis. Detected CTCs underwent single cell, low pass whole genome sequencing. Prevalence and concordance of BRCA2 copy-loss, regardless of whether single copy or homozygous, was compared in matched tissue and CTC samples. Results: BRCA2 copy-loss was identified in 21% (23/108) and 50% (58/115) of successfully sequenced tissue and CTC samples, respectively. In the 58 patients with CTC-detected BRCA2 loss, BRCA2 loss was detected in 36% (220/565) of the sequenced CTCs, representing a median of 46% (range 4-100%) of CTCs found in each individual sample. When both sequencing assays were successful, BRCA2 loss was detected in CTCs in 84% (16/19) of the tissue-positive cases, whereas tissue sequencing detected BRCA2 loss in 35% (16/46) of CTC-positive cases. Conclusions: Data from this study supports the notion that single-cell CTC sequencing can detect BRCA2 copy-loss at a high frequency, including cases that were negative in tissue, while also characterizing inter-cellular heterogeneity. Further studies will investigate whether CTC BRCA2 copy-loss can predict the likelihood of response to PARPi.

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Single Cell RNA Profiling of Circulating Tumor Cells in Patients With Prostate Cancer

International Journal of Radiation Oncology*Biology*Physics ◽

10.1016/j.ijrobp.2015.07.250 ◽

2015 ◽

Vol 93 (3) ◽

pp. S104 ◽

Cited By ~ 3

Author(s):

D.T. Miyamoto ◽

Y. Zheng ◽

B. Wittner ◽

R.J. Lee ◽

H. Zhu ◽

...

Keyword(s):

Prostate Cancer ◽

Single Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Rna Profiling

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Size and deformability based separation of circulating tumor cells from castrate resistant prostate cancer patients using resettable cell traps

Lab on a Chip ◽

10.1039/c5lc00226e ◽

2015 ◽

Vol 15 (10) ◽

pp. 2278-2286 ◽

Cited By ~ 48

Author(s):

Xi Qin ◽

Sunyoung Park ◽

Simon P. Duffy ◽

Kerryn Matthews ◽

Richard R. Ang ◽

...

Keyword(s):

Prostate Cancer ◽

Spectral Analysis ◽

Single Cell ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Castrate Resistant Prostate Cancer ◽

Castrate Resistant

Separation of CTCs using resettable cell traps followed by single-cell spectral analysis.

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Single‐cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT‐related genes in metastatic prostate cancer

The Prostate ◽

10.1002/pros.22625 ◽

2012 ◽

Vol 73 (8) ◽

pp. 813-826 ◽

Cited By ~ 146

Author(s):

Chun‐Liang Chen ◽

Devalingam Mahalingam ◽

Pawel Osmulski ◽

Rohit R. Jadhav ◽

Chiou‐Miin Wang ◽

...

Keyword(s):

Prostate Cancer ◽

Single Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Metastatic Prostate Cancer ◽

Single Cell Analysis ◽

Expression Patterns ◽

Cell Analysis

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Single Cell Characterization of Prostate Cancer-Circulating Tumor Cells

10.21236/ada596639 ◽

2013 ◽

Author(s):

James D. Brooks

Keyword(s):

Prostate Cancer ◽

Single Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Cell Characterization

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Genomic heterogeneity of circulating tumor cells in castration-resistant prostate cancer (CRPC) revealed by single-cell sequencing.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.5030 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 5030-5030

Author(s):

Allison Welsh ◽

Daniel Costin Danila ◽

Aseem Anand ◽

Jude Kendall ◽

Charles L. Sawyers ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Sequencing ◽

Single Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Single Cells ◽

Dna Amplification ◽

Copy Number Variations ◽

Castration Resistant Prostate Cancer ◽

Blood Draw

5030 Background: Circulating tumor cells (CTC) provide an opportunity to sample multiple metastatic tumor sites through a single blood draw – a ”fluid biopsy.” NextGen DNA sequencing provides the means to obtain detailed genetic information from captured cells prior to and during treatment. Here we demonstrate the use of DNA sequencing to interrogate genome-wide copy number variations (CNV) at the single-cell level in CTC isolated from pts with CRPC. Methods: Pre- and post-treatment blood samples were obtained from pts treated at MSKCC. EpCAM+ events were collected singly and in groups by cytometric flow sorting and were subjected to DNA amplification and Illumina NextGeneration sequencing. Parallel samples were assayed using the Veridex CellSearch method to ensure the presence of malignant cells. Results: Samples with up to 50 EpCAM+ events analyzed in bulk displayed CNV patterns expected from published CRPC data. Subsequent single cell analyses showed that the method could reliably detect common genomic markers in CRPC, including AR amplification, PTEN and RB1 loss, and the TMPRSS-ERG fusion. Individual genomic CNV profiles obtained from 125 single cells isolated from 15 patients were then analyzed. Using unsupervised clustering, cells from each pt showed a closely related lineage structure, consistent with an evolution from a common ancestor. The degree of genomic heterogeneity within CTC from an individual pt was highly variable, with R2 correlation coefficients ranging from >0.92 (nearly homogeneous) to <0.75 (mixed populations). Two pts harbored separate subpopulations with both amplified AR and non-amplified AR cells and another displayed mixtures of genetic markers that changed over the course of treatment. Conclusions: The observed variation in complexity of CTC populations in CRPC pts underscores the importance of being able to sample and analyze multiple cells from an individual pt on multiple occasions and with real time analytics. Doing so is essential to understand and identify mechanisms of resistance so that they can be targeted effectively. Supported by STARR Cancer Consortium, NCI SPORE in Prostate Cancer; Department of Defense; Prostate Cancer Foundation.

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