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Highlights • CTCs in NSCLC correlate with the presence of Treg cells expressing checkpoint markers in the tumor microenvironment. • Treg cells in NSCLC express CCR5, a new class of drug targets in inhibiting tumor growth and metastasis. • NSCLC tumors have a signature that is predictive of the presence of CTCs. • Highly multiplexed immune profiling reveals different patterns of expression in diploid compared to aneuploidy tumor cells from the same tumor. • Highly sensitive CTC enumeration methodology., Non-small cell lung cancer (NSCLC) has a poor prognosis. Targeted therapy and immunotherapy in recent years has significantly improved NSCLC patient outcome. In this study, we employed cell-by-cell immune and cancer marker profiling of the primary tumor cells to investigate possible signatures that might predict the presence or absence of circulating tumor cells (CTCs). We performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples. The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization and stained with a total 25 immune, cancer markers and DNA content dye and analyzed with high-parameter flow cytometry. CTCs were isolated and analyzed from the paired peripheral blood. We investigated a total of 74 biomarkers for their correlation with CTC number. Strong correlations were observed between CTC number and the frequency of immune checkpoint marker expressing lymphocytes (CTLA-4, LAG3, TIM3, PD-1), within the CD103+CD4+ T lymphocyte subset. CTC number is also correlated with the frequency of PD-L1 expressing cancer cells and cancer cell DNA content. In contrast, CTC number inversely correlated to the frequency of CD44+E-cadherin− cancer cells. Unsupervised clustering analysis based on the biomarker analysis separated the CTC negative patients from the CTC positive patients. Profiling multiple immune and cancer markers on cancer samples with multi-parametric flow cytometry allowed us to obtain protein expression information at the single cell level. Clustering analysis of the proteomic data revealed a signature driven by checkpoint marker expression on CD103+CD4+ T cells that could potentially be predictive of CTCs and targets of therapy.

The potential of circulating tumor cells (CTCs) in the diagnosis and prognosis of cancer patients has become increasingly attractive. However, molecular analysis of CTCs is hindered by low sensitivity and a high level of background leukocytes in CTC enrichment technologies. We have developed a novel protocol using a microfluidic device, which enriches and retrieves CTCs from blood samples. The principle of CTC capturing is that tumor cells are larger and less deformable than normal blood cells. To evaluate the potential of utilizing Celsee PREP100 in CTC molecular analysis, we prepared prostate cancer cell lines PC3 and LNCaP, retrieved the captured cells and analyzed them using PCR amplicon sequencing. We were able to recover an average of 79% of 110‑1,100 PC3 and 60‑1,500 LNCaP cells, and detect the p.K139fs*3 deletion of the p53 gene in PC3 cells and p.T877A mutation of the androgen receptor gene in LNCaP cells. Next, we spiked these two types of cells into normal donor blood samples, captured the cells and analyzed them using PCR amplicon sequencing. The PC3 and LNCaP cells were captured and retrieved with the ratio of captured CTCs to the background leukocytes reaching 1:1.5 for PC3 and 1:2.9 for LNCaP cells. We further revealed that the p.K139fs*3 deletion and p.T877A mutation can be detected in the captured PC3 and LNCaP cells, respectively. We successfully validated this approach using clinical blood samples from patients with metastatic prostate cancer. Our results demonstrated a novel approach for CTC enrichment and illustrated the potential of CTC molecular characterization for diagnosis, prognosis and treatment selection of patients with metastatic malignancy.

Molecular analysis of circulating tumor cells (CTCs) is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment technologies. We have developed a novel device to enrich and retrieve CTCs from blood samples by using a microfluidic chip. The Celsee PREP100 device captures CTCs with high sensitivity and allows the captured CTCs to be retrieved for molecular analysis. It uses the microfluidic chip which has approximately 56,320 capture chambers. Based on differences in cell size and deformability, each chamber ensures that small blood escape while larger CTCs of varying sizes are trapped and isolated in the chambers. In this report, we used the Celsee PREP100 to capture cancer cells spiked into normal donor blood samples. We were able to show that the device can capture as low as 10 cells with high reproducibility. The captured CTCs were retrieved from the microfluidic chip. The cell recovery rate of this back-flow procedure is 100% and the level of remaining background leukocytes is very low (about 300-400 cells). RNA from the retrieved cells are extracted and converted to cDNA, and gene expression analysis of selected cancer markers can be carried out by using RT-PCR assays. The sensitive and easy-to-use Celsee PREP100 system represents a promising technology for capturing and molecular characterization of CTCs.

109 Background: Non-small cell lung cancer (NSCLC) has a poor prognosis as most patients are at advanced stage when diagnosed. Targeted therapy and immunotherapy in recent years has significantly improved NSCLC patient outcome. In this study, we employed cell-by-cell immune and cancer marker profiling of the primary tumor cells to investigate possible signatures that might predict the presence or absence of circulating tumor cells (CTCs). Methods: We performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples. The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization. The single cell suspensions were stained with a total 25 immune, cancer markers and a DNA content dye and analyzed with advanced, high-parameter flow cytometry. CTCs were isolated and analyzed from the paired peripheral blood. Results: Out of the 26 unique cell markers stained, we investigated a total of 72 biomarkers for their correlation with CTC number. Strong correlations were observed between CTC number and the frequency of immune checkpoint marker expressing lymphocytes, especially with the immune checkpoint marker expressing CD103+CD4+ T lymphocytes. CTC number is also correlated with the frequency of PD-L1 expressing cancer cells and cancer cell DNA content. In contrast, CTC number inversely correlated to the frequency of CD44+E-cadherin- cancer cells. Unsupervised clustering analysis based on the biomarker analysis separated the CTC negative patients from the CTC positive patients. Conclusions: Profiling multiple immune and cancer markers on cancer samples with multi-parametric flow cytometry allowed us to obtain protein expression information at the single cell level. Clustering analysis of the proteomic data revealed a signature driven by checkpoint marker expression on CD103+CD4+ T cells that could potentially be predictive of CTCs.

For effective targeted therapeutics, it is critical to identify cancer mutations in cells that are often regional and heterogenic. Recent advances in single-cell mRNA sequencing (scRNA-seq) have made it possible to study intratumor heterogeneity and its role in cancer progression, metastasis, and treatment resistance. Next-generation sequencing (NGS)-based studies have identified critical genetic alterations in a variety of malignancies. However, the current workflow of existing technologies leads to significant loss of cells and thus loss of critical information. In this study we present a technology that decreases cell loss and increases efficiency of scRNA-seq. C-PREP GENESIS is a fully automated, random-access, multiple-sample processor for single-cell sequencing preparation. C-PREP GENESIS allows automated fluid dispensing and controlled pumping through Nanochips. The instrument utilizes unitized reagent cartridge for dispensing reagents for wash, lysis, cDNA synthesis and oil (for partitioning). With its automated thermocycling (4oC- 100oC), it can achieve the single cell to cDNA synthesis on beads with unique cell barcode and UMI tags in under 2 hours with an efficiency of ~80% of the 50,000 single cells captured on the nano-PREP 250K sequencing chip. The cDNA can then be retrieved from the chip for downstream library preparation. Alternatively, users can perform a targeted library prep PCR on the same instrument. Here we present scRNA-seq analysis of single cells obtained from patient tumor biopsy samples and cancer tissue culture single cells to discuss the critical mutations observed in single cells of the heterogenic tumor populations. Citation Format: Vishal Premdev Sharma, William Chiro Chow, Brian Boniface, Kyle Gleason, Austin Payne, Priya Gogoi, Kalyan Handique. High-throughput scRNA-seq analysis using Celsee Nano-prep 250K sequencing chip on Celsee C-PREP GENESIS [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 2347.

Detection of PD-L1 expression in NSCLC cancer patients is increasingly becoming a widely used molecular diagnostic tool. Evidence also suggests that PD-L1 can be induced with radiotherapy and may be an immune escape mechanism in cancer. Current methods analyze PD-L1 expression in either solid tumor biopsies or circulating tumor cells (CTC), independent of each other. In order to design a comprehensive PD-L1 treatment regime, it is imperative to look at the PD-L1 expression in cancer cells of the tumor as well as in CTCs. In this study we present a comprehensive profiling of PD-L1 expression in solid tumor biopsy and CTC of 10 lung cancer patients. We further extend the study by evaluating the co-relation of the PD-L1 expression with other immune cell infiltration markers in the lung biopsy samples. There is no single technology that can provide a comprehensive profiling of PD-L1 expression in lung biopsies as well as the CTCs isolated from the peripheral blood. Using two very different and unique molecular diagnostic technologies, IncellDx OncoTect iO Single Cell Quantitative PD-L1 assay and automated CTC analysis Celsee C-PREP GENESIS, we present the first report discussing the PD-L1 expression in tumor cells of the solid and liquid biopsies and its correlation with DNA content and infiltrating immune cell markers. Nine out of the ten patient samples evaluated showed strong correlation between DNA content and PD-L1 expression in tumor-associated cells and CTC. One aneuploid patient sample showed very low expression of PD-L1 in tumor-associated cancer cells; however, it still showed a positive staining for PD-L1 in CTC. Obtaining a comprehensive PD-L1 expression and associated immune cell marker profile in solid tumor biopsy and circulating tumor cells may be needed to design a more effective and lasting immunotherapy to treat NSCLC lung cancer patients. The results discussed in this study will strongly impact the current understanding and implementation of the molecular diagnostics tools to evaluate cancer patients in order to define a robust therapeutic regimen. Citation Format: Fariba Fazeli, Vishal Premdev Sharma, William Chiro Chow, Amanda Chargin, Priya Gogoi, Kalyan Handique, Bruce Patterson. Characterizing PD-L1 expression in tumor-associated cells and CTC using IncellDx and Celsee molecular diagnostic technologies [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 635.

Tumors are comprised of heterogeneous cells. Only a specific or rare population of tumor cells is capable of metastasizing through blood. While mapping out the genome of cancer cells, gene expression variations indicative of more aggressive cancer cells might be missed among the noise. Therefore, single cell gene expression and mutation analysis provide a more comprehensive and most precise information about the heterogeneity of cancer progression and metastasis. Although there has been a surge in technologies used for selection and analysis of single cells, single cell analysis of rare cells such as circulating tumor cells (CTCs) is fraught with technical challenges. Among many used techniques to isolate single cells, the most common cell separation method is cell sorting by flow cytometry, which requires cell labeling. The numerous steps involved in cell labelling and centrifugation result in significant cell loss and changes in gene expression. In addition, centrifugation may also cause shear-induced gene expression changes in viable cells. For other single cell analysis platforms, there is a minimum input of cells ranging from 200 - 1000 cells. Rare cells like CTCs are found in the range of 1-10 per mL of blood. Another challenge is that the input volume required for these systems is very low usually in microliters. Here, we introduce a novel microfluidic chip with 250,000 microwells which captures cells at 100% efficiency down to one cell with a dynamic range over 5 logs. The underlying principle of this technology is that the geometric pattern of the microfluidic chip captures a single cell from an initial volume of up to 1 mL of fluid due to cell settling. The unique microwell structure promotes easy fluid exchange without perturbing the cells from their captured locations, thereby eliminating cell loss during multistep downstream processing. Preliminary tests have shown that single or cluster of spiked cancer cells in blood can be immunostained, both live or fixed and can also be retrieved very efficiently from this chip using a micro capillary. The retrieved single cells can further be used for several single cell genetic analysis. We will present data describing cancer gene expression and gene mutation analysis in isolated single cells. Citation Format: Kalyan Handique, Priya Gogoi, William Chow, Kyle Gleason, Auston Payne, Vishal Premdev Sharma, Yixin Wang. Celsee™ SingleCell Chip can retrieve rare single cells with 100% efficiency for genetic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4030. doi:10.1158/1538-7445.AM2017-4030

Molecular analysis of circulating tumor cells (CTCs) is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment technologies. The Celsee PREP100 device captures CTCs with high sensitivity and allows the captured CTCs to be retrieved for molecular analysis. To evaluate the potential of utilizing PREP100 in CTC molecular analysis, we first prepared mock samples using cultured prostate cancer cells PC3 and LnCAP and analyzed captured cells by PCR amplicon sequencing. Using Celsee PREP100, we were able to recover the average of 79% (ranging from 40% to 100%) of 110 - 1,100 PC3 cells and 60 - 1,500 LnCAP cells and detect p.K139fs*3 deletion in PC3 cells and T877A mutation in LnCAP cells. In addition, we also spiked in the two cell lines into normal donor blood samples and analyzed the captured cells by PCR amplicon sequencing. Preliminary experiments showed that 40% of 250 spiked PC3 cells and 74% of 50 spiked LnCAP cells were captured. Further, we were able to detect p.K139fs*3 deletion in the retrieved 50 - 1,000 spiked PC3 cells. Our results illustrate that Celsee PREP100 system represents a promising technology for capturing and molecular characterization of CTCs. Citation Format: Ruqin Kou, Eric Zhao, Conrad Leung, Priya Gogoi, Will Chow, Yixin Wang. Circulating tumor cell (CTC) enrichment and DNA mutation detection by sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1720. doi:10.1158/1538-7445.AM2017-1720

e23031Background: Current technologies for the analysis of circulating tumor cells (CTCs) are hindered by sub-optimal sensitivity and specificity as well as the incorporation of clinically relevant...