Kalyan Handique

A novel microchannel-based device to capture and analyze circulating tumor cells (CTCs) of breast cancer

Circulating tumor cells (CTCs) have been shown in many studies as a possible biomarker for metastasis and may be instrumental for the spread of the disease. Despite advances in CTC capturing technologies, the low frequency of CTCs in cancer patients and the heterogeneity of the CTCs have limited the wide application of the technology in clinic. In this study, we investigated a novel microfluidic technology that uses a size- and deformability-based capture system to characterize CTCs. This unique platform not only allows flexibility in the selection of antibody markers but also segregates the CTCs in their own chambers, thus, enabling morphological, immunological and genetic characterization of each CTC at the single cell level. In this study, different breast cancer cell lines including MCF7, MDA-MB-231 and SKBR3, as well as a panel of breast cancer biomarkers were used to test the device. The technology can capture a wide range of cells with high reproducibility. The capturing efficiency of the cells is greater than 80%. In addition, the background of leukocytes is minimized because individual cells are segregated in their own chambers. The device captured both epithelial cancer cells such as MCF7 and SKBR3 and mesenchymal cells such as MDA-MB-231. Immunostaining of the captured cells on the microchannel device suggests that a panel of breast cancer biomarkers can be used to further characterize differential expression of the captured cells.

Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples

Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.

Microfluidic-Based Enrichment and Retrieval of Circulating Tumor Cells for RT-PCR Analysis

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.

Abstract 4030: Celsee™ SingleCell Chip can retrieve rare single cells with 100% efficiency for genetic analysis

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

Abstract 1391: Gene expression profiling for cancer classification in circulating tumor cells

Background: Enumeration and molecular characterization of circulating tumor cells (CTCs) offer a non-invasive method for tumor analysis, particularly in cases where the biopsy is difficult to obtain or has been exhausted by conventional diagnostic methods. The 92-gene assay (CancerTYPE ID®, bioTheranostics, Inc.) is a clinically-validated cancer classifier based on the collective expression of 87 informative genes, many of which are involved in lineage commitment and signal transduction. The objective of this study was to determine the feasibility of a blood-based application using differential gene expression. Methods: Technical feasibility studies for cancer cell enrichment and RNA viability were conducted by purifying cancer cell lines that were spiked into donor blood using a microfluidic chip (Celsee PREP™100 system, Celsee Diagnostics) followed by immunodepletion of leukocytes. Quantitative RT-PCR and immunoflourescence were performed on cells harvested at different steps of the purification workflow. Quantitative RT-PCR on individual genes specific to each lineage compartment was used to estimate cell types and cell numbers that were co-purified by the workflow. Gene expression was evaluated by the 92-gene assay. Results: An integrated workflow was established that allows for the molecular characterization of cancer cells purified from blood. Cell enrichment on the microfluidic chip followed by purification using immunodepletion resulted in efficient recovery (> 80% of input cells) and viable RNA from cancer cells spiked into whole blood. A key result of this study was the demonstration that the level of cancer-specific gene expression in purified samples was similar to the level of expression from an equivalent number of unspiked cancer cells suggests that significant cell loss does not occur during the purification workflow. A comparison of gene expression profiles from purified and mixed cell populations allowed for the identification of subsets of genes that are differentially expressed in cancer cells compared with leukocytes. Conclusion: A functional workflow with viable RNA and efficient recovery of cancer cells that are within the technical specifications of the 92-gene assay was established. Findings from this feasibility study are fundamental to the potential for CTC-based cancer classification. Citation Format: Harris S. Soifer, Ranelle C. Salunga, Tristan G. Harris, Jose Ramirez, Priyadarshini Gogoi, Yixin Wang, Saedeh Sepehri, Kalyan Handique, Catherine A. Schnabel. Gene expression profiling for cancer classification in circulating tumor cells. [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 1391.

Abstract P4-01-20: A novel microfluidic system for the detection, enumeration and molecular analysis of circulating tumor cells (CTCs) in metastatic breast cancer (MBC)

Background: In recent years blood testing for circulating tumor cells (CTC) has gained increasing interest in cancer research. CTC detection and enumeration can serve as a ‘liquid biopsy’ and an early marker of response to systemic therapy. Different analytical systems for CTC detection and isolation have been developed, but the CellSearch® is currently the only FDA-approved technology. We aimed to evaluate CTCs detection by a novel microfluidific technology, a size- and deformability-based capture system. This unique platform not only allows flex[not]ibility in the selection of antibody markers but also segregates the CTCs in their own chambers, thus, enabling morphological, immunological and genetic characterization of each CTC at the single cell level. Methods: We performed a prospective study to compare the detection of CTCs using the CellSearch® (Janssen Diagnostics) vs. the new microfluidic platform. Enumeration by the CellSearch® was performed according to standard protocol. For the microfluidific-device capture (De Novo Sciences, MI) peripheral blood from MBC patients was diluted 1:1 with PFA 0.8% and PBS 1%. Prior to sample loading, the microfluidic device was coated with priming buffer. After cells were fixed using 4.0% PFA and were subsequently stained with pancytokeratin, Zym 5.2 and CD45. Nuclei were coun[not]terstained with Hoechst-33342. CTCs were identified as round and bright green cells not stained with red (CK+/CD45-/DAPI+). Data were analyzed using non-parametric methods: Cohen’s kappa, Chi2 test, Spearman rank correlations and Mann-Whitney test. Associations with CTCs were calculated in two ways: CTCs as a continuous variable normalized for ml of blood and CTCs categorized as Results: The two methods was concordant in 88.2% of patients with a Cohen’s kappa of 0.743 when the detection of a single CTC is considered like positive. We also found a concordance of 85% (k=0.70) when we use the CTC cut-off level of ≥5 cells per 7.5 ml of blood to identify patient with higher risk for disease progression. Thirty-one patients with MBC were tested for CTCs using both methods. CTC detection by microfluidific platform was positively associated with Her2 positive patients if we consider as categorical variable; a weak association was seen also with the CellSearch®. Conclusions: The enumeration of CTCs showed strong prognostic significance in MBC raising interest in a more accurate molecular characterization to achieve the possibility for a dynamic molecular monitoring. Introducing novel methodologies should demonstrate comparable detection rate for epithelial cells. This new microfluidic system showed accuracy and flexibility to move to the next phase of molecular testing with the intent of assessing the value as liquid biopsy. Citation Format: Carmela Paolillo, Zhaomei Mu, Angela Toss, Priyadarshini Gogoi, Saedeh Sepehri, Yi Zhou, Kalyan Handique, Ye Zhong, Hushan Yang, Ettore Capoluongo, Massimo Cristofanilli, Paolo Fortina. A novel microfluidic system for the detection, enumeration and molecular analysis of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-20.