Eric Kaldjian

The influence of tumor size and environment on gene expression in commonly used human tumor lines

BackgroundThe expression profiles of solid tumor models in rodents have been only minimally studied despite their extensive use to develop anticancer agents. We have applied RNA expression profiling using Affymetrix U95A GeneChips to address fundamental biological questions about human tumor lines.MethodsTo determine whether gene expression changed significantly as a tumor increased in size, we analyzed samples from two human colon carcinoma lines (Colo205 and HCT-116) at three different sizes (200 mg, 500 mg and 1000 mg). To investigate whether gene expression was influenced by the strain of mouse, tumor samples isolated from C.B-17 SCID and Nu/Nu mice were also compared. Finally, the gene expression differences between tissue culture and in vivo samples were investigated by comparing profiles from lines grown in both environments.ResultsMultidimensional scaling and analysis of variance demonstrated that the tumor lines were dramatically different from each other and that gene expression remained constant as the tumors increased in size. Statistical analysis revealed that 63 genes were differentially expressed due to the strain of mouse the tumor was grown in but the function of the encoded proteins did not link to any distinct biological pathways. Hierarchical clustering of tissue culture and xenograft samples demonstrated that for each individual tumor line, the in vivo and in vitro profiles were more similar to each other than any other profile. We identified 36 genes with a pattern of high expression in xenograft samples that encoded proteins involved in extracellular matrix, cell surface receptors and transcription factors. An additional 17 genes were identified with a pattern of high expression in tissue culture samples and encoded proteins involved in cell division, cell cycle and RNA production.ConclusionsThe environment a tumor line is grown in can have a significant effect on gene expression but tumor size has little or no effect for subcutaneously grown solid tumors. Furthermore, an individual tumor line has an RNA expression pattern that clearly defines it from other lines even when grown in different environments. This could be used as a quality control tool for preclinical oncology studies.

Abstract 1601: Clinical performance of the AccuCyte® - CyteFinder® System, a dual-technology platform for comprehensive collection and high resolution imaging of circulating tumor cells

High numbers of circulating tumor cells (CTC) predict poor prognosis. In addition, serial monitoring of CTC numbers may be used to assess response to therapy or progressing disease. The AccuCyte® - CyteFinder® (AC/CF) system combines density-based collection of all nucleated blood cells with high-resolution digital microscopic imaging, image analysis and single-cell retrieval. The AccuCyte® kit contains a novel separation tube, transfer device, and spreader for simple collection of nucleated cells onto microscopic slides compatible with automated immunostaining. CyteFinder® is a high-resolution fluorescent scanner with image analysis, review software and integrated CytePicker™ module for single-cell retrieval. In a blinded study we directly compared the AC/CF platform to the CellSearch® System (Veridex), the only FDA-cleared CTC platform, which relies on magnetic capture of cells that express EpCAM. We first compared recovery of model CTCs (mCTCs) using tumor cell lines with high EpCAM expression (MCF7 and LNCaP) or low EpCAM expression (PC3 and A549) in paired spike-in samples. CTCs were identified by positive cytokeratin and nuclear staining and an absence of CD45 staining. AC/CF and CellSearch® detected similar numbers of mCTCs from high-EpCAM lines (AC/CF average: 91.5% for MCF7 and 92% for LNCaP; CellSearch® average: 87% for MCF7 and 85% for LNCaP). AC/CF detected more cells from low-EpCAM lines, finding an average of 69% more PC3 cells and 57% more A549 cells. These results suggest that CellSearch® captures low-EpCAM cells inefficiently. We next compared the assays using samples from cancer patients. Paired blood samples were obtained from patients with metastatic cancer under the University of Washington (UW) IRB. Samples were processed by UW using the CellSearch® assay and the AC/CF assay was performed in parallel by RareCyte using AC/CF. RareCyte was blinded to CellSearch® counts until after AC/CF analysis was performed and documented. On average the AC/CF System found 34% more CTCs in 17 prostate cancer patients, 47% more in 15 breast cancer patients, and 70% more in 8 lung cancer patients. In samples where there was more than a 10% difference in CTC count, AC/CF detected >60% more cells with these combined indications. Very low EpCAM expression was confirmed in CTCs from several of the patients where the AC/CF system found greater numbers of cells. These results demonstrate that the AC/CF platform is capable of high CTC recoveries in clinical samples, outperforming the current FDA-cleared platform, especially when CTCs express low levels of EpCAM. Citation Format: Jackie L. Stilwell, Nick Drovetto, Arturo B. Ramirez, Daniel Campton, Joshua Nordberg, Paulina Varshavskaya, Alisa Clein, Steve Quarre, Barry Friemel, Daniel E. Sabath, Eric P. Kaldjian. Clinical performance of the AccuCyte® - CyteFinder® System, a dual-technology platform for comprehensive collection and high resolution imaging of circulating tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1601. doi:10.1158/1538-7445.AM2015-1601

Abstract 3072: High-recovery multiplex analysis of circulating tumor cells by density-based enrichment, automated platform immunofluorescence staining, and digital microscopy

Background: Analysis of circulating tumor cells (CTC) is an intense area of diagnostic research that is rapidly evolving from prognostic to therapeutic applications. We applied three sequential technologies - density-based enrichment, automated immunofluorescence staining, and digital microscopy with image analysis to the investigation of recovery rates from blood containing spiked-in tumor cells as a model of CTC. Materials and Methods: Cultured MCF7, PC3, A549 and LNCaP cells were counted using Vitrotube visualization. ∼100 cells per line were spiked into normal blood samples in two experiments. Density-based enrichment was performed using the AccuCyte tube, float and collector system in a single-tube, two-spin process. Collected cells were processed with an adherence solution and applied to charged microscopic slides with a simple spreading device. After drying, slides were placed onto the Ventana Discovery Ultra automated platform and fluorescently stained with antibodies to cytokeratin, CD45, and EpCAM or EGFR, as well as a nuclear dye. Slides were scanned on a CyteFinder digital microscope and candidate CTCs were identified using CyteMapper image analysis software. CTCs were verified by a reviewer by appropriate morphology and expression of both epithelial and nuclear stains without CD45 expression. Results: In a total of seven samples the recovery of spiked-in CTCs ranged from 88 to 98% and averaged 91%. This method was successfully applied to samples of blood from patients with triple-negative breast cancer. Conclusions: Density-based enrichment combined with automated immunofluorescence staining, digital microscopy and confirmation of candidate cells by a trained reviewer, produces exceptional recovery of CTCs and can be used to assess clinical samples. Citation Format: Daniel Campton, Arturo Ramirez, Joshua Nordberg, Anthony Blau, Jackie Stilwell, Eric Kaldjian. High-recovery multiplex analysis of circulating tumor cells by density-based enrichment, automated platform immunofluorescence staining, and digital microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2014-3072

Reliable detection of subchromosomal deletions and duplications using cell‐based noninvasive prenatal testing

To gather additional data on the ability to detect subchromosomal abnormalities of various sizes in single fetal cells isolated from maternal blood, using low‐coverage shotgun next‐generation sequencing for cell‐based noninvasive prenatal testing (NIPT).

The RareCyte® platform for next-generation analysis of circulating tumor cells: RareCyte platform CTC analysis

Circulating tumor cells (CTCs) can reliably be identified in cancer patients and are associated with clinical outcome. Next‐generation “liquid biopsy” technologies will expand CTC diagnostic investigation to include phenotypic characterization and single‐cell molecular analysis. We describe here a rare cell analysis platform designed to comprehensively collect and identify CTCs, enable multi‐parameter assessment of individual CTCs, and retrieve single cells for molecular analysis. The platform has the following four integrated components: 1) density‐based separation of the CTC‐containing blood fraction and sample deposition onto microscope slides; 2) automated multiparameter fluorescence staining; 3) image scanning, analysis, and review; and 4) mechanical CTC retrieval. The open platform utilizes six fluorescence channels, of which four channels are used to identify CTC and two channels are available for investigational biomarkers; a prototype assay that allows three investigational biomarker channels has been developed. Single‐cell retrieval from fixed slides is compatible with whole genome amplification methods for genomic analysis.

Abstract 5190: Development of a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype

The epithelial-mesenchymal transition (EMT) is understood to be an important step in invasion and metastasis of cancer. It is of increasing investigational interest to identify circulating tumor cells (CTCs) that express mesenchymal markers that indicate entrance into EMT. Such cells may not express surface epithelial markers (such as EpCAM) which are often used to capture CTCs. RareCyte has developed a platform for automated visual identification and retrieval of rare cells in blood by immunofluorescence (IF) that does not rely on surface marker capture. We developed a 5-parameter assay to identify epithelial CTCs with or without mesenchymal differentiation.

RareCyte ® CTC Analysis Step 2: Detection of Circulating Tumor Cells by CyteFinder ® Automated Scanning and Semiautomated Image Analysis

The RareCyte CyteFinder instrument is an automated scanner that allows rapid identification of circulating tumor cells (CTCs) on microscope slides prepared by the AccuCyte process (see Chapter 13 ) and stained by immunofluorescence. Here, we present the workflow for CyteFinder scanning, analysis, and CyteMapper scan review which includes CTC confirmation and report generation.

RareCyte ® CTC Analysis Step 3: Using the CytePicker ® Module for Individual Cell Retrieval and Subsequent Whole Genome Amplification of Circulating Tumor Cells for Genomic Analysis

The CytePicker module built into the RareCyte CyteFinder instrument allows researchers to easily retrieve individual cells from microscope slides for genomic analyses, including array CGH, targeted sequencing, and next-generation sequencing. Here, we describe the semiautomated retrieval of CTCs from the blood processed by AccuCyte (see Chapter 13) and amplification of genomic DNA so that molecular analysis can be performed.

Abstract B21: Probing tumor heterogeneity and immune infiltration with Cyclic Immunofluorescence (CycIF), a robust, multiplexed imaging method

Tumor microenvironment, defined by surrounding stromal/immune cells as well as blood vessels, plays an important role in disease progression and therapy resistance. Increasing understanding of the heterogeneity in both tumor and its microenvironment will be crucial to development more effective therapies. Recently, several studies employing state-of-the-art single-cell sequencing methods reveal enormous complexity in tumor microenvironment. However, the spatial information and cell-to-cell interaction could not be preserved in these dissociated cells. Immunofluorescence has been widely used in different fields of biological and medical research for decades. The ability to obtain in situ and single-cell information makes this technique particularly important in tumor biology. However, biochemical and optical constraints limit the number of signals that could be captured simultaneously within the same sample. We have developed the CycIF (Cyclic Immunofluorescence), an easy and low-cost method to increase the multiplexity of conventional immunofluorescence. The CycIF method has been first applied in pre-clinical drug discovery, cancer and stem cell biology with adherent cell cultures. In here, we modified the original CycIF method for IHC/IF on FFPE samples, and used that to probe tumor heterogeneity, microenvironment and immune infiltration in various types of tumors. Up to 30 different antigens/markers could be simultaneously detected in different tumor samples, and these makers represent a wide range of biological processes, including the key molecules for lymphocyte surface makers (CD45, CD4, CD8, CD20, CD11c), immune checkpoints (PD-1, PD-L1), stromal/EMT proteins (E-Cadherin, Vimentin), cell cycle regulators (CycD1, PCNA, Ki67, pRB, p21/CIP), signaling proteins (EGFR, pERK, pS6) and apoptosis mediators (p53, Bax, Bcl-2, Survivin). Our study not only provides the first detailed map of tumor and its immune microenvironment, but also illustrates a robust multiplexed imaging platform for probing tumor heterogeneity. Citation Format: Jia-Ren Lin, Benjamin Izar, Sabrina Hawthorne, Josh Nordberg, Eric Kaldjian, Peter Sorger. Probing tumor heterogeneity and immune infiltration with Cyclic Immunofluorescence (CycIF), a robust, multiplexed imaging method. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B21.

Abstract 2757: Using liquid biopsies and NGS as tools to analyze mutation burden and copy number variation in the blood of a patient with triple negative breast cancer to better inform therapeutic targets

The ability to characterize molecular features of cancer from liquid biopsies is resulting in the development of innovative health care for patients. Longitudinal changes in the mutational profiles of DNA isolated from liquid biopsies are being used to better understand and monitor the development, progression, and evolution of therapy resistance in cancer patients. To define changes in the mutational landscape and predict drug susceptibilities in Triple Negative Breast Cancer (TNBC) patients, we used whole exome analysis to profile circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) from eight selected time points of a patient enrolled in the Intensive Trial of OMics in Cancer clinical Trial (ITOMIC-001). The patient initially received weekly cisplatin infusions followed by additional targeted therapy. Peripheral blood samples were collected at specific time points over a period of 272 days following enrollment in the clinical trial. Our data indicates that the identified mutations in genomic DNA isolated from CTCs and ctDNA can be used to understand and mitigate the impact of tumor heterogeneity in addition to identifying clinically relevant mutations at these selected time points. To further increase the resolution of our analysis, we profiled ctDNA from these samples to a higher depth targeting only clinically relevant genes. These analyses increased the sensitivity of detection and identified additional targets that could have been used for therapeutic intervention. In addition to sequence variants, copy number variations (CNVs) have also been significantly associated with the development of metastasis and changes in CNVs have been used to monitor disease progression. We performed a bioinformatics analysis of genomic instability and CNVs across 32 different time points from ctDNA from the same patient throughout the treatment period. The genomic instability number (GIN) calculated for each of the 32 time points seems to mirror the overall CTC burden in the patient at each time point tested. CNV analysis is ongoing and these data sets are being further analyzed in combination with TCGA data to define possible cancer driver genes for the functional prediction of significant TNBC candidate alterations and the results of these analyses will be presented. Citation Format: Kellie Howard, Kimberly Kruse, Brianna Greenwood, Elliott Swanson, Mathias Ehrich, Christopher K. Ellison, Taylor Jensen, Sharon Austin, Arturo Ramirez, Debbie Boles, John Pruitt, Elisabeth Mahen, Jackie L. Stilwell, Eric P. Kaldjian, Michael Dorschner, Sibel Blau, Marcia Eisenberg, Steve Anderson, Anup Madan. Using liquid biopsies and NGS as tools to analyze mutation burden and copy number variation in the blood of a patient with triple negative breast cancer to better inform therapeutic targets [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 2757. doi:10.1158/1538-7445.AM2017-2757