Expression Profiling Of Circulating Tumor Cells Research Articles

Abstract 5365: Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay

Abstract Introduction and objective: The presence of circulating tumor cells (CTC) is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. The aim of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by gene profiling using the NanoString low RNA input nCounter assay to achieve reliable CTC gene profiling. Methods: Blood samples were collected from 4 mPCa patients and 3 healthy donors. In the LP procedure, different blood elements were separated based on density. The mononuclear cell layer (presumed to contain any CTCs) was collected. The Alere™ CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method for isolation of EpCAM+ CTCs, followed by RT-qPCR for PSA, PSMA, and HOXB13 was used to detect CTCs from LP. The Ct value thresholds for positive CTC signal by RT-qPCR were set based on healthy donor LP subjected to AdnaTest as a negative control (PSA 36.3, PSMA 36.82, HOXB13 40). The cDNA of cells captured by EpCAM+ selection (including putative CTCs) and of the EpCAM+ depleted white blood cells (WBC) were assayed by the nCounter PanCancer Progression Panel to determine expression of 770 selected mRNAs. The NanoString nCounter Low RNA Input Kit with the multiplex 770-gene primer pool was used for the pre-amplification of cDNA and overnight hybridization with the PanCancer Progression panel. To validate the assay, healthy donor LPs were spiked with known numbers of PCa cell line LN95 cells. Results: For mPCa patients, the LP procedure took 136.2±10.6 min, with a screening volume of 10L of peripheral blood. The mean volume of mPCa LP product was 123.64±12.23 ml. The WBC concentration was 69.75±28.2×109/L. All samples were subjected to AdnaTest in triplicate and CTCs were identified in 3 out of 4 patients. The positive Ct values of PSA were 33.86±0.81, 32.14±0.31 and 33.09±0.34; PSMA were 35.55±1.02, 33.55±0.83 and 35.30±0.65; HOXB13 were 36.25±0.34, 37.15±0.22 and 37.09±0.33. To assess profiling of CTCs isolated from LP using the nCounter platform, 100, 500, and 1000 LN95 cells were spiked in healthy LPs and were analyzed with the AdnaTest. Within each spiking group, pre-spiking WBC, captured CTC, post-capture WBC, and LN95 cells in PBS alone were subjected to the Nanostring assay. Eisen Cluster analysis separated pre- and post- WBCs, captured CTCs, LN95 cells into three groups. The gene expression of captured CTCs correlated with those of LN95 cells, and the correlation increased with the spiking cancer cell number (R2=0.45, 0.57, and 0.70, respectively). The gene expression of pre-spiked WBCs were highly correlated with those of post-capture WBCs regardless of spiking cancer cell number (R2=0.83, 0.84 and 0.85). Conclusions: Analyzing CTCs in LP samples by AdnaTest is feasible and offers a promising method for liquid biopsy of mPCa patients. The NanoString low RNA input nCounter assay can provide reliable gene expression profiling of CTC. Citation Format: Liang Dong, Chung-Ying Huang, Changxue Lu, Diane Reyes, Sarah R. Amend, Jun Luo, Kenneth Pienta. Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5365.

Abstract 5362: Analysis of circulating tumor cells from prostate cancer patients

Abstract Background: Intermediate and high-risk prostate cancer patients are candidates for radiotherapy (RT). Evidence has shown that the presence of circulating tumor cells (CTCs) correlates with poor overall survival for patients with metastatic prostate cancer. CTCs have been reported as a better earlier predictor of treatment response than evaluation of prostate-specific antigen (PAS). The aim of our study was to identify and quantify circulating tumor cells from the peripheral blood of patients enrolled in two randomized Phase II radiotherapy clinical trials (BLaStM, NCT02307058 and CoMBINe, NCT02997709). Moreover, we investigated the genomic profiling of single CTCs using 10X Genomics to explore prognostic factors. Design/Methods: CTCs were successfully harvested and enumerated from more than 215 patients and 500 blood samples using a microfilter system, the Circulogix FaCTChecker, and the cells of interest enriched onto CyteCatch slides. Circulating tumor cells were visualized by staining, identified and quantified for epithelial and prostate specific markers. The number of single CTCs, CTC clusters and their sum were recorded. Unfixed CTCs were enriched from whole blood from 13 prostate cancer patients at high risk of metastasis (median age of 63.5 years and median CTC value of 12 (range 0-42)) using The Parsortix™ Cell Separation System (ANGLE Inc.), a microfluidic-based platform able to capture and harvest CTCs, and single-cell RNA sequencing was then performed with Chromium (10X Genomics) and Illumina NGS sequencing technologies. Initial bioinformatic and data analysis was performed by the Biostatistics and Bioinformatics Shared Resource (BBSR) at SCCC. Subsequent principal component analysis (PCA) and gene set enrichment analysis (GSEA) were performed. Meanwhile, patient demographic and disease characteristic variables (PSA, Gleason Score (GS), T stage, % tumor in biopsies, age, race/ethnicity) were also collected and compared. Results: Single CTCs or CTC clusters were evident in over 70% of patients analyzed using Immunofluorescence staining, with a median number of 30 (range 0-346). CTC transcriptomes at the single cell level was performed using the 10X platform, and principal component analysis discovered 16 distinct cell clusters of differential gene signatures within these cells in a t-SNE plot. Gene ontology analysis found some of these cell clusters contain differential expression pathways such as mTOR signaling (p=5.82E-05), EIF2 signaling (p=7.90E-05) and regulation of eIF4 and p70S6K signaling (p= 1.04E-04) which have been associated with prostate cancer. Gene Set Enrichment Analysis also identified distinct differential expression pathways in prostate CTCs comparing with non-CTCs. Conclusion: We have developed a novel pipeline to harvest CTCs and analyze transcriptomes at the single-cell level. Our results suggest that gene expression profiling of CTCs can be a promising way to explore prognostic markers for predicting radiation sensitivity and metastatic risk. Citation Format: Teresa Giret, Wensi Tao, Robert Suter, Saba Ansari, Sion Williams, Nagi Ayad, Radka Stoyanova, Brian Marples, Alan Pollack. Analysis of circulating tumor cells from prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5362.

Abstract P1-01-11: Something from nothing? The case for quality control in liquid biopsy studies

Abstract Background: Circulating tumor cells (CTCs) as a liquid biopsy strategy are currently being studied as a surrogate biomarker that may reflect metastatic tumor biology. Given the rarity of CTCs, target enrichment is commonly used to profile the gene signatures of valuable clinical samples to evaluate for multiplexed gene panels of interest. The aim of our study was to evaluate if the NanoString PAM50 could be used for accurate gene expression profiling of CTCs and controls using the research-use-only probeset. Methods: We collected two 7.5mL EDTA tubes of blood from 12 healthy female volunteers. CTC assays were performed using the ANGLE Parsortix system as a microfluidics filter that separates cells based on size and deformability. The cell lines Hs578T (basal-like) and SK-BR-3 (HER2 amplified) were used to spike 20 cells into n=6 blood tubes per cell line (termed spiked samples). N=12 7.5mL tubes of blood (termed unspiked samples) and n=12 spiked samples were processed using Parsortix for CTC harvesting and lysis using a 10 micron cassette. From each lysate, 5uL was taken for cDNA amplification, multiple target enrichment for 14 cycles, followed by NanoString PAM50 assays. From each of the 12 peripheral blood (PB) samples, we extracted RNA and used 100ng for NanoString PAM50 assays. For cell line controls, 100ng of Hs578T or SK-BR-3 were subjected to NanoString Assays. Results: Low PAM50 gene expression was observed in all 12 PB samples. Unspiked PB harvested from the CTC assay showed a higher level of PAM50 gene expression compared to PB, suggesting that the target enrichment amplification produces false positive detection of expected breast cancer related transcripts. On ANOVA testing, 10/12 (83%) of unspiked, sorted, target enriched samples had significant differential expression (p<0.0001) of the mean log normalized counts for the PAM50 genes compared to PB. In spiked experiments using n=20 cells in 7.5mL of PB, sorted Hs578T were found to be triple negative in only 3/6 (50%) while sorted SK-BR-3 were found to be HER2 positive in only 3/6 (50%). On ANOVA testing, the spiked/sorted and bulk were found to have a difference among the mean log normalized counts for the PAM50 genes across all samples for both cell lines (p<0.0001). However, 3/6 (50%) samples had a difference in mean PAM50 gene expression when compared to bulk Hs578t on multiple comparison testing while 2/6 (33%) were statistically significantly different when comparing spiked, sorted SK-BR-3 versus bulk cell line. Conclusions: Unspiked blood processed via a CTC assay and subjected to target enrichment showed high expression of genes in the NanoString PAM50 assay, likely due to amplification bias. When working with enriched but not ultra-pure CTC samples, amplified gene expression of background leukocytes may influence read counts. This is important to consider in assays that enrich for CTCs but retain a leukocyte background. Further studies will address the effect of the CTC assay procedure and number of leukocytes on accuracy of gene expression of rare CTC mimics. This study emphasizes the importance of selecting genes that are not expressed in PB or performing background subtraction or normalization as strategies for accurate gene expression profiling of CTCs. Citation Format: Porras TB, Bains PK, Ring A, Carrasco S, Forte V, Punj V, Lang JE. Something from nothing? The case for quality control in liquid biopsy studies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-11.

Abstract P1-01-04: Comprehensive genomic characterization of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) sheds light on the biology of blood-borne metastasis

Abstract Background: CTCs offer a relatively non-invasive source of metastatic tissue for molecular analysis. To elucidate the underlying biology of blood-borne metastasis, we profiled CTCs from MBC patients (pts). Methods: CTCs were isolated by IE/FACS (immunomagnetic enrichment/fluorescence-activated cell sorting). Expression of 64 cancer-related genes in CTCs was analyzed via microfluidic-based multiplex QPCR. Genome-wide copy number (CN) analysis by array comparative genomic hybridization (ACGH) was performed on CTCs isolated from the same tumor-enriched blood samples. The Illumina platform was utilized for next generation sequencing and data was analyzed using NantOmics analysis pipeline and Nexus 8.0 software. Mutations were confirmed by Sanger sequencing or by digital droplet PCR. Results: Expression profiles of CTCs from 105 MBC pts clustered away from those of blood, indicating high-purity isolation of CTCs by IE/FACS. In addition to EPCAM, tumor-related genes, e.g., CCND1, MUC1, and TTF3 were upregulated in CTCs. Approximately 70% of the CTC samples were considered ER-positive, of which 47% were ER+HER2+, and 22% ER+HER2-. Among the ER+HER2- samples, about two-thirds (68%) had low proliferative (MKI67) status. HER2-positive and triple-negative CTCs accounted for 27% and 30% of the samples, respectively. Furthermore, 30% of the samples were assigned to luminal A, 6% to luminal B, 13% to Her2-enriched, 33% to basal-like, and 12% to normal-like subtypes. Expression profiling of CTCs in 74 serial blood samples from 28 pts showed fluctuations in expression at the gene-level, while subtype calls were mostly consistent across time points. CTCs from 49 of the 105 pts analyzed by ACGH revealed numerous genomic aberrations such as 1q/8q gains and 8p/16q losses, consistent with breast cancer origin. CN profiles grouped into three major clusters: CTCs exhibiting low genomic instability, 8q gain, and 1q gain/11q loss. ERBB2 and CCND1 were upregulated in CTCs showing increased genomic alterations. Changes in ER (n=102) and HER2 (n=130) status between CTCs and matched primary tumors (PT) were observed in 27% and 23% of the pts, respectively, indicating that biomarker status may change during disease progression. Comparative analysis of CN data from low-pass whole genome sequencing (WGS) of CTCs vs. matched PTs (n=7 pairs) demonstrated clonal-relatedness as well as some genetic divergence. WGS (38x) and whole exome sequencing (140x) analysis of CTCs from an index pt diagnosed with invasive lobular carcinoma detected numerous genomic aberrations, including a copy loss and a frameshift mutation in E-cadherin (CDH1). Interestingly, analysis of CN and mutation data revealed that CTCs were more closely related to the lymph node metastases than to the PT. Single-cell sequencing of CTCs revealed uniformity in genome-wide CN alterations, while cell-to-cell heterogeneity was observed only when single-cell expression profiles were analyzed. Conclusions: Comprehensive molecular characterization provided novel insights into the biology of breast CTCs. Further CTC profiling may open avenues for discovery and development of novel biomarkers for personalized medicine and strategies to prevent metastasis. Citation Format: Magbanua MJ, Hauranieh L, Roy R, Wolf D, Benz S, Vaske C, Pendyala P, Sosa E, Scott J, Lee JS, Ordonez A, Ho B, Solanki T, VantVeer L, Rugo H, Park J. Comprehensive genomic characterization of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) sheds light on the biology of blood-borne metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-04.

Abstract P1-04-07: Isolation and expression profiling of EpCAM-negative circulating tumor cells in human and xenograft models of breast cancer

Abstract Background: While several assays currently exist to enumerate circulating tumor cells (CTCs), limitations to existing assays include reliance on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM) and difficulty isolating pure populations of rare CTC RNA from peripheral blood without background subtraction of leukocytes. We evaluated the biomarkers CD146 and CD49f as a strategy to isolate CTCs that underwent epithelial to mesenchymal transition (EMT). We hypothesized that using immunomagnetic separation and fluorescence activated cell sorting (IE/FACS) it is possible to isolate RNA derived from EpCAM-negative CTCs. Methods: 10 breast cancer cell lines were acquired from the ATCC, authenticated by short tandem repeat profiling, and stratified according to subtype: HER2 positive (SKBR3, MDA-MB-453, SUM190), luminal A (T47D, MCF7, ZR751), luminal B (BT474) and basal-like (SUM149, MDA-MB-231, Hs578T). IE/FACS with EMT gates was performed using CD146 ferrofluid particles via a magnetic separator followed by incubation with Thioflavin T, CD 31 and CD45 PE-Cy7 (for human or mouse blood), CD146-V450, and CD49f-A647. FACS sorting was performed using a FACS Aria II (BD Biosciences). Known quantities of cells were spiked into PBS or peripheral blood (PB) from healthy female donors and subjected to IE/FACS with EMT gates. Absolute cell counts and recovery rates were determined using the TruCOUNT method (BD Biosciences) with acquisition of 35,000 beads. MDA-MB-231 xenografts were established in 4 male NOD/SCID/IL2 gamma chain deficient mice; cardiac punctures were performed to acquire a complete xenograft PB volume for IE/FACS. Sorted xenograft CTCs, human PB and MDA-MB-231 were used to isolate RNA, amplify and profile via Affymetrix human gene ST 1.0 arrays. The effect of sample processing with IE/FACS was assessed using our EpCAM gates (EpCAM ferrofluid, EpCAM-PE, thioflavin, CD45 PE-Cy7) to sort BT474 and profiled via next generation sequencing (NGS) using the Helicos Single Molecule sequencing approach. Results: MDA-MB-231 spiked into PB from an unaffected mouse and negative mouse PB controls were used to establish gating thresholds. This gating strategy recovered 14% of MDA-MB-231 cells from spiked human PB via the TruCOUNT method, which represented a 200% yield versus EpCAM based IE/FACS. CTCs were successfully isolated from 4/4 mice bearing xenografts (ranging from 193-6352 cells captured per ∼1mL mouse blood). Principal component analysis demonstrated that gene expression may differentiate xenograft CTCs from PB and MDA-MB-231. Recovery rates using the EMT gates for 10 cell lines spiked into PBS and human PB will be reported and compared to our typical EpCAM gating strategy. Pearson's correlation coefficient of BT474 spiked into PB and recovered via IE/FACS using an EpCAM gating strategy was 92% by NGS. Conclusions: We have established a method for the isolation and expression profiling of EpCAM-negative CTCs as well as EpCAM-positive CTCs. Future studies are required to determine if expression profiling of CTCs can be used as prognostic, predictive and discovery tools. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-07.

Abstract P1-04-11: Recovery rates of circulating tumor cells in breast cancer cell lines spiked into peripheral blood

Abstract Background: Circulating tumor cells (CTCs) have been demonstrated to be prognostic in all stages of breast cancer. Immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) isolates enriched populations of CTCs derived from peripheral blood without the need for background subtraction of leukocytes. We previously reported IE/FACS as a strategy for gene expression profiling of CTCs. We hypothesized that variable expression of the epithelial cell adhesion marker (EpCAM) may bias IE/FACS recovery rates and composition. Patients and Methods: 10 breast cancer cell lines were acquired from the ATCC, authenticated by short tandem repeat profiling, and stratified according to subtype: HER2 positive (SKBR3, MDA-MB-453, SUM190), luminal A (T47D, MCF7, ZR751), luminal B (BT474) and basal-like (SUM149, MDA-MB-231, Hs578T). Unknown quantities of cell lines were spiked into PBS or peripheral blood (PB) from healthy female donors. IE/FACS was performed using EpCAM (MJ37) ferrofluid particles via a magnetic separator followed by incubation with Thioflavin T, CD45 PE-Cy7, and EpCAM (EBA-1) mAb conjugated to phycoerythrin. FACS sorting was performed using a FACS Aria II (BD Biosciences) and a gating strategy devised based on negative controls (n = 23). Absolute cell counts and recovery rates were determined using the TruCOUNT method (BD Biosciences) with acquisition of 35,000 beads. Two-way ANOVA was used to analyze variation in recovery rates between groups (Prism GraphPad 6.0). Results: Overall mean recovery rates for the 10 cell lines were 51.4% from PBS and 39.5% from PB. The specific cell type being analyzed was a more significant source of variation (p = 0.03) than was whether measurements were made from PBS or PB (p = 0.2). However, analysis by molecular subtype did not show differences between intrinsic groups (p = 0.23) nor did it show differences in recovery rates from PBS or PB (p = 0.26). The two inflammatory breast cancer cell lines (SUM 149 and SUM190) showed no differences in recovery rates compared to other cell lines (p = 0.41) nor in recovery rates from PBS versus PB (p = 0.75). Recovery rates for the 10 cell lines are shown in Table 1. Recovery Rate Recovery from PBS (%)Recovery from PB (%)SKBR368.051MDA-MB-45361.270.6T47D28.911.4MCF794.024.4BT47497.147.6ZR75152.850.8SUM1496372.9SUM19048.358.3MDA-MB-2310.927.04Hs578T0.250.84Recovery rate of immunomagnetic enrichment/fluorescence activated cell sorting of cell lines spiked into saline (PBS) or peripheral blood (PB) Conclusions: Significant variation occurred in recovery rates of spiked, sorted cells depending on cell line type. Negative control PB specimens from healthy individuals could successfully define a consistent gating strategy that captured zero CTCs from healthy individuals and permitted acquiring a portion of CTCs from each of the 10 cell lines. Further experiments will characterize the gene expression of the sorted cells compared to bulk RNA for each cell line. Better biomarkers are needed to improve upon the recovery rates of CTCs while minimizing selection bias. Future studies are required to determine if expression profiling of CTCs is an informative biomarker that may be applied clinically as a prognostic or predictive tool. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-11.

Utility of microsieve device for enumeration and miRNA expression profiling of CTCs for personalized cancer management.

e22107 Background: Although a rare peripheral cell population, circulating tumor cells (CTCs) represent a potential alternative to serial invasive biopsies as a source of tissue in monitoring clinical states of non-hematological malignancies. CTCs harvested from a single patient are a heterogeneous mixture of cells differing in their transcriptome expressional profiles, metastatic potential and drug resistance properties. Mere identification and enumeration of CTCs are unable to provide qualitative information about individual CTC biology. The presence of a few highly malignant, metastasis-capable CTCs and cancer stem cells among the other less aggressive CTCs in the circulating population will not be apparent from CTC counts alone. Methods: We developed a high-throughput size-exclusion microsieve device (CellSievo, Singapore) to isolate CTCs (defined as DAPI-positive, CD45-negative cells), regardless of the cell surface receptors expressed, from 74 breast cancer patients. Using CTCs eluted from the microsieve post-capture, we performed microRNA expression profiling to further characterize breast cancer CTCs. Results: Using CellSievo’s microsieve device, we obtained an average mean CTC count of 21.2 (+/- 31.8; range=0-112.5) per 7.5ml peripheral blood, for breast cancer patients, as opposed to 1.3 (+/-1.6, range 0-5.0) for healthy subjects. Viable CTCs from breast cancer patients were successfully harvested for downstream microRNA profiling. CTCs from breast cancer patients were routinely found to express microRNA profiles mediating epithelial-to-mesenchymal transition, tumor invasion, and regulation of chemotherapeutic drug resistance to tamoxifen, anthracyclines, taxanes, and trastuzumab. Serial monitoring of microRNA expression pattern of CTCs could identify emergence of novel tumor populations with distinct microRNA expression patterns during or after anticancer therapy (detailed microRNA expression data to be presented). Conclusions: Our results suggest that microRNA biomarkers derived from peripheral CTCs can yield highly useful information and offer prognostic and predictive applications for therapeutics and companion diagnostic development.

Abstract P3-02-13: Is HER2 Evaluation with the CellSearch System a Method Reliable for Detecting HER2 Overexpression?

Abstract Background. Circulating tumor cells (CTCs) detected in patients with both localized and metastatic breast cancer are significantly associated with a worse outcome. In addition to enumeration, an exciting area of CTC research involves the phenotyping and expression profiling of CTCs. In this regard, in patients with metastatic breast cancer, the evaluation of CTCs could be considered as a “real-time” biopsy allowing the detection of possible changes in tumor phenotype, such as a shift in patients HER2- negative on the primary tumor to HER2-positive CTCs. This could be of relevance as these patients may become suitable to targeted anti-HER2 therapy. Currently, there is no standardized and widely accepted method available for the determination of HER2 status on CTC. Aims. Objectives of this study were: 1. verifying the feasibility and reliability of HER2 determination on cells from scraping of breast cancer tissue by FISH analysis, 2. evaluating the concordance of HER2 status determined on primary breast tumor by immunohistochemistry (IHC) and on scraped cells, obtained from the same breast tumor and spiked in blood from healthy subjects, using the CellSearch System, and finally 3. evaluating the concordance of HER2 expression determinated by FISH analysis and by CellSearch on the same scraped cells. Methods. Cells from scraping of fresh breast cancer tissues with different level of HER2 expression were spiked in 18 healthy subjects blood samples. The determination of the HER2 expression on these cells was performed with the CellSearch System (Veridex, USA) by the addition of a fluorescein conjugated monoclonal antibody to be used in conjunction with the CellSearch™ Epithelial Cell Kit to phenotype CTCs for the presence of HER-2/neu. The HER2 characterisation of the primary breast tumors was performed by IHC by FISH analysis according to standard procedures. FISH was also performed on cells from scraping of fresh breast cancer tissues after CellSearch enumeration and characterization, by removing them from the “MagNest” cartridge. Tumors with a score of 3+ were considered positive. Results. The results of the FISH analysis performed on the cells aspirated from the cartridge demonstrated a 100% concordance with the FISH performed on fresh tissue (9 not amplified and 9 amplified). The evaluation of HER2 expression on scraped cells by CellSearch System and by IHC on the corresponding tumor showed that the CellSearch method is reliable in identifying HER2 overexpression, as in all the 3+ tumors it was possible to detect variable percentage of scraped cells overexpressing HER2. Finally, different number of HER2+ scraped cells were found in 16 out of the 18 samples: the only 2 negative samples were both IHC negative and FISH not amplified. On the contrary 2 of the 4 remaining negative/1+ IHC samples, showed some scraped cells HER2+ which resulted FISH amplified. Conclusion. This study demonstrates that FISH analysis is feasible and the results are reliable when performed on cells after CellSearch procedure. Moreover HER2 expression may be evaluated with the CellSearch System and it may be used as a preliminary method to indicate possibly HER2 positive samples which may be confirmed by FISH analysis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-13.

Abstract P3-03-01: Minimal Residual Disease Monitoring in Breast Cancer Patients

Abstract BACKGROUND: Breast cancer (BC) patients still harbor a considerable risk of metastatic relapse caused by minimal residual disease (MRD) despite of complete removal of primary tumor. The aim of this study was to identify single disseminated tumor cells (DTC) in the bone marrow and circulating tumor cells (CTC) in the peripheral blood as potential biomarkers for therapy response monitoring and metastatic relapse risk prediction. Finally, the gene expression profiles of CTCs have been analyzed. METHODS: A total of 87 patients with diagnosed BC at stage I to III and 115 metastatic patients were enrolled into a prospective study between years 2008 - 2010. Peripheral blood (5ml) for CTC-detection was collected from primary BC patients before chemotherapy (CHT), after 2 cycles of CHT and after the CHT. Metastatic BC patients have been examined for CTCs before starting a new line of the treatment. Bone marrow aspirates from 16 premenopausal BC patients (mean age 31) with primary tumor were analyzed for the presence of DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 (Epimet™, AS Diagnostik, Germany) before surgery. CTC detection in blood was performed by AdnaTest BreastCancerTM(AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. cDNA from isolated CTCs has been further pre-amplified and used for multimarker qPCR gene expression profiling using Biomark® microfluidic 48x48 GE Dynamic arrays (Fluidigm, USA). qPCR results have been analyzed by GENEX vs. 5.2 software (MultiD Analysis). RESULTS: 286 CTC samples have been analyzed in total. The analysis has shown that the expression profiles of CTCs from primary BC patients have been significantly different comparing them to the CTC-profiles from metastatic BC patients for several of tested genes (e.g., CK19, GA7332, MLFIP1, SATB1, PTEN). Interestingly, before surgery of primary tumor DTCs were found in 5/16 patients (31 %) and CTCs in 7/16 (43 %). Both DTCs and CTCs together were found in 4/16 (25%) patients. In 18% of the primary BC patients no dissemination markers were found CONCLUSIONS: Information based on the CTCs-gene expression profiles could provide an additional support for therapy management. Metastatic potential of enriched CTCs will be further evaluated on the single cell level. This work has been supported by Grant Agency of Ministry of Health, Czech Republic (IGA NS9976) and Grant Agency of Charles University in Prague, Czech Republic no. 7709 and 59410. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-01.

Expression profile of CTC and corresponding tumors in primary breast cancer patients

538 Background: The target of adjuvant therapy in breast cancer is minimal residual disease (MRD). However, treatment decisions are based on expression of predictive markers (ER; PR, HER-2) of the primary tumor. Based on current studies, presence of MRD may be reflected by the detection of circulating tumor cells (CTC). Therefore, the aim of the study was to characterize CTC by multiplex-PCR for the expression of HER-2, ER, and PR and compare the expression profiles of CTCs with those of the corresponding primary tumors. Methods: CTC from blood of 431 patients with primary breast cancer were analyzed for EpCAM, MUC1, and HER-2 transcripts with the AdnaTest BreastCancer (AdnaGen AG, Germany). A blood samples was CTC positive when at least one of the markers was detected. Expression of the ER and PR receptor was assessed in an additional RT-PCR. The ER, PR and HER2 receptor status of the primary tumors was determined by immunohistochemistry. The DAKO-Score for the expression of HER-2 was determined with the HercepTest. Results: The overall detection rate for CTC was 13% (58/431 patients) with the expression rates of 38% for HER-2 (22/58 patients), 25% for ER (12/48) and 4% for PR (2/48), respectively. ER positivity of the primary tumor was demonstrated in 45/58 (78%) of these patients, PR positivity in 41/58 (71%) patients and HER-2 in 9/58 (16%) patients, respectively. The concordance rate between ER, PR, and HER-2 status of CTCs and the primary tumor was 29%, 25%, and 53%, respectively Interestingly, the spread of CTC was mostly found in triple negative tumors (p = 0.01) and CTC in general were mostly found to be triple-negative regardless of the ER, PR, and HER-2 status of the primary tumor. Conclusions: Since the expression profile between CTC and the primary tumor differs, the consequence for the selection of adjuvant treatment targeting minimal residual disease has to be further evaluated in clinical trials. No significant financial relationships to disclose.

Acquisition of HER2/neu over-expression on circulating tumor cells (CTCs) in patients (pts) with advanced breast cancer (ABC) during chemotherapy

11017 Background: CTCs can be detected and isolated from the peripheral blood of women with ABC. In addition to enumeration, an exciting area of research involves the phenotyping and expression profiling of CTCs. In this regard, assessing the presence of target antigens on CTCs could be considered as a real-time biopsy allowing the possibility to evaluate the change in tumour phenotype during the clinical course of the disease. HER2/neu status of tumor cells detected in metastatic sites may differ from the original primary tumor in about 5–20% of cases. Furthermore, there is evidence that nearly one third of pts whose primary tumors were HER2/neu negative might have amplified HER2/neu on CTCs. Methods: We collected 7.5 ml blood samples from each of 40 pts with ABC. CTCs were isolated with the CellSearch System® (Immunicon Co.) by means of immunomagnetic separation, using ferrofluid nanoparticles binding anti-epithelial cell adhesion molecules (EpCAM), and fluorescently stained with Epithelial Cell Kit®. C...

Gene expression profiling of circulating tumor cells from breast cancer patients

10769 Background: Levels of circulating tumor cells (CTC) have prognostic and predictive significance in metastatic breast cancer. However, since CTCs are extremely rare, little is known about the actual phenotype of these cells. In order to characterize these cells, we performed cDNA microarray analyses of CTC isolated from peripheral blood (PB) of breast cancer patients. Methods: CTCs were directly isolated via immunomagnetic enrichment (IE) followed by fluorescence activated cell sorting (FACS). Total RNA was then subjected to two rounds of linear amplification and hybridized to cDNA microarrays (∼40,000 cDNAs). Validation studies used spiked BT474 cells. Clinical studies used PB (10–20 ml) from patients with metastatic breast cancer. Results: Rare spiked tumor cells (e.g., 320 cells in 10 mL PB) were efficiently recovered by IE/FACS (50% yield). Expression profiles of recovered cells, both by TaqMan of a 37 gene panel as well as by global gene expression analysis, matched that of BT474 cells in culture. In contrast, these profiles were clearly distinct from that of normal PB, ruling out significant contamination from blood elements. In clinical studies, IE/FACS isolated small numbers of CTCs (10–1000 cells). Expression profiles of CTCs were compared to that of normal blood, primary breast tumors, and normal epithelial samples. Unsupervised hierarchical clustering revealed that CTC profiles were readily distinguished from that of normal blood and normal epithelium; and further analysis revealed that CTC cluster with a subset of primary breast tumors, particularly the basal-like phenotype. Candidate genes associated with the CTC phenotype were also identified. Conclusions: We have developed and validated a method to isolate rare CTCs and profile them via cDNA microarray analysis. In addition, our gene expression analyses of CTC further provide evidence to the malignant nature of these cells. Further expression profiling of CTC may yield insights into their phenotype, pathophysiology and potential as biomarkers. [Table: see text]