EpCAM-positive Circulating Tumor Cells Research Articles

Evaluating blood levels of neuron specific enolase, chromogranin A, and circulating tumor cells as Merkel cell carcinoma biomarkers.

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer. Although used to monitor MCC patients, the clinical utility of neuron-specific enolase (NSE) and chromogranin A (ChrA) blood levels is untested. EpCAM-positive circulating tumor cells (CTC) reflect disease status in several epithelial tumors. Here we investigate the use of NSE and ChrA blood levels and CTC counts as biomarkers for MCC disease behavior. NSE and ChrA blood levels from 60 patients with MCC were retrospectively analyzed; 30 patients were additionally screened for CTC. Biomarker values were correlated to clinical parameters. Despite routine use by some physicians, NSE and ChrA blood levels did not correlate with progression free survival, disease specific survival, or MCC recurrence. We found CTC in 97% of tested MCC patients. CTC counts were elevated in patients with active disease, suggesting their potential use in monitoring MCC. NSE and ChrA levels were not effective in predicting outcomes or detecting recurrences of MCC. In contrast, CTC counts have potential utility as a biomarker for MCC disease behavior.

PIK3CA mutational status in circulating tumor cells can change during disease recurrence or progression in patients with breast cancer.

Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while PIK3CA somatic mutations play a crucial role in therapy response. We investigated the presence of PIK3CA mutations in CTC and whether this is associated with clinical outcome. We developed and validated an ultrasensitive methodology for the detection of PIK3CA mutations that is based on a combination of allele-specific, asymmetric rapid PCR and melting analysis. We analyzed PIK3CA hotspot mutations in: (i) a training group consisting of EpCAM-positive CTC fraction from 37 patients with clinically confirmed metastasis, and 26 healthy female volunteers and 15 primary breast tumor tissues and (ii) an independent group consisting of EpCAM-positive CTC fraction from 57 metastatic and 118 operable breast cancer patients and 76 corresponding primary tumors. The assay could detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons (9 and 20) and was highly specific (0/26 healthy donors). PIK3CA mutations were identified in EpCAM-positive CTC in 20 of 57(35.1%) and in 23 of 118 (19.5%) patients with metastatic and operable breast cancer, and in 45 of 76(59.2%) corresponding FFPEs. Our data indicate that PIK3CA mutational status in CTCs can change during disease progression and is associated with worse survival (P = 0.047). PIK3CA hotspot mutations are present at a relatively high frequency in CTCs and their presence is associated with worse survival in patients with breast cancer with metastasis. Evaluation of PIK3CA mutational status in CTCs is a strategy with potential clinical application.

Abstract 4813: DNA methylation of tumor suppressor and metastasis suppressor genes in primary tumors, circulating tumor cells and cell free DNA in the same breast cancer patients

Abstract Introduction: Blood based specimens such as Circulating Tumor Cells (CTCs) and cell free DNA (cfDNA) provide a very promising and non-invasive liquid biopsy approach. In our study, we first compared the promoter methylation status of BRMS1 and SOX17 genes in DNA samples isolated from primary tumors, matched CTCs and cfDNA isolated from the same breast cancer patients. Methods: We first developed and validated real time Methylation Specific PCR (MSP) assays for studying BRMS1, CST6 and SOX17 promoter methylation. We further analysed 165 paired DNA samples isolated from: a) the EpCAM positive CTC fraction from 92 operable, 61 metastatic breast cancer patients and 12 healthy individuals, b) the corresponding plasma from the same individuals. All CTC were also checked for CK-19 mRNA expression by RT-qPCR. Additionally, we investigated the promoter methylation status of BRMS1 and SOX17 in corresponding primary tumors (n=75). Results: In operable breast cancer, comparison of SOX17 promoter methylation status in CTCs and corresponding cfDNA revealed a direct association (P = 0.001), while it was also highly correlated with CK-19 mRNA expression (P=0.006). In patients with verified metastasis, there was a direct connection between SOX17 promoter methylation in CTCs and cfDNA (P=0.046), while SOX17 promoter methylation in cfDNA was highly correlated with CK-19 mRNA expression (P=0.04). Our results are shown below Conclusion: Our findings indicate a direct connection between SOX17 promoter methylation in CTCs and cfDNA both in patients with operable breast cancer, and verified metastasis. resultsBreast CancerGenesCTC fractioncfDNAFFPEsOperable (N=92)CST624/92(26.1%)33/92(35.9%)not analyzedSOX1719/92(20.7%)24/92(26.1%)26/43(60.5%)BRMS120/92(21.7%)22/92(23.9%)19/43(44.1%)Verified metastasis (N=61)CST621/61(34.4%)26/61(42.6%)not analyzedSOX1726/61(42.6%)22/61(36.1%)21/32(65.6%)BRMS126/61(42.6%)4/61(6.6%)5/32(15.6%) Citation Format: Maria Chimonidou, Areti Strati, Nikos Malamos, Vassilis Georgoulias, Evi Lianidou. DNA methylation of tumor suppressor and metastasis suppressor genes in primary tumors, circulating tumor cells and cell free DNA in the same breast cancer patients. [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 4813. doi:10.1158/1538-7445.AM2014-4813

Prognostic value of circulating tumor cells with podoplanin expression in patients with locally advanced or metastatic head and neck squamous cell carcinoma.

Podoplanin (PDPN) is a prognostic factor for head and neck squamous cell carcinoma (HNSCC). However, PDPN expression in circulating tumor cells (CTCs) and its prognostic value are not clear. The PowerMag system was used to enumerate CTCs from 53 patients with HNSCC prechemotherapy and 61 healthy donors. PDPN expression was determined by immunofluorescence staining. Results were correlated with clinicopathological parameters and clinical outcome, such as patient survival by receiver operating characteristic (ROC) and univariate and multivariate analyses. PDPN was expressed in a subset of CTCs. Both EpCAM-positive CTC and PDPN-positive CTC counts were statistically different between the disease and nondisease groups (p < .0001) with no prognostic value. After a median follow-up of 10.5 months (range, 6.6-18.5 months), the PDPN-positive/EpCAM-positive CTC ratio >20% was a significant prognostic factor for death within 6 months (p = .011) and was correlated with poor progression-free survival (p = .016) and overall survival (p = .015). PDPN-positive/EpCAM-positive CTC ratio is a prognostic factor and defining the ratio in patients with HNSCC might be valuable to clinical management.

Gemcitabine inhibits the micrometastasis of non-small cell lung cancer by targeting the EpCAM-positive circulating tumor cells via the HGF/cMET pathway.

Recurrence and metastasis are responsible for the death of non-small cell lung cancer (NSCLC) patients. Circulating tumor cells (CTCs) in the metastatic pathway have proven to be essential. This pilot study evaluated the sensitivity of gemcitabine in micrometastasis and CTCs from NSCLC patients. EpCAM-positive CTCs were detected in forty patients with NSCLC at treatment initiation and disease evaluation time-points. EpCAM-positive CTCs were defined as EpCAM-positive and CD45-negative. Total RNA was isolated from EpCAM-enriched CTCs and cytokeratin levels were detected by PCR. The HGF/cMET pathway was evaluated in CTCs from patients with different treatments and in A549 cells. The EMT-related markers were analyzed by IHC. We further explored the predictive value of baseline CTCs in patients that were receiving different treatments. The median number of CTCs in NSCLC patients was 65 CTCs/ml more than in the healthy 23?fold (median, 5.2 CTCs/ml). The mean change in cell count was significantly different for patients with gemcitabine compared to patients with non-gemcitabine treatments (-86.28 vs. -15.23/ml; P<0.05). A significant decrease was noted in the expression of cytokeratin in the CTCs of the two groups (P<0.05). The HGF/cMET pathway was inactivated in CTCs and A549 cells treated with gemcitabine, and the cell migration and invasion abilities were inhibited by gemcitabine via the HGF/cMET pathway. Furthermore, the decreased cell migration and invasion abilities may also be involved in the inhibition of the epithelial-mesenchymal transition (EMT) by gemcitabine. At a median follow-up of 36 months, the CTC count was confirmed to be a robust prognostic marker in the NSCLC population (CTCs >151, median: 15.0 months and CTCs <151, median: 32.0 months). Additionally, the survival rate in the gemcitabine group (24 months) was better than in non-gemcitabine group (21 months), suggesting a therapeutic benefit for NSCLC patient survival with the common therapy plus gemcitabine. Gemcitabine treatment decreased EpCAM-positive CTCs in NSCLC patients and inhibited EMT by the HGF/cMET pathway.

Retaining the long-survive capacity of Circulating Tumor Cells (CTCs) followed by xeno-transplantation: not only from metastatic cancer of the breast but also of prostate cancer patients.

We investigated whether Circulating Tumor Cells (CTCs) isolated from epithelial tumors could survive and grow in xenotransplants. To this purpose, EpCAM-positive CTCs were enriched by CellSearch platform the only FDA-cleared automated platform that quantifies tumor burden in peripheral blood and provides clinical evidence of predictive and prognostic value. The CTCs were isolated from metastatic prostate (n=6) and breast (n=2) cancer patients. The xenograft assay was developed in 8-week-old NOD/SCID mice that were subcutaneously injected with increasing amounts of CTCs (ranging from 50 to 3000). Human CTCs were found in 8 out of 8 murine peripheral blood (muPB) and in 6 out of 8 murine bone marrow (muBM) samples, after a median follow-up of 10.3 months. Six out of 8 spleens were positive for human cytokeratin. Our assay showed higher successful rate than those previously reported in breast cancer and hepatocellular carcinoma. The role of EpCAM during carcinogenesis is controversial. The identification of human CTCs in muPB, muBM and spleen demonstrates that the EpCAM-positive fraction of CTCs retains the migratory capacity. This is the first experimental evidence that as few as 50 EpCAM-positive prostate cancer CTCs putatively contain metastasis-initiating-cells (MIC).

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.

Presence of EpCAM‐positive circulating tumor cells as biomarker for systemic disease strongly correlates to survival in patients with hepatocellular carcinoma

Current imaging technologies do not sufficiently detect micrometastasis and therefore do not allow adequate stratification of patients with hepatocellular carcinoma (HCC) for curative or systemic therapy. In HCC, presence of stem cell-like, epithelial cell adhesion molecule (EpCAM)-positive cells correlates with tumor aggressiveness and formation of metastasis. Therefore, we investigated the prognostic relevance of EpCAM-positive circulating tumor cells (CTCs) in patients with HCC. Blood from 78 patients (19 patients in the control cohort and 59 patients with HCC) was tested for CTCs with the CellSearch™ system. Correlation analysis to overall survival (OS), the Barcelona Clinic Liver Cancer (BCLC) staging system, macroscopic and microscopic vascular invasion and alpha-fetoprotein (AFP) levels were performed. We detected ≥1 CTC in 18/59 HCC patients and in 1/19 patients with cirrhosis or benign hepatic tumor (p = 0.026). OS was significantly shorter (460 vs. 746 days) in the CTC-positive cohort (p = 0.017). Comparing BCLC stages, significant differences in CTC detection rates were also observed: BCLC stages A 1/9, B 6/31 and C 11/19 (p = 0.006). Ten of 18 patients with macroscopic and 10/16 patients with microscopic vascular invasion exhibited positive findings in CTC testing (p = 0.004 and p = 0.006). Furthermore, CTC results correlated to AFP (cutoff > 400 ng/mL) levels (p = 0.050). Our study demonstrates frequent presence of EpCAM-positive CTC in patients with intermediate or advanced HCC and its prognostic value for OS with possible implications for future treatment stratification.

Abstract 4135: Isolation of circulating tumor cells (CTCs) from breast cancer patients in EpCAM independent manner using parallel multi-orifice flow fractionation (p-MOFF).

Abstract It is generally accepted that circulating tumor cells (CTCs) in the peripheral blood of cancer patients are highly correlated with the "invasive behaviors" of a proportion of cancer cells. Therefore the precise measurement or isolation of CTCs can serve as a powerful tool for cancer prognosis, diagnosis of minimal residual disease, assessment of tumor sensitivity to anticancer drugs, and personalization of anticancer therapy. In a previous study, we introduced a novel hydrodynamic method for size-based particle separation multi-orifice flow fractionation (MOFF), in which the animal cellis moved laterally according to their size due to hydrodynamic inertial force. This technique is advantageous for CTC separation because of its simple experimental set up and high operational flow rate (100-300 μL/min). However, it has a limitation that the blood sample should be diluted over 400 times to make a suitable inter-particle distance in the channel. Therefore, we designed the parallel MOFF chip (p-MOFF) which is connected by four single MOFF channel to isolate CTC from cancer patient blood. Using breast cancer cell line, we separated 93.75% of MCF-7 cells and 91.60 % of MDA_MB_231 cells, and eliminate 90.8 % of WBCs from individual experiment at 600 μL/min inlet flow rate and 240 μL/min outlet flow rate. Based on these results, we have attempted to isolate CTCs from whole blood (7.5ml) of the breast cancer patients under the same conditions. After processing of blood from metastatic breast cancer patients on the p-MOFF device, cells were fixed on a slide glass and stained with DAPI for DNA content, PE conjugated pan-Cytokeratin for CTC, and APC conjugated CD45 for WBC. Following imaging the slide glass, captured images were examined carefully with predefined criteria. Cell size and shape were also considered in cell characterization. We found the CTCs which are positive for DAPI, negative for CD45 and positive for Cytokeratin. When the blood samples of 24 breast cancer patients were subjected to our device, 1 to 21 CTCs were identified in 19 patients. Subsequently, we have conducted the multi-parameter analysis to investigate the EpCAM expression level of CTCs. The blood samples of 24 metastatic breast cancer patients were analyzed. With our device, 17 patients had at least one CTC in 7.5 ml of blood. Among them, when we stained the cells using multi-parameter (DAPI for DNA content, phycoerythrin conjugated pan-Cytokeratin for CTC, APC conjugated EpCAM for EpCAM positive CTC, and FITC conjugated CD45 for WBC), we observed that the 47.1% of patients had both of EpCAM positive and negative CTCs simultaneously. So,the most advantageous feature is that p-MOFF is able to detect heterogeneous CTCs because the method is based on label-free and hydrophoresis Citation Format: Hyo-Il Jung, Kyung-A Hyun, Hyunju Han, Seung-Il Kim. Isolation of circulating tumor cells (CTCs) from breast cancer patients in EpCAM independent manner using parallel multi-orifice flow fractionation (p-MOFF). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4135. doi:10.1158/1538-7445.AM2013-4135

Abstract P2-01-08: Different numbers and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes.

Abstract Introduction: The enumeration of circulating tumour cells (CTCs) with the EPCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). However, breast cancer has been shown to be a molecularly heterogeneous disease. The aim of this study was to assess potential differences in the detection and prognostic significance of CTCs according to the immunohistochemically defined molecular subtypes of breast cancer. Methods: CellSearch CTC counts were obtained from 110 patients with MBC prior to first line systemic treatment, treated at GZA Hospitals Sint-Augustinus between november 2007 and december 2011. Clinicopathological variables were prospectively entered in a database. Based on the St-Gallen surrogate definitions of intrinsic breast cancer subtypes (Goldhirsch et al. Ann Oncol 2011), patients were divided in 5 groups: luminal A (ER/PR+, HER2−, Bloom-Richardson histological grade I-II), luminal B – HER2 negative (ER/PR+, Her2−, grade III), luminal B – HER2 positive (ER/PR+, HER2+, any grade), HER2 positive – non luminal (ER/PR−, HER2+), and triple negative (TN) (ER/PR−, HER2−). Differences in progression free survival (PFS) and overall survival (OS) according to the FDA approved prognostic cut-off of ≥5 CTC/7.5 ml blood were estimated using Kaplan Meier and Cox proportional hazard statistics. Results: CTC were detected in 78 of 110 (71%) patients. Higher detection rates and numbers of CTC were observed in patients with luminal A and TN breast cancer as compared to patients with luminal B and HER2 positive disease. However, no differences in positivity rates were observed between molecular subtypes according to the 5 CTC prognostic cut-off point (table 1). After a median FU time of 3.1 years, 39 patients had died. In the total study population, the presence of ≥5 CTC was an independent predictor of PFS and OS in multivariate analysis (PFS: HRCTC≥5=2.236 (1.366–3.658), p = 0.001; OS: HRCTC≥5=3.180 (1.553–6.509), p = 0.002). When analyzing subgroups separately, a lower prognostic power was observed in the HER2 positive and luminal B subgroups. Conclusion: Significant differences were observed in the detection and prognostic significance of EPCAM positive CTC according to the immunohistochemically defined breast cancer subtypes. Interestingly, CTC were detected more frequently in patients with luminal A and TN tumors. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2 positive patients with MBC. Our data independently confirm those reported by Giordano et al. (Ann Oncol 2010) in a large clinically uniform population of patients with MBC before the start of first-line treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-08.

A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire

The isolation of circulating tumor cells (CTCs) from the blood of patients afflicted with solid malignant tumors becomes increasingly important as it may serve as a ‘liquid biopsy’ with the potential of monitoring the course of the cancer disease and its response to cancer therapy, with subsequent molecular characterization. For this purpose, we functionalized a structured medical Seldinger guidewire (FSMW), normally used to obtain safe access to blood vessels and other organ cavities, with a chimeric monoclonal antibody directed to the cell surface expressed epithelial cell surface adhesion molecule (EpCAM). This medical device was optimized in vitro and its biocompatibility was tested according to the regulations for medical devices and found to be safe with no noteworthy side effects. Suitability, specificity and sensitivity of the FSMW to catch and enrich CTCs in vivo from circulating peripheral blood were tested in 24 breast cancer or non-small cell lung cancer (NSCLC) patients and in 29 healthy volunteers. For this, the FSMW was inserted through a standard venous cannula into the cubital veins of healthy volunteers or cancer patients for the duration of 30 min. After removal, CTCs were identified by immunocytochemical staining of EpCAM and/or cytokeratins and staining of their nuclei and counted. The FSMW successfully enriched EpCAM-positive CTCs from 22 of the 24 patients, with a median of 5.5 (0–50) CTCs in breast cancer (n=12) and 16 (2–515) CTCs in NSCLC (n=12). CTCs could be isolated across all tumor stages, including early stage cancer, in which distant metastases were not yet diagnosed, while no CTCs could be detected in healthy volunteers. In this observatory study, no adverse effects were noted. Evidently, the FSMW has the potential to become an important device to enrich CTCs in vivo for monitoring the course of the cancer disease and the efficacy of anticancer treatment.

A new medical device for in vivo capturing of circulating tumor cells in breast cancer (BC) patients.

10537 Background: In BC, the number of circulating tumor cells (CTCs) is discussed as a prognostic and stratification biomarker, and could also reflect treatment efficacy. Currently, CTCs are isolated ex vivo from a small volume of blood. Results from a larger volume of blood are scarce. The aim of the study was to assess a functionalized and structured medical wire (FSMW) for in vivo capturing of CTCs directly from the blood stream of BC patients. Methods: The device was inserted in a cubital vein through a standard cannula for thirty minutes. The interaction of target CTCs with the FSMW was mediated by antibodies directed against the epithelial cell adhesion molecule (EpCAM). To confirm binding of CTCs to the wire, the immunohistochemical positive staining against EpCAM as well as negative staining for CD45 was performed. There were 54 applications of the wire in 42 stage I-IV BC patients (12 double applications). Enumeration data from 37 BC patients with 49 applications (5 failed subsequent analyses) were assessed. CTC counts on 23 devices were directly compared to counts by CellSearch. Results: The device was well tolerated in all 54 applications without side effects. We obtained in vivo isolation of CTCs in 44 of 49 applications to BC patients (89.7 %). The sensitivity was similar for early and late stage BC patients. The median (range) of isolated EpCAM-positive CTCs was 5 (0-515). The CellSearch method reached a sensitivity of 18.5%. In all paired samples the number of CTCs detected with the FSMW was higher or equal to CellSearch, regardless of the disease stage. Linear regression of the data of the double application of the FSMW showed a very good concordance (r2 = 0.97, p&lt;0.0001). Conclusions: Whilst well tolerated without side effects, the CTC detection rate of the FSMW in BC patients was nearly 90 %. CTC detection was obtained in 18.5% by the CellSearch. Double application of FSMWs in the same patient indicates ample precision. This proof of concept study may have important clinical implications, as the device may improve early detection, prognosis and therapy monitoring of BC patients. The molecular analysis of the captured CTCs could become a breakthrough in personalized medicine.

P4-07-08: Subsets and Molecular Signatures of Circulating Tumor Cells in Breast Cancer Brain Metastasis.

Abstract Background: Circulating tumor cells (CTCs) represent the “seeds” of intractable brain metastatic breast cancer (BMBC); however, properties of CTCs targeting the brain remain elusive. For example, the FDA-approved CTC platform (CellSearch™, Veridex, LLC) detects only CTCs positive for epithelial cell adhesion molecule (EpCAM) and cytokeratins (CKs), but is unable to capture any other CTC subtypes or analyze biomarkers of brain-homing CTCs. We hypothesized that profiling CTCs from BMBC patients might result in the identification of brain-colonizing CTC signatures. Materials and Methods: We employed CellSearch™ and a novel technology that uses analysis of specific antigenic markers by immunofluorescence, coupled with detecting gene amplification by fluorescence in situ hybridization on the same cells; and quantification of the signal via automated scanning (FICTION; BioView Duet-3™ system). Results: We established that our approach was feasible by performing CTC analyses on peripheral blood mononuclear cells isolated from BMBC patients or patients not possessing overt metastatic disease. We detected a differential gene amplification for human epidermal growth factor receptor1 and 2 (EGFR and HER2, respectively). Second, the number of EpCAM-positive CTCs visualized by the BioView™ platform was at least three orders of magnitude higher than one obtained from CellSearch™ CTC analyses using the same specimen. Third, we identified the presence of CTCs positive for CKs but negative for EpCAM. Conversely, high levels of prometastatic heparanase, in conjunction with the expression of aldehyde dehydrogenase-1 (ALDH1), a known cancer stem-cell marker, were detected in CTCs from BMBC patients; with a correlation between heparanase, ALDH1, and high EGFR amplification. Finally, extensive flow cytometric/FACS analyses validated the presence of CTC subsets negative for EpCAM and CD45, a hematolymphoid marker, however enriched for heparanase/ALDH1 expression. Discussion: These findings indicate that the BioView™ platform not only captures more EpCAM-positive CTCs than CellSearch™ but also allows the detection of novel CTC subtypes possessing varying EpCAM levels. Importantly, they suggest that profiling CTC subtypes in patients with BMBC can be relevant towards the discovery of BMBC founder CTCs. Work is ongoing to further characterize these CTC subtypes, and to assess their abilities to metastasize to brain in xenotransplantation studies using immunodeficient mice. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-08.

Abstract 5172: Fluorescence labeling of cytokeratin-negative circulating tumor cells in peripheral blood: A new paradigm for the study of cancer progression

Abstract Introduction: The measurement of circulating tumor cells (CTCs) in blood generally requires either anti-EpCAM for capture, anti-cytokeratin (CK) for detection, or both. However, EpCAM and CK are absent in some tumor cells, and both may be down-regulated during neoplastic progression or during epithelial-to-mesenchymal transition (EMT). Here we show capture on a micro-fluidic system using single or multiple antibodies, and CTC detection with CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to the CTCs. Methods: Buffy coat cells isolated from 8 mL of blood were pre-incubated with either anti-EpCAM alone or with a mixture of antibodies to epithelial and mesenchymal cell surface antigens. CTCs were captured on a micro-fluidic channel. Identification of CTCs was determined with anti-CK and with CE to detect cells with the capture antibodies bound to the cell surface antigens. All cells scored as positive for CK or CE were, by definition, CD45-negative and DAPI-positive. Results: Using anti-EpCAM alone for capture, significantly more CTCs were detected by CE staining than with anti-CK in breast and prostate cancers. This indicated that CK-negative CTCs were captured but not detected and that some EpCAM-positive CTCs were CK-negative. Results using capture antibody mixtures varied from sample to sample but gave up to 2-fold higher CK-positive cells than anti-EpCAM only, and as much as 4-fold higher CE-positive cells. Control blood from healthy donors was CK and CE-negative. All CK-positive cells co-stained with CE, as determined with different fluorescent labels. In a clinical study of stage IV breast cancer, CTCs were isolated with an antibody mixture and sequentially stained with CK and CE. Fifteen of 24 samples (63%) contained CK-positive cells (range 1-60 CTCs) while 24 of 24 samples (100%) contained additional CE-positive cells (range 1-41; median=11; Wilcoxon test, p=0.02). The modest correlation coefficient (r = 0.57, p=0.004) for the number of CE-positive and CK-positive cells in each sample suggests that one or more different phenotypes of CTCs were being detected. Amplified Her2 was detected by FISH in isolated CK-positive CTCs, and also in CK-negative, CE-positive CTCs from Her2-positive patients, indicating these were tumor cells. Conclusions: The CEE-Enhanced staining technology enables the detection of CK-negative CTCs. This novel detection method, based on the in situ labeling of antibodies used for capture, greatly expands single and multi-antibody approaches to the study of rare circulating cells. Specifically, this allows the exploration and study of circulating cells not expressing CK such as highly de-differentiated tumor cells, stem cells or those tumor cells undergoing EMT. The clinical significance of these CK-negative CTCs is under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5172. doi:10.1158/1538-7445.AM2011-5172

Relationship between the detection rate of EpCAM-positive circulating tumor cells (CTCs) in metastatic breast cancer patients and the CTC enrichment technology.

e21066 Background: Immunomagnetic EpCAM-based methods are successfully used to enrich and enumerate circulating tumor cells (CTCs) in metastatic breast cancer patients (mBC pat). In vitro data raised concerns whether EpCAM dependent CTC assays may enrich EpCAM negative (EpCAM-) CTCs. Methods: Tumor cells were identified with the anticytokeratin antibody, A45-B/B3. (i) We evaluated the EpCAM expression of 3 cell lines MCF-7, ZR-75-1 and Hs587T with flow cytometry. (ii) An anti-EpCAM immunomagnetic enrichment method, MACS HEA MicroBeads (MACS) was compared to a non-anti-EpCAM based new density centrifugation method, OncoQuick plus (OQ+), using spiking experiments. (iii) To validate these data in vivo, peripheral blood from 26 mBC pat was collected to compare the two methods. (iv) To explore EpCAM expression of the respective patients' CTCs, blood was enriched with the OQ+ procedure. An aliquot of enriched blood was immunocytochemically stained with a fluorescence-labeled EpCAM antibody. The other aliquot was used to verify EpCAM expression with RT-qPCR. Results: (i) MCF-7 and ZR-75-1 cell lines showed a strong (>90%), Hs587T tumor cells had a low (< 5%) or no EpCAM expression, respectively. (ii) MACS recovered significantly more of a definite number of spiked tumor cells than OQ+ in EpCAM positive (EpCAM+) cell lines (p = 0.002 and p = 0.007, respectively). OQ+ recovered significantly more EpCAM- Hs587T cells (p < 0.0001). (iii) With MACS 42.3% of mBC pat. had CTCs detected (median 0). With OQ+ CTCs were spotted in 69.2% of pat (median 8). The positivity rate of detected CTCs did not differ significantly between MACS and OQ+ (p = 0.065). A significantly higher number of CTCs was enumerated with OQ+ (p = 0.018). (iv) Irrespectively of the EpCAM detection method, 19% of mBC pat had EpCAM+ CTCs. MACS detected significantly (p = 0.001) more EpCAM+ CTCs. No association was found between CTCs detected by OQ+ and their EpCAM expression. Conclusions: The CTC enrichment method should be chosen according to the respective CTCs' EpCAM expression. Further investigations have to determine whether the accuracy of prognostication of mBC pat depends on the detection of EpCAM± CTC subpopulations. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca