Positive Circulating Tumor Cells Count Research Articles

Clinical significance of circulating tumor cells and their expression of cyclooxygenase-2 in patients with nasopharyngeal carcinoma.

To explore the expression of circulating tumor cells (CTCs) and cyclooxygenase-2 (COX-2) in CTCs and to assess their association with clinical parameters and treatment. Peripheral blood samples from 50 patients with nasopharyngeal carcinoma (NPC) were included. We applied advanced CanPatrolTM CTC enrichment technique and in situ hybridization assay to isolate, identify, and classify CTCs and COX-2 in CTCs. Epstein-Barr virus DNA was detected by Real Time-quantitative PCR (RT-qPCR). No CTCs were identified in ten healthy volunteers (100%). Of the total patients, 48 (96%) had positive CTCs counts and 36 (72%) had positive mesenchymal CTCs counts before treatment. CTCs cells were highly expressed in different NPC stages, and the positive ratio of mesenchymal CTCs in stage IV was higher than that in other stages. The proportion of mesenchymal cells was higher expressed in metastasis patients. The expression of COX-2 was different in different types of CTCs. The positivity of COX-2 in CTCs was higher in stage IV patients than that in stage II and stage III patients. Decreased mesenchymal CTCs and that express COX-2 indicated a favorable curative effect in NPC patients. The positivity of mesenchymal CTCs and COX-2 was higher in EBV DNA positive patients compared with EBV DNA negative patients (p<0.05). Meanwhile, the mean number of CTCs, mesenchymal CTCs, CTCs that express COX-2, hybrid CTCs that express COX-2 and mesenchymal CTCs that express COX-2 was significantly higher in the EBV DNA positive patients than negative patients before treatment (p<0.05). CTCs and their expression of COX-2 were correlated with NPC clinical characteristics, and have a relation with Epstein-Barr virus DNA. Decreased mesenchymal CTCs and that express COX-2 indicated a favorable curative effect in NPC patients.

Prognostic value of circulating tumor cells in non-small cell lung cancer.

e20520 Background: Quantitative analysis of circulating tumor cells (CTCs) is increasingly investigated as a novel predictor of prognosis in non-small cell lung cancer (NSCLC). The primary aim of this study was to further investigate the prognostic value of CTCs enumeration in patients with advanced NSCLC. Methods: The medical records of patients with chemotherapy-naïve, advanced NSCLC and at least one available measurement of CTCs were retrospectively reviewed. The presence of CTCs was evaluated using the CellSearch system. A cut-off point of 2 CTCs ( &lt; 2 vs ≥2 ) was used to discriminate between negative and positive samples, respectively. The detection of CTCs was correlated with the clinicopathological features of patients and outcome. Results: Our study population comprised a total of 43 patients (mean age 67.1±9.9 years; male/female ratio: 39/4). Eight out of 43 patients (18.6%) harbored CTCs. No significant associations were found between treatment response and baseline count of circulating tumor cells (CTCs) (p = 0.067). Positive CTC count was associated with the presence of five or more metastatic sites (p = 0.018). Neither PFS nor OS were found to differ significantly between CTC-positive and CTC-negative cases (log-rank p = 0.079 and 0.549, respectively). The presence of five or more metastatic sites and worse PS were recognized as independent predictors of reduced PFS [HR = 2.9, p = 0.042 and HR = 2.7, p = 0.035, respectively], and reduced OS [HR = 7.8, p = 0.003 and HR = 16.9, p &lt; 0.001, respectively]. Conclusions:CTCs were detected in a relatively small percentage of our patient population and their presence was associated with increased metastatic burden. In contrast, we failed to observe any significant associations between baseline CTC count and the prognostic variables evaluated. Our results support the suggested role of CTCs in the metastatic dissemination of cancer and underline the need for more efficient methods of CTCs detection.

Predictive and prognostic value of folate receptor-positive circulating tumor cells in small cell lung cancer patients treated with first-line chemotherapy.

To assess the predictive and prognostic significance of folate receptor (FR)-positive circulating tumor cells (CTCs) in patients with small cell lung cancer (SCLC) received first-line chemotherapy. Eligible patients with chemotherapy-naïve, unresectable SCLC were enrolled and blood samples were collected. CTCs were enumerated using ligand-targeted polymerase chain reaction (LT-PCR) at baseline, after two cycles of chemotherapy regimen and on disease progression. In total, 80 patients were enrolled and 67 (83.8%) had positive CTC count at baseline (CTCs ≥ 8.7 FU/3mL). The baseline CTC counts in patients with partial response (PR) were significantly higher than those with progression disease (PD) (P = 0.0365). An obvious reduction of CTC enumeration after two cycles of chemotherapy was significantly correlated with PR (P = 0.0380), instead of SD (P = 0.4934). Among positive CTC count group, patients with relative low CTC level had significantly longer progression-free survival (PFS) and overall survival (OS) than those with high CTC level (PFS: 9.1 vs 6.9 months, P = 0.0458; OS: 11.1 vs 8.6 months, P = 0.056). In multivariate analysis, distant metastases (HR = 1.466, P = 0.021) and relative low CTC level (HR = 0.656, P = 0.049) were the independent predictive factors for patients with SCLC received first-line chemotherapy. The present results demonstrated that baseline CTC counts could be the valuable predictive and prognostic biomarker for patients with SCLC received first-line chemotherapy. The reduction of CTC enumeration after two cycles of chemotherapy was a potential predictor of chemotherapeutic response in SCLC.

Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer.

Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3–15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0–2 cells/1.88 mL of blood). The latter range was validated as the ‘false positive’ cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing.

Limited prognostic value of preoperative circulating tumor cells for early biochemical recurrence in patients with localized prostate cancer

IntroductionThe presence of circulating tumor cells (CTCs) is an established marker for prognosis in men with castration-resistant prostate cancer. A cutoff of ≥5 CTCs/7.5ml blood in the CellSearch Epithelial Cell Test has been shown to stratify prognostic groups and predict outcome of abiraterone treatment. In contrast, the value of CTC detection in men with localized prostrate cancer before radical prostatectomy (RP) is unknown. Materials and methodsA total of 152 patients treated with RP between 06/2009 and 09/2009 were included. Peripheral venous blood drawn the day before RP was evaluated for CTCs by the CellSearch system. The detection of CTCs was correlated with prostate-specific antigen (PSA) and the histopathological outcome of the RP specimen. A cutoff of 0 vs. ≥1 CTC/7.5ml blood was defined as the threshold for positive vs. negative CTC status. ResultsMedian age was 62 years and median PSA was 6.7ng/dl. Staging revealed 62.5% pT2, 26.3% pT3a, and 11.2% pT3b tumors, and high-grade disease (≥Gleason 4+3) was determined in 25.6% of patients. CTCs were detected in 17 patients (11%) with a median CTC count/7.5ml of 1 (range: 1–clusters with>100 epithelial cells) without significant correlations to PSA levels, pT stage, or Gleason scores. Postoperative pT stage was a significant predictor of biochemical recurrence (BCR) in univariable logistic regression models and as a composite measure together with positive CTC counts (P<0.0001). CTC positivity alone tended to have a higher hazard ratio for BCR, but this was not statistically significant (P = 0.1). After a median follow-up of 48 months, there was no significant difference in BCR-free survival between patients with or without CTCs (P = 0.7). ConclusionUsing the CellSearch system, we infrequently detected CTCs in patients with localized tumors before RP. The detection of CTCs did not correlate significantly with PSA, disease characteristics, or the development of BCR. However, larger cohorts with extended follow-up are needed to validate our findings.

Abstract LB-305: Circulating tumor cells as a novel model to test efficacy of individualized therapy in breast cancer

Abstract Background: Recent reports suggest that circulating tumor cells (CTCs) mimic cancer progenitor cells, which are commonly resistant to various treatment modalities and are capable of invasion and tumor initiation at a single cell level. We hypothesized that CTCs isolated from metastatic breast cancer (MBC) patients can be propagated in 3-D in vitro systems, representing a clinically relevant model to study MBC biology and to test efficacy of multiple anti-cancer drugs. Methods: CTCs were isolated from MBC patient peripheral blood by depleting leucocytes and red blood cells. Enriched CTCs were immunostained for detection as CD45-negative (-) and nuclear counter stain-positive (+) cells, and for expression of estrogen receptor (ER) and HER-2. In order to obtain a large and continuous supply of CTCs for our culture studies, we screened 19 primary transplantable xenograft lines, established by directly transplanting human breast tumors into epithelium-free mammary fat pads of SCID/Beige mice, for their production of CTCs. The CTCs were isolated together with mouse mononuclear blood cells by either gradient centrifugation or red blood cell lysis, and identified by human pan-cytokeratin immunostaining. Viable CTCs isolated from both patients and xenograft mice were plated in mammosphere conditions either alone or in co-culture with mammary fibroblasts tagged with green fluorescent protein (GFP). the propagated CTCs were identified as GFP-, cytokeratin 19+ cells. Results: To date, we have collected and processed blood samples from 14 MBC patients. CTCs were detected in 5 out of the 8 samples (62%) already analyzed. Interestingly, in 2 out of 4 patients (50%) with positive CTC counts and ER+ primary tumor, all the isolated CTCs were ER-. Moreover, we found HER-2+ CTCs in 1 out of 4 patients (25%) with HER-2 negative disease. CTC count and characterization of the additional collected samples is ongoing and will be presented at the conference. In the mice bearing primary xenografts, we detected CTCs in 13 out of 19 lines (68%) and in 26 out of 54 mice (48%). When CTCs isolated from either human patients or primary xenografts were cultured alone, no mammospheres were observed. In contrast, when CTCs were cultured in direct contact with fibroblasts we observed the formation of mixed CTC/fibroblast spheres that may reproduce the tumor cell/stroma interaction observed in vivo. Moreover, in the presence of GFP+ fibroblasts, the numbers of GFP- cells and cell aggregates were clearly higher at all time points compared to the single culture. Conclusions: Our study suggests that viable CTCs isolated from human patients and primary xenograft bearing mice can be propagated in vitro in presence of mammary fibroblasts. The successful development of a CTC-based cell culture platform will provide a unique model to better understand the biological mechanisms of the metastatic process and to guide treatment decisions in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-305. doi:1538-7445.AM2012-LB-305

Abstract 2370: Development of microcavity array system for size- and deformability-based isolation of circulating tumor cells

Abstract Background: EpCAM-based enumeration of circulating tumor cells (CTCs) has prognostic value in solid tumors such as advanced breast, colon and prostate cancers. However, currently poor sensitivity has limited the use of CTCs in other types of cancers including non-small cell lung cancer (NSCLC). We have developed a microcavity array (MCA) system integrated with microfluidic device for recovery and enumeration of CTC regardless of EpCAM expression level, allowing isolation of tumor cells on the basis of differences in the size and deformability between tumor and hematologic cells. Shapes and sizes of the cavity were optimized in order to trap tumor cells while letting blood cells flow through the microcavities during whole blood filtration. Enrichment of CTCs, fixation, permeabilization, staining, and counting process were implemented in a microfluidic assay within one integrated device. In this study, we evaluated the sensitivity of the MCA system in detecting CTCs with a preclinical model using cell lines and conducted a clinical feasibility study in NSCLC patients. Methods: A wide range of cancer cell lines derived from breast (MCF-7, Hs578T), colon (SW620), gastric (AGS, SNU-1) and lung (A549, HCC-827, NCI-H358, NCI-H441, NCI-H1650, NCI-H1975, NCI-H69, NCI-H82) were used for spike-in experiments. Cells were spiked into 1 mL of healthy donor blood and then introduced into the MCA system. Trapped cells were stained with Hoechst 33342, FITC-labeled anti-pan cytokeratin antibody and PE-labeled anti-CD45 antibody for subsequent imaging analysis. CTCs were defined as cells with round to oval morphology, a visible nucleus, positive staining for pan-cytokeratin and negative staining for CD45. For the clinical evaluation, 16 advanced NSCLC patients were enrolled into the study and we conducted a head-to-head comparison study with CellSearch system. Results: We obtained a quite high recovery rate regardless of tumor types ranging from 80 to 99% in the cell line spike-in experiments containing EpCAM-negative cell lines (Hs578T, SNU-1). Most of recovered cells were viable and were able to proliferate even after isolation process, suggesting the potential for further biological and molecular analyses of CTCs. In the clinical part of the study, CTCs were detectable in 12 out of 16 patients (count β1 per 7.5 ml) with our system. More CTCs were detected by MCA system (median 13, range 0-313 cells/7.5ml blood) than by CellSearch system (median 0, range 0-37 cells/7.5ml blood) demonstrating statistical superiority (p=0.0132, Wilcoxon test). It is also noteworthy that among patients who had negative CTC count by CellSearch system, several patients had positive CTC count by MCA system, suggesting better detection of EpCAM-negative CTCs. Conclusion: Our results suggest the potential of our MCA system for detection of CTCs in solid tumors including NSCLC and further clinical development should be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2370. doi:1538-7445.AM2012-2370

Abstract B20: EpCAM-independent ApoStream™ technology isolates circulating tumor cells from blood of patients with various types of cancer.

Abstract Introduction: Referred to as a “liquid tumor biopsy”, the ability to enrich Circulating Tumor Cells (CTCs) from blood samples allows for the analysis of cancer cells undergoing metastatic dissemination and has been a hallmark of biomarker discovery. However, the use of EpCAM-based enrichment platforms limits the type of tumor cells that can be recovered, which subsequently limits the use of CTCs as surrogates for the classical tumor biopsy. Therefore an EpCAM-independent enrichment platform is critically needed. Specific Aims: The performance of a novel antibody-independent dielectrophoretic field-flow fractionation based CTC isolation technology ApoStream was demonstrated in spiked cell model and cancer patient blood. Experimental Procedures: We used ApoStream, a novel antibody-independent technology, to enrich CTCs from prostate and breast cancer patient blood (high EpCAM expression), non-small cell lung cancer patient blood (NSCLC, low EpCAM expression) and melanoma (no EpCAM expression). Cells isolated from ApoStream were stained for cytokeratin (CK), CD45, and DAPI, and melanoma CTCs with tumor marker S100, CD45 and DAPI. Imaging and CTC enumeration were done using laser scanning cytometry (LSC). CTC morphology in lung cancer specimens was confirmed with H&amp;E staining. To further demonstrate performance of ApoStream platform, EpCAM-negative ovarian cancer cells SKOV3 were spiked into peripheral blood mononuclear cells (PBMCs) from normal donor blood and isolated using the ApoStream device. Results: High CTC recovery from cancer patient blood samples was achieved with counts ranging from 0 − 2104 (lung, n=33), 0 − 3490 (prostate, n=15), 176 − 968 (breast, n=3), and 4 − 3120 (melanoma, n=11) CTCs per 7.5 ml blood. Positive CTC counts were obtained in ninety percent of NSCLC samples, 93% of prostate cancer samples, 100% breast cancer and melanoma specimens. There were no false-positive CTCs from normal donor blood controls demonstrating ApoStream's specificity. In cell spiking experiments, 80 ± 3% cells were recovered by ApoStream after spiking of EpCAM-negative ovarian cancer cells (SKOV3) into human peripheral blood mononuclear cells (PBMCs). EpCAM-negative CTCs were also recovered in all tested breast cancer patient specimens, confirming that ApoStream isolates cancer cells independent on their EpCAM status. Conclusions: Our data demonstrate that the ApoStream platform recovers large numbers of CTCs from the blood of patients with metastatic NSCLC, prostate, breast cancer and melanoma. Thus, the ApoStream CTCisolation platform provides a new effective tool with broad applications in cancer biomarker discovery and personalization of cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B20.