Isolation By Size Of Epithelial Tumor Research Articles

Detection of circulating stage III–IV gastric cancer tumor cells based on isolation by size of epithelial tumor: using the circulating tumor cell biopsy technology

Background: To explore the correlations between circulating tumor cells (CTCs) and the patients’ clinical characteristics and other tumor markers in stage III–IV gastric cancer patients, and assess the value of CTCs as a predictor of the efficacy of chemotherapy. Methods: CTCs were measured in 42 patients with stage III–IV gastric cancer using the isolation by size of epithelial tumor (ISET) method. We analyzed the correlations between the number of CTCs, patients’ pathological characteristics and peripheral blood tumor markers. Then divided the 42 patients into two groups, the progressive disease group (PD group) and the disease control rate group (DCR group), according to the efficacy of chemotherapy, and analyzed the differences in the CTC expression between the two groups. Results: The threshold number of CTCs was closely related to the clinical stage (P=0.044), and was positively correlated with the value in U/mL of CA724 (P Conclusions: CTCs technology based on ISET method has a high detection rate. CTCs are promising predictor for the evaluation and prediction of treatment responses in stage III–IV gastric cancer.

Detection of circulating stage III-IV gastric cancer tumor cells based on isolation by size of epithelial tumor: using the circulating tumor cell biopsy technology.

BackgroundTo explore the correlations between circulating tumor cells (CTCs) and the patients’ clinical characteristics and other tumor markers in stage III–IV gastric cancer patients, and assess the value of CTCs as a predictor of the efficacy of chemotherapy.MethodsCTCs were measured in 42 patients with stage III–IV gastric cancer using the isolation by size of epithelial tumor (ISET) method. We analyzed the correlations between the number of CTCs, patients’ pathological characteristics and peripheral blood tumor markers. Then divided the 42 patients into two groups, the progressive disease group (PD group) and the disease control rate group (DCR group), according to the efficacy of chemotherapy, and analyzed the differences in the CTC expression between the two groups.ResultsThe threshold number of CTCs was closely related to the clinical stage (P=0.044), and was positively correlated with the value in U/mL of CA724 (P<0.05). The treatment response to cytotoxic chemotherapy in the high threshold number group was significantly poorer.ConclusionsCTCs technology based on ISET method has a high detection rate. CTCs are promising predictor for the evaluation and prediction of treatment responses in stage III–IV gastric cancer.

Abstract B07: CTC and CTM detection in sarcoma patients

Abstract Background: Sarcomas represent 1% of all cancers in adults, 8% in adolescents and young people, and 10% in children. Even though it is a rare cancer, it contributes to a significant loss of years of life in comparison with other types of cancer (Amankwah et al., 2013). The presence of isolated circulating tumor cells (CTCs) or circulating tumor microemboli (CTM) in the blood of patients with sarcoma may be early markers of tumor invasion, because it is known that these cells circulate in the blood for months or years before the development of metastases (Paterlini-Brechot and Benali, 2007; Klain, 2009; Rhim et al., 2012). Objective: To isolate and quantify CTCs and CTM from sarcoma patients, evaluating their presence and correlation with progression-free survival (PFS). Methods: Blood (approximately 8 mL) was prospectively collected from patients with different types of high-degree sarcoma. Blood was collected before the beginning of chemotherapy. The samples were processed and filtered on ISET (Isolation by Size of Epithelial Tumor Cells, Rarecells, France) system for the isolation and quantification of CTCs and CTMs. Immunocytochemistry (ICC) was later performed with anti-CD45 antibody and counterstained with hematoxylin-eosyn for leucocytes population exclusion. The cutoff was estimated using the maximum of the standardized log-rank statistic proposed by Lausen and Schumacher (1992). Results: We analyzed 11 high-degree sarcoma patients after metastasis diagnosis, before the beginning of treatment. The median age was 53 years (18-77) and 54.5% were male. The median CTCs number was 2.0 CTCs/mL (1.0-11.0) at baseline. By Kaplan-Meier test, we observed that patients with CTC number below the established cutoff (4.5 CTCs/mL) had better PFS compared to those above the cutoff (7.4 vs. 1.0 months, respectively; P= 0.006). Moreover, patients without CTM had also better PFS compared to those patients with the presence of CTM (7.6 vs. 2.7 months, respectively; P= 0.13). Conclusion: This is the first study to demonstrate, besides the CTC and CTM presence in sarcoma patients, its real prognostic value, even in a limited sample. Our next step is to characterize these cells to better understand its capability to promote tumor progression. Citation Format: Alexcia C. Braun, Celso A L Mello, Marcelo P. Corassa, Alves S. Vanessa, Emne A. Abdallah, Nicolau R. Ulisses, Mônica T. M. Díaz, Bianca T. C. P. Flores, Marcelo F. Fanelli, Patrizia Paterlini-Brechót, Ludmilla T. D. Chinen. CTC and CTM detection in sarcoma patients [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B07.

Circulating Epithelial Cells in Intraductal Papillary Mucinous Neoplasms and Cystic Pancreatic Lesions.

Circulating epithelial cells (CECs) are identified in the blood of patients with intraductal papillary mucinous neoplasms (IPMNs) despite the absence of malignancy. We assessed the blood of patients undergoing resection for IPMN or other benign pancreatic lesions for CECs. Peripheral blood was collected from 26 patients prior to pancreatic resection and filtered by the ISET (Isolation by Size of Epithelial Tumor Cells) method. Circulating epithelial cells were identified with antibodies to cytokeratin and Pdx1 (pancreas and duodenal homeobox protein 1), a pancreas marker. Nineteen patients underwent resection of an IPMN without associated malignancy. Eleven patients (58%) had cytokeratin-positive CECs. Circulating epithelial cells were significantly more likely to be found in patients with IPMNs with high-grade dysplasia (P = 0.04). In addition, 10 of the 11 patients with cytokeratin-positive CECs also had separate populations of cytokeratin-positive, Pdx1-positive CECs, suggesting a pancreatic source. Dual-staining CECs were more frequently found in patients with high-grade dysplasia (P = 0.04). Patients with IPMNs were significantly more likely to have pan-cytokeratin CECs in the blood compared with those without IPMNs (P = 0.01). Circulating epithelial cells staining with potential pancreas-specific markers have been found in patients with IPMNs, even without malignancy. Circulating epithelial cells may help to differentiate patients with high-grade IPMN from lower grades of dysplasia and other pancreatic cysts.

Abstract 1728: CTCs and CTM as prognostic factors in sarcoma patients

Abstract Background: Sarcomas represent 1% of all cancers in adults, 8% in adolescents and young people, and 10% in children. Even though it is a rare cancer, it contributes to a significant loss of years of life in comparison with other types of cancer (Amankwah, et al., 2013). The presence of isolated Circulating Tumor Cells (CTCs) or Circulating Tumor Microemboli (CTM) in the blood of patients with sarcoma may be early markers of tumor invasion, because it is known that these cells circulate in the blood for months or years before the development of metastases (Paterlini-Brechot and Benali, 2007; Klain, 2009; Rhim et al., 2012). Objective: To isolate and quantify CTCs and CTM from sarcoma patients, evaluating their presence and correlation with progression free survival (PFS). Methods: The blood (approximately 8mL) was prospectively collected from patients with different types of high degree sarcoma. Blood was collected before the beginning of chemotherapy. The samples were processed and filtered on ISET (Isolation by Size of Epithelial Tumor Cells, Rarecells, France) system for the isolation and quantification of CTCs and CTMs. Later, was performed immunocytochemistry (ICC) with anti-CD45 antibody and counterstained with hematoxylin-eosyn for leucocytes population exclusion. The cutoff was estimated using the maximum of the standardized log-rank statistic proposed by Lausen &amp; Schumacher (1992). Results: we analyzed 11 high degree sarcoma patients before the beginning of treatment (any treatment line). The median age was 53 years-old (18-77) and 54,5% were male. The median CTCs number was 2.0 CTCs/mL (1.0-11.0) at baseline. By Kaplan-Meier test, we observed that patients with CTC number below the established cutoff (4.5 CTCs/mL) had better PFS compared to those above the cutoff (7.4 vs. 1.0 months respectively; P= 0.006). Moreover, patients without CTM had also better PFS compared to those patients with the presence of CTM (7.6 vs. 2.7 months respectively; P= 0.13). Conclusion: this is the first study to demonstrate, besides the CTC and CTM presence in sarcoma patients, its real prognostic value, even in a limited sample. Our next step is to characterize these cells to better understand its capability to promote tumor progression. Citation Format: Alexcia C. Braun, Celso A. Mello, Marcelo P. Corassa, Vanessa S. Alves, Emne A. Abdallah, Ulisses R. Nicolau, Mônica Taiane de Macedo Díaz, Bianca de Cássia Troncarelli de Campos Parra Flores, Marcelo F. Fanelli, Patrizia Paternili-Brechót, Ludmilla T. Chinen. CTCs and CTM as prognostic factors in sarcoma patients [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 1728. doi:10.1158/1538-7445.AM2017-1728

Circulating Tumor Cells Expressing Markers of Tumor-Initiating Cells Predict Poor Survival and Cancer Recurrence in Patients with Pancreatic Ductal Adenocarcinoma.

Purpose: Circulating tumor cells (CTCs) have been identified in the blood of patients with pancreatic adenocarcinoma (PDAC), but little is known about the exact phenotype of these cells. We assessed expression of aldehyde dehydrogenase (ALDH), CD133, and CD44 as markers of CTCs with a tumor-initiating cell (TIC) phenotype in patients with PDAC and the relationship of this expression to patient outcomes.Experimental Design: Peripheral blood from 60 consecutive patients with PDAC undergoing surgical resection was obtained and processed using the Isolation by Size of Epithelial Tumor (ISET) method. Immunofluorescence was used to identify CTCs expressing cytokeratin, CD133, CD44, and ALDH.Results: Forty-seven patients (78%) had epithelial CTCs staining positive for pan-cytokeratin and at least one TIC marker. Forty-six patients (77%) had epithelial CTCs that labeled with antibodies to cytokeratin and ALDH. By separate analysis, 34 (57%) had cytokeratin-positive, CD133-positive, and CD44-positive (triple-positive) CTCs, whereas 40 (67%) had cytokeratin-positive, CD133-positive, CD44-negative CTCs. The remaining 13 patients did not have CTCs, as defined by cytokeratin expression. ALDH-positive CTCs and triple-positive CTCs were significantly associated with worse survival by univariate analysis, even when accounting for other significant prognostic factors (all, P ≤ 0.01). ALDH-positive CTCs, triple-positive CTCs, and dual cytokeratin- and CD133-positive CTCs were independent predictors of tumor recurrence by logistic regression analysis and associated with decreased disease-free survival (all, P ≤ 0.03).Conclusions: CTCs labeling with one or more markers of TICs are found in a majority of patients with PDAC and are independently predictive of decreased disease-free and overall survival. Clin Cancer Res; 23(11); 2681-90. ©2016 AACR.

The association between circulating tumor cells and Epstein-Barr virus activation in patients with nasopharyngeal carcinoma

ABSTRACTBackground: Circulating tumor cells (CTCs) and microemboli (CTM) are attracting increasing attention in medical biology and clinical practice. However, the clinical relevance of CTCs in nasopharyngeal carcinoma (NPC) has not yet been ascertained, and no study has focused on the influence of Epstein-Barr virus (EBV) status on CTCs in NPC patients. These issues were therefore examined. Methods: Peripheral blood samples were prospectively obtained from 33 NPC patients before treatment. CTCs and CTM were captured using the Isolation by Size of Epithelial Tumor (ISET) method. Immunohistochemistry on CK5/6 (cytokeratin5/6) and P63, as well as in situ hybridization of EBERs (EBV-encoded RNAs) were used to validate the harvested tumor cells. Results: Out of 33 NPC patients, CTCs were detected in 22 cases (66.7%), and CTM were observed in 2 cases (6.1%). CTCs were presented in all stages of NPC patients but had no association with the TNM stages (all P > 0.05). The presence of CTCs appeared to correlate with EBV activation status. Among the total NPC patients, the EBV VCA-IgA levels in CTC-positive cases were higher than that in CTC-negative cases (negative vs. positive: 3.88 vs. 4.86, P = 0.016). A similar result was observed in the patients without distant metastasis (negative vs. positive: 3.76 vs. 4.95, P = 0.009). When the number of CTCs was considered, CTC counts were found to correlate with EBV VCA-IgA (R = 0.382, P = 0.041) and EBV DNA load (R = 0.396, P = 0.033) for all NPC patients as well as NPC patients without distant metastases. Conclusions: These findings strongly suggested detectable CTCs/CTM in all stages of NPC patients and support a positive correlation between CTCs and EBV activation in NPC patients.

Method for semi-automated microscopy of filtration-enriched circulating tumor cells.

BackgroundCirculating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients.MethodsSpiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner.ResultsTwo combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45− cells, cytomorphological staining, then scanning and analysis of CD45− cell phenotypical and cytomorphological characteristics. CD45− cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 μm2. The second assay sequentially combined fluorescent staining, automated selection of CD45− cells, FISH scanning on CD45− cells, then analysis of CD45− cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 μm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals.ConclusionsThe semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2461-4) contains supplementary material, which is available to authorized users.

Abstract 5188: Immunostaining of MRP-1 protein in circulating tumor cells predicts shorter progression free survival in metastatic colorectal cancer patients

Abstract Background: colorectal cancer (CRC) is the third leading cause of cancer death in both men and women in the United States (Siegel et al., 2014). So, it is important to look for novel biomarkers to identify and define the best treatment for patients with this disease. Circulating Tumor Cells (CTCs) have been appointed for many researchers as a manner to follow up patients at current status. MRP-1 protein acts as an ATP-dependent efflux pump, naturally protecting cells from both toxic and therapeutic compounds. It works as a drug resistant protein, generally with irinotecan-based chemotherapy (Cole, 2014; Gazzaniga et al., 2010). Objective: to study the presence of MRP-1 protein in CTCs and its predictable value in treatment resistance, evaluating the time of progression free survival (PFS). Methods: This present cohort (n = 18) belongs to a larger cohort of patients (n = 54) treated with many chemotherapeutic agents combined with 5-FU. The results of 5-FU resistance were presented previously in AACR annual meeting 2014. The blood was collected prospectively from patients with metastatic CRC. Blood was collected before the beginning of chemotherapy. The sample was filtered on ISET (Isolation by Size of Epithelial Tumor Cells, Rarecells, France) according to manufacture procedure. CTCs fixed on spots from ISET membranes were stained by immunocytochemistry with anti- MRP-1 antibody (1/100) and counterstained with hematoxylin-eosyn. Leucocytes were identified by anti-CD45 antibody. CTCs were analyzed by light microscope and quantified by 1 mL of blood. Results: here we analyzed blood samples of 18 mCRC patients who underwent irinotecan-based chemotherapy, with or without 5-FU. The median age was 54 years-old (32 - 80) and 77.7% were male. All the patients included were clinical stage IV with tumors classified as adenocarcinoma. The median CTC number detected was 1.5 CTC/ml (0 - 13.75) at baseline. Immunostaining of MRP-1 in CTCs was performed in all samples and 4 (21%) were found positive. By Kaplan-Meier test we observed that patients with CTC MRP-1 positive had shorter PFS than patients CTC MRP-1 negative (2.1 vs. 9.8 months respectively; P = 0.003). Interestingly, patients irinotecan resistant do not belong to the same group of patients 5-FU resistant (CTC TYMS positive, link: http://www.abstractsonline.com/Plan/ViewAbstract.aspx?mID = 3404&amp;sKey = f39b6e91-7d91-4e03-a37e-3bc3724bade3&amp;cKey = 1fa1e08e-af70-461a-9275-d8cdc2dd7b14&amp;mKey = 6ffe1446-a164-476a-92e7-c26446874d93). Conclusion: the necessity of novel tools to follow up and to predict treatment response has been increasing. Here, we proposed a potential protein found in CTCs, MRP-1, which might represent a powerful tool to manage and triage patients candidate to receive treatment with irinotecan. Furthermore, we are the first group to demonstrate the role of MRP-1 in CTCs as a predictive factor for patients with metastatic CRC. Citation Format: Emne A. Abdallah, Ludmilla T. D. Chinen, Virgílio S. Silva, Natalia B. Mingues, Marcelo C. Machado Netto, José Luiz Gasparini Júnior, Bruna M. M. Rocha, Juliana V. Romero, Marcilei E. C. Buim, Mitzi Brentani, Marcello F. Fanelli. Immunostaining of MRP-1 protein in circulating tumor cells predicts shorter progression free survival in metastatic colorectal cancer patients. [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 5188. doi:10.1158/1538-7445.AM2015-5188

Abstract LB-194: The presence of TYMS immunostaining in circulating tumor cells is a predictive biomarker in colorectal cancer

Abstract Background: colorectal cancer (CRC) was the third most commonly diagnosed cancer in both men and women in the last three years in the United States (Siegel et al., 2011, 2012, 2013). The first strategy for treatment is complete resection of the lesion. However, some patients experience recurrence, believed to be due to residual resistant micrometastases. Circulating tumor cells (CTCs) have been considered an important prognostic biomarker and crucial for tumor dissemination. Traditional diagnostic methods are unable to detect CTCs present in these sites and released into the circulation. Objective: to count CTCs levels and search for Thymidylate synthase (TYMS), a drug resistance gene, in these cells from patients with metastatic or advanced colorectal cancer. Methods: prospective study made by blood collection of patients with metastatic or advanced CRC. Blood was collected before the beginning of chemotherapy and after 60 days, in accordance with image exams. The sample was filtered on ISET (Isolation by Size of Epithelial Tumor Cells) according to manufacture procedure. CTCs fixed on spots from ISET membranes were stained by immunocytochemistry with anti-TYMS antibody and counterstained with hematoxylin-eosyn. Leucocytes were identified by anti-CD45 antibody. CTCs were analised by light microscope and quantified by 1 mL of blood. Results: there were included 42 patients. 78.5% were treated with FOLFOX or FOLFORI. The median age was 58 years-old (30-81). The majority of patients was men (59.5%) and included at stage IV of disease (88%). The median CTCs numbers detected in all patients were 2.18 CTCs/ml (0- 31.25) at baseline. Immunostaining in CTCs with TYMS was performed in 41 samples. Nine (21.9%) were found positive. Six of these patients had tumor progression in a follow-up of 12 months after treatment with 5-FU. Among patients with CTC TYMS positive and disease progression, 4 had also tumor tissue positive. Conclusion: It is been related in literature that high tissue expression of TYMS shows worse response to 5-FU-based chemotherapy (Lurje et al., 2009). Our results with CTCs expressing TYMS strongly contribute with these findings and suggest that maybe these cells can be used as a predictor biomarker of chemotherapy response. Furthermore, CTCs levels may offer a novel approach to follow up patients in an individualized manner. Citation Format: Ludmilla T. Chinen, Emne Ali Abdallah, Virgilio S. Silva, Marcilei E. Buim, Marcelo Calil Neto, Jose Luiz Gasparini Junior, Bruna Maria Malagoli Rocha, Juliana R. Romero, Natalia Breve Mingues, Fernando Augusto Soares, Mitzi Brentani, Marcello Ferretti Fanelli. The presence of TYMS immunostaining in circulating tumor cells is a predictive biomarker in colorectal cancer. [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 LB-194. doi:10.1158/1538-7445.AM2014-LB-194

Abstract 1471: Characterization of the molecular heterogeneity of circulating tumour cells in metastatic prostate cancer.

Abstract Purpose: CTCs represent an alternative source of tumor material potentially usable as a surrogate tissue marker in the context of targeted treatments. The objective of the present study was to characterize the status of key molecular markers such as ERG gene rearrangement, and AR and c-myc amplifications, according to epithelial and/or mesenchymal marker expression in CTCs of patients with metastatic prostate cancer (PCa). Patients and Methods: CTCs were isolated from the blood of 40 patients at day 0 and day 30 of abiraterone treatment. CTCs were detected using two methods, CellSearch® (Veridex LLC, Raritan, NJ, USA) and ISET (Isolation by Size of Epithelial Tumor cells). Molecular characterization of CTCs was performed on ISET filters by combining four color immunofluorescent staining and Filter Adapted-Fluorescent In situ Hybridization (FA-FISH®). To optimize pan-keratin detection, four pancytokeratin antibodies, including AE1/AE3, KL1, C11, and A45-B/B3, were tested on the LnCAP prostate cell line spiked in normal blood cells and on PCa CTCs enriched by ISET. Epithelial (cytokeratins, E-cadherin, EpCAM (Epithelial Cell Adhesion Molecule)), mesenchymal (vimentin, N-cadherin), PTEN, androgen receptor (AR) and Ki67 marker expression was detected by immunofluorescent staining on ISET filters according to a method we previously established. FA-FISH® was optimized for the detection of ERG gene rearrangement and AR and c-myc amplification or gain of copy number. A two step method combining immunofluorescent staining and FA-FISH® was established on an ARIOL automated scanner (LEICA) to precisely relocate FISH signals in phenotypically different CTC subsets. Results: Higher CTC levels were detected by ISET compared to CellSearch®. Quantitative analysis of the intensity values for each tested pan-keratins antibodies showed that KL1 and A45-B/B3 clones gave the strongest immunofluorescent signals both on the LnCAP cell line and PCa CTCs. CTCs expressing epithelial, mesenchymal and hybrid (both epithelial and mesenchymal) markers were detected in most patients suggesting the existence of CTCs at different stages of the epithelial to mesenchymal transition (EMT) process. The loss of PTEN expression was also observed using hematopoietic cells present on ISET filters as a positive control of PTEN expression. ERG gene rearrangement, AR and c-myc amplifications were detected. The presence of these markers within CTC subsets expressing epithelial and/or mesenchymal markers is currently studied. Conclusion: Several molecular markers such as ERG gene rearrangement, AR and c-myc amplifications were detected in PCa CTCs. The presence of these markers in CTCs according to their EMT status will be presented. Citation Format: Marianne Oulhen, Christophe Massard, Alexander Valent, Rachel Young, Sylvestre Le Moulec, Karim Fizazi, Philippe Vielh, Françoise Farace. Characterization of the molecular heterogeneity of circulating tumour cells in metastatic prostate cancer. [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 1471. doi:10.1158/1538-7445.AM2013-1471

Detection of circulating tumor cells as a prognostic factor in patients undergoing radical surgery for non‐small‐cell lung carcinoma: comparison of the efficacy of the CellSearch Assay™ and the isolation by size of epithelial tumor cell method

Comparison of the efficacy of different enrichment methods for detection of circulating tumor cells (CTCs) before radical surgery is lacking in non-small-cell lung carcinoma (NSCLC) patients. Detection and enumeration of CTCs in 210 consecutive patients undergoing radical surgery for NSCLC were evaluated with the CellSearch Assay™ (CS), using the CellSearch Epithelial Cell Kit, and by the isolation by size of epithelial tumor (ISET) method, using double immunolabeling with anti-cytokeratin and anti-vimentin antibodies. CTCs were detected in 144 of 210 (69%) patients using CS and/or ISET and in 104 of 210 (50%) and 82 of 210 (39%) patients using ISET and CS, respectively. Using ISET, 23 of 210 (11%) patients had vimentin-positive cells with cytological criteria of malignancy. Disease-free survival (DFS) was worse for patients with CTCs compared to patients without CTCs detected by CS alone (p < 0.0001; log rank = 30.59) or by ISET alone (p < 0.0001; log rank = 33.07). The presence of CTCs detected by both CS and ISET correlated even better with shorter DFS at a univariate (p < 0.0001; log rank = 42.15) and multivariate level (HR, 1.235; 95% CI, 1.056-1.482; p < 0.001). CS and ISET are complementary methods for detection of CTCs in preoperative radical surgery for NSCLC. CTC detection in resectable NSCLC patients using CS and/or ISET could be a prognostic biomarker of great interest and may open up new avenues into improved therapeutic strategies for lung carcinoma patients.

Effect of laparoscopy versus laparotomy on circulating tumor cells using isolation by size of epithelial tumor cells.

To assess the effect of laparoscopy on circulating tumor cell (CTC) detection in case of carcinosis. We compared laparoscopy versus laparotomy on tumor cell blood release in an animal model of ovarian carcinosis obtained by intraperitoneal inoculation of IGR-OV1 cells in nude rats. Animals were randomly assigned to one of the following groups: CO(2) laparoscopy (L), gasless laparoscopy (GL), midline laparotomy (ML), or general anesthesia as control (C). A 0.5 ml blood sample was taken in each case before and after experiment and tested with a novel assay, ISET (isolation by size of epithelial tumor cells), which isolates CTC by filtration on account of their size. Statistics were performed with the Fisher's and the Chi-square tests. Ten rats were included in each group. We did not find any significant difference in CTC prevalence before and after surgery (2/14 versus 3/19, respectively, P = 1). Similarly, the three surgical accesses were equivalent with one post-experiment detection per group: 1/5 for L, 1/7 for ML, 1/7 for GL, and 1/6 for C (P = 0.9). This trial did not show any deleterious effect of laparoscopy on CTC when compared to laparotomy.

Effect of laparoscopy versus laparotomy on circulating tumor cells using isolation by size of epithelial tumor cells

AimTo assess the effect of laparoscopy on circulating tumor cell (CTC) detection in case of carcinosis.Material and methodsWe compared laparoscopy versus laparotomy on tumor cell blood release in an animal model of ovarian carcinosis obtained by intraperitoneal inoculation of IGR-OV1 cells in nude rats. Animals were randomly assigned to one of the following groups: CO2laparoscopy (L), gasless laparoscopy (GL), midline laparotomy (ML), or general anesthesia as control (C). A 0.5 ml blood sample was taken in each case before and after experiment and tested with a novel assay, ISET (isolation by size of epithelial tumor cells), which isolates CTC by filtration on account of their size. Statistics were performed with the Fisher's and the Chi-square tests.ResultsTen rats were included in each group. We did not find any significant difference in CTC prevalence before and after surgery (2/14 versus 3/19, respectively,P= 1). Similarly, the three surgical accesses were equivalent with one postexperiment detection per group: 1/5 for L, 1/7 for ML, 1/7 for GL, and 1/6 for C (P= 0.9).ConclusionThis trial did not show any deleterious effect of laparoscopy on CTC when compared to laparotomy.

Isolation by Size of Epithelial Tumor Cells: A New Method for the Immunomorphological and Molecular Characterization of Circulating Tumor Cells

We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.