Size Of Epithelial Tumor Cells Research Articles

Pilot study to evaluate isolation by size of circulating tumour cells in canine oral melanoma.

Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size-based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan-A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan-A-positive, 3/9 PNL2-positive, and 8/9 S100-positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative-stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p < .05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours.

Use of circulating tumor cells and microemboli to predict diagnosis and prognosis in diffuse glioma.

Circulating tumor cell (CTC) detection is a promising noninvasive technique that can be used to diagnose cancer, monitor progression, and predict prognosis. In this study, the authors aimed to investigate the clinical utility of CTCs in the management of diffuse glioma. Sixty-three patients with newly diagnosed diffuse glioma were included in this multicenter clinical cohort. The authors used a platform based on isolation by size of epithelial tumor cells (ISET) to detect and analyze CTCs and circulating tumor microemboli (CTMs) in the peripheral blood of patients both before and after surgery. Least absolute shrinkage and selector operation (LASSO) and Cox regression analyses were used to verify whether CTCs and CTMs are independent prognostic factors for diffuse glioma. CTC levels were closely related to the degree of malignancy, WHO grade, and pathological subtypes. Receiver operating characteristic curve analysis revealed that a high CTC level was a predictor for glioblastoma. The results also showed that CTMs originate from the parental tumor rather than from the circulation and are an independent prognostic factor for diffuse glioma. The postoperative CTC level is related to the peripheral immune system and patient survival. Cox regression analysis showed that postoperative CTC levels and CTM status are independent prognostic factors for diffuse glioma, and CTC- and CTM-based survival models had high accuracy in internal validation. The authors revealed a correlation between CTCs and clinical characteristics and demonstrated that CTCs and CTMs are independent predictors for the diagnosis and prognosis of diffuse glioma. Their CTC- and CTM-based survival models can enable clinicians to evaluate patients' response to surgery as well as their outcomes.

Peripheral arterial rather than venous blood is a better source of circulating tumor cells in early lung cancer.

Circulating tumor cells (CTCs) play a crucial role in the early diagnosis and prognosis of lung cancer. Identification of a more suitable sample source could be a breakthrough towards enhancing CTC detectability in early-stage lung cancer. We investigated the differences in detectable CTCs between peripheral arterial and venous blood in early- and mid-stage lung cancer patients undergoing surgery and analyzed the association between clinicopathological factors and detectable CTCs in peripheral arterial and venous blood. Peripheral arterial and venous blood was collected in 5-mL samples from 56 patients with surgically resected and pathologically clear at early- or mid-stage lung cancer. Blood specimens were enriched for CTCs based on isolation by size of epithelial tumor cells. The CTCs were identified using Swiss Giemsa staining and immunohistochemistry for CD45/CD31. In stage I lung cancer, CTC-positive rate was significantly higher in peripheral arterial than in venous blood (45.45% vs. 17.39%). There was no significant difference in the number of detectable CTCs between peripheral arterial and venous blood. A low degree of differentiation was associated with a high positive rate of CTCs in peripheral venous blood. The number of circulating tumor microemboli was significantly higher in patients with tumor size >3 cm compared with ≤3 cm. CTC levels in peripheral arterial and venous blood differed little in lung cancer patients.Compared to peripheral venous blood, peripheral arterial blood had a higher CTC positivity rate in early-stage lung cancer.This study was favorable for early detection and monitoring of lung cancer.

Significance of circulating tumor cells in the portal vein regarding metastases and vascular invasion in hepatocellular carcinoma patients.

Vascular invasion is an important risk factor of poor prognosis in hepatocellular carcinoma (HCC) patients. The detection of circulating tumor cells (CTCs) in the blood is direct evidence of tumor presence. There are few reports on CTCs and metastasis and vascular invasion of HCC. The purpose of this study was to analyze the significance of CTCs in the portal vein regarding metastases and vascular invasion in HCC patients. A total of 104 HCC patients diagnosed and treated in Zhengzhou University People's Hospital were enrolled. Surgery was performed in 60 individuals. Portal vein blood samples were collected before treatment for CTCs detection. We used the isolation by size of epithelial tumor cells (ISET) and fluorescence in situ hybridization (FISH) to enrich and classify CTCs from blood samples. The patients were divided into metastasis and nonmetastasis groups according to the metastasis status before treatment. Differences in clinical indicators such as alpha-fetoprotein (AFP) levels, tumor size, CTCs count, and macrovascular tumor thrombus between the two groups were analyzed as well as the associations of CTCs count with the above indicators. For individuals with postoperative pathology, the relationship between CTCs counts and microvascular invasion (MVI) was analyzed. The amounts of portal vein CTCs were higher in patients with metastases compared with the nonmetastases group (20 vs. 7; z=3.795; P<0.001). Multivariate logistic regression analysis showed that the CTC count was a risk factor for HCC metastasis [odds ratio (OR) =1.044; 95% CI: 1.011-1.079]. The sensitivity and specificity of CTC count in predicting HCC metastasis were 82.93% and 52.38%, respectively. CTC count was significantly correlated with tumor size (rs=0.308; P=0.001), vascular invasion (z=4.211; P<0.001), and MVI (z=12.763; P=0.002). A threshold CTC count of seven showed the most significant power for predicting metastasis. Vascular invasion positivity was closely related to HCC metastasis. Portal vein CTC count before treatment was correlated with vascular invasion and could be considered one of the factors affecting HCC metastasis. However, the ability of CTC count was limited in predicting HCC metastasis due to insufficient specificity.

Prognostic Significance of Circulating Tumor Cells for Locally Advanced Pancreatic Cancer with High-dose Hypofractionated Radiotherapy

The aims of the present study were to investigate the circulating tumor cells (CTCs) in peripheral blood in order to assess their prognostic value in locally advanced pancreatic cancer treated with high-dose hypofractionated radiotherapy (HDHR). Patients with newly diagnostic locally advanced pancreatic cancer were enrolled in our study. Peripheral blood samples were obtained from these patients before HDHR and concurrent chemotherapy, and at three months after radiotherapy. CTCs were identified and enumerated from 5 mL of venous blood by isolation by size of epithelial tumor cells (ISET). The dose delivered to PTV, CTV and GTV of the pancreatic lesions was 50Gy, 60Gy, and 70-75Gy in 15 or 20 fractions, respectively. Associations between CTCs enumeration and clinical outcomes were prospectively analyzed. 54 patients with pancreatic cancer were enrolled. CTCs were identified in 23 (42.6%) patients before treatment. CTCs were detected in 14 out of 49 patients (28.6%) at three months after HDHR. Incidences of CTCs positivity after HDHR in patients with disease progression and disease control were 62.5% and 22.0%, respectively. Change of CTCs before and after HDHR was a univariate predictor of overall survival (P = 0.022), as well as a strong independent predictor of overall survival on multivariate analysis (P = 0.036). Our work suggested that CTCs decline status is a prognostic biomarker for locally advanced pancreatic cancer with HDHR.

Clinical value and feasibility of ISET in detecting circulating tumor cells in early breast cancer.

BackgroundPatients with operable breast cancer have a better prognosis for recovery. However, once distant organ metastasis occurs, the chance of a long-term survival or a cure is limited. The collection and counting of circulating tumor cells (CTCs) by reliable detection techniques are of increasing importance in the diagnosis of early metastasis and prognosis of disease progression. Isolation by size of epithelial tumor cells (ISET) has the advantage of simplicity of operation and high homogeneity. It is practical for large-scale clinical detection showing cell abundance. The value of ISET in the detection of circulating breast cancers in the blood has not been determined. The purpose of this study is to explore the feasibility of applying ISET to detect CTCs by determining the detection rate of ISET in operable breast cancer and by evaluating the correlation between detection rate, cell count and clinical factors such as molecular typing and pathological staging.MethodsThe experiment included 193 breast cancer patients who were diagnosed by core needle biopsy before the operation. 10 mL of venous blood was collected from the patients preoperatively, and CTCs in their blood samples were counted and analyzed by ISET.ResultsPatients were divided into groups according to pathology and immunohistochemistry. The overall detection rate of CTCs by ISET was 41.24%. The detection rate, the number of overall CTCs and the average number of CTCs in each group were analyzed individually. No significant differences were observed between the different groups.ConclusionsAlthough ISET has a relatively good detection rate for circulating breast cancer cells, it fails to provide more information on pathological staging, molecular classification and so forth.

Prognostic impact of baseline circulating tumor cells (CTCs) detected by the isolation by size of epithelial tumor cells (ISET) in locally advanced head and neck squamous cell carcinoma (LAHNSCC): Results of a prospective study.

6061 Background: The prognostic impact of CTCs in LAHNSCC is yet to be determined, with conflicting results in previous trials, the majority utilizing cytokeratin dependent techniques for identification and counting of CTCs. The primary objective of this study is to determine the detection rates using the ISET method, and the prognostic role of CTCs in LAHNSCC patients (pts) treated with a curative intent. Methods: In this prospective study, peripheral blood samples of pts with non-metastatic LAHNSCC, stages III/IV, were analyzed for CTCs using the ISET method, in two scenarios: curative surgical resection and adjuvant radiotherapy (CTCs before RT) and candidates for a non-surgical strategy (unresectable or organ preservation) either with upfront RT concurrent with chemotherapy (CT) or cetuximab (CTCs before RT), or preceded by induction CT (CTCs before ICT). Results: Eighty-three pts were included, the majority males (83%), with oropharynx primary (50%) and submitted to ICT (40%). The detection rate of baseline CTCs was 94% (78/83), and CTCs counts were significantly correlated with survival. For each increase of 1 CTC at baseline there was a relative increase of 18% in the risk of death (HR = 1.18; 95%CI: 1.06-1.31; p &lt; 0.001), 16% in the risk of progression (HR = 1.16; 95%CI: 1.04-1.28; p = 0.004) and a reduction of 26% in the odds of complete response to treatment (non-surgical group only - OR = 0.74; 95%CI: 0.58-0.95; p = 0.022). Using the maximum of the standardized log-rank statistic proposed by Lausen and Schumacher we establish cut off points for overall survival (OS) and progression-free survival (PFS). Pts with CTCs &lt; 6.5/ml had an estimated 2y OS of 85.6% versus 22.9% for CTCs ≥ 6.5/ml (HR = 0.18; 95%CI: 0.06-0.49; P &lt; 0.0001) and pts with CTCs ≤ 3.8/ml had an estimated 2y PFS of 71.8% versus 37% for CTCs &gt; 3.8/ml (HR = 0.32; 95%CI:0.15-0.67; p = 0.001). Conclusions: The detection rate of baseline CTCs using the ISET method was very high in LAHNSCC and the counts of CTCs were strongly correlated with survival and response to treatment.

The Potential Clinical Implications of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer.

Gastric adenocarcinoma (GAC) is the third deadliest malignant neoplasm worldwide, mostly because of late disease diagnosis, low chemotherapy response rates, and an overall lack of tumor biology understanding. Therefore, tools for prognosis and prediction of treatment response are needed. Quantification of circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) and their expression of biomarkers has potential clinical relevance. Our aim was to evaluate CTCs and CTM and their expression of HER2 and plakoglobin in patients with nonmetastatic GAC, correlating the findings to clinicopathological data. CTC enrichment was performed with isolation by size of epithelial tumor cells, and the analysis was performed with immunocytochemistry and microscopy. Two collections were made: one at diagnosis (55 samples before neoadjuvant treatment) and one after surgery and before adjuvant therapy (33 samples). A high detection rate of CTCs (90%) was observed at baseline. We evaluated HER2 expression in 45/55 biopsy samples and in 42/55 CTC samples, with an overlap of 36 subjects. Besides the good agreement observed for HER2 expression in primary tumors and paired CTCs for 36 cases (69.4%; κ = 0.272), the analysis of HER2 in CTCs showed higher positivity (43%) compared with primary tumors (11%); 3/5 patients with disease progression had HER2-negative primary tumors but HER2-positive CTCs. A significant CTC count drop in follow-up was seen for CTC-HER2-positive cases (4.45 to 1.0 CTCs per mL) compared with CTC-HER2-negative cases (2.6 to 1.0 CTCs per mL). The same was observed for CTC-plakoglobin-positive cases (2.9 to 1.25 CTCs per mL). CTC analysis, including their levels, plakoglobin, and HER2 expression, appears to be a promising tool in the understanding the biology and prognosis of GAC. The analysis of circulating tumor cell levels from the blood of patients with gastric adenocarcinoma, before and after neoadjuvant treatment, is useful to better understand the behavior of the disease as well as the patients more likely to respond to treatment.

Isolation of circulating tumor cells in patients undergoing surgery for esophageal cancer and a specific confirmation method.

The clinical significance of circulating tumor cells (CTCs) in patients with esophageal squamous cell carcinoma (ESCC) who have undergone radical surgery was investigated. A novel confirmation method for identifying CTCs or circulating tumor microemboli (CTM) in ESCC was also investigated. Blood samples from 55 patients with ESCC were collected 1–3 days prior to surgery and 7 days post-surgery. All patients underwent curative thoracic esophagectomy and lymphadenectomy. Blood samples from 20 healthy volunteers were obtained as controls. Isolation by size of epithelial tumor cells (ISET) was performed also. The overall CTC detection rate was 52.7% preoperatively and 49.1% postoperatively. The presence of CTCs correlated with the Tumor-Node-Metastasis stage and the Log odds of positive lymph nodes. No significant difference in perioperative CTC transformation was discovered between the thoracoscopic and laparoscopic approach, and the open approach. The P40+/cluster of differentiation (CD)45− phenotype was confirmed in the CTCs and CTM. ISET appeared to have high sensitivity for detecting CTCs within ESCC patients. Immunofluorescence staining for CD45 and P40 was a specific, accurate and convenient method for confirming the presence of CTCs or CTM in patients with ESCC, and is strongly recommended as a supplement to morphological analysis.

Clinical significance of tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma.

Circulating tumor cells (CTCs) are suspected of predicting the prognosis of malignant tumor, but there are few relevant reports specific to esophageal squamous cell carcinoma (ESCC). This study investigated the clinical significance of CTCs in patients with ESCC.Sixty patients with ESCC were enrolled, from whom CTCs had been tested by our team previously. Peripheral blood samples were obtained from these patients before treatment; and CTCs were assayed by isolation by size of epithelial tumor cells (ISET). Associations between the presence of CTCs and patients’ clinicopathological parameters and clinical outcomes were analyzed.CTCs were detected in 20 patients (33.3%), who experienced significantly shorter progression-free survival (PFS) than did the CTC-negative patients. Overall, PFS was negatively associated with the number of CTCs. Multivariate analyses showed that a CTC count >2 was a strong independent prognostic indicator of tumor recurrence (hazard ratio [HR] 5.63; 95% confidence interval [CI] 1.77–17.89; P = .003). In the subgroup of 50 patients who underwent R0 resection and postoperative adjuvant radiotherapy or chemotherapy, CTC was a strong, independent, and prognostic indicator of tumor recurrence (HR 10.70; 95% CI, 1.40–81.91; P = .022). The number of CTCs correlated with the T stage (r = 0.26, P = .043) but not with the N or M stage. For subgroups in stages II or I-IIIB or T3 or T3 + T4, the PFS of patients with CTCs > 1 or > 2 was significantly shorter than that of the patients with CTCs ≤ 1 or CTCs ≤ 2. In the stage III or T3 + T4 groups, the PFS of patients with CTCs > 0 was significantly shorter than that of patients with CTC = 0.This is the first study to report that the CTC detected by ISET is an independent and prognostic indicator of patients’ outcome in ESCC. Consideration of CTCs may improve the accuracy of preoperative staging in ESCC.

Circulating tumor cells as marker of poor prognosis in metastatic lung cancer: a pilot study

BackgroundCirculating tumor cells (CTCs) play an important role in progression and metastasis, particularly in form of cluster, which is called circulating tumor microemboli (CTM), and can be found in the peripheral blood of cancer patients. The aim of this study was to evaluate the presence of CTCs and CTM, and its influence on tumor progression in lung cancer patients.MethodsCTCs were isolated by Isolation by Size of Epithelial Tumor cells (ISET®) Technology. The samples of metastatic lung cancer patients were collected before the beginning of systemic chemotherapy. CTCs and CTM were identified according to their morphological features.ResultsFifteen patients were analyzed. Patients who had CTM in blood previously the beginning of systemic therapy had poor progression-free survival (PFS) compared to those with absence of CTM, although without statistical significance (median PFS 3.1 months × 6.7 months, p = 0.29). Moreover, patients without any prior treatment had less than 3 CTCs/mL compared to patients previously exposed to chemotherapy, who had 3 CTCs/mL or more (p = 0.31). Additionally, these patients, with prior treatment, showed poor PFS, compared to chemo-naive patients, although without statistical significance (mean PFS: 4.6 months × 7.3 months, p = 0.47).ConclusionsWe identified, even in a limited number of samples, that an elevated baseline levels of CTCs and the presence of CTM were associated with poor prognosis in patients with metastatic lung cancer. In addition, we showed that an increase of CTCs counts could indicate a pre-existing resistance. However, further studies with a large cohort are needed to support this information.

Abstract B06: Circulating tumor cell analysis in locally and advanced colon cancer: A pilot study

Abstract Colorectal cancer presents high incidences (9.7%) and mortality rates (8.5%) worldwide in men and women. Although the early detection of colon cancer promotes high cure rates (30-40%), there are still patients who experience local recurrence as well as distant metastases. The main treatment is surgery-based and then, after risk evaluation, patients can undergo or not undergo adjuvant chemotherapy. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) seem to play an important role in these processes, being detached from tumor and circulating in blood vessels before surgery intervention. Our objective, therefore, was to evaluate the role of CTCs and CTM in stages I-III colon cancer patients. Here, we collected 10 mL of blood from presurgery colon cancer and filtrated the sample in ISET® (Isolation by Size of Epithelial Tumor cells) device. This study started on July 2016. CTCs counts were assessed and correlated with clinical and pathologic data. We included 15 patients. However, one patient was identified as metastatic after surgery and therefore excluded from these analyses. From 14 patients analyzed, 8 (57.1%) were male, with a median age of 65 years (53-87). The most prevalent cancer sites were right colon (n= 6;42.9%) and left colon (n=4;28.6%). CTCs detection rate was 85.7%, and the mean and median of CTCs were 5.1/mL and 2.6/mL, respectively (0-30.3/mL). We found CTM in 4/14 patients (28.5%). Histopathologic analysis showed that no patient had blood vessel invasion, 5 (35.7%) had lymphatic invasion, and 4 (28.6%) had perineural invasion. Four patients (28.4%) were classified as stage I and 10 (71.4%) were classified as stage II and III. From patients with RAS mutational analysis available (12), 9 (75%) presented tumor mutation. Although we present a limited sample, exact Fisher's test showed an association between lymphatic invasion and CTC number above the median (P= 0.03), and the presence of CTM was most frequent in this group of patients, although without statistical significance (P= 0.09). CTM absence was associated with pathologic T= 2 and 3 (P= 0.06), when compared to pathologic T= 4. Our next steps are to evaluate some proteins in CTCs that could confer an invasive phenotype, resistance to adjuvant treatment as well as verify the pattern of mutations in CTCs and CTMs. Moreover, this is the first study to show the efficacy of ISET method in detecting high rates of CTCs from stage I-III colon cancer patients. Citation Format: Emne Abdallah, Virgílio Silva, Bianca Flores, Alexcia Braun, Ana Urvanegia, Vanessa Alves, Marcello Fanelli, Samuel Aguiar, Ludmilla Chinen. Circulating tumor cell analysis in locally and advanced colon cancer: A pilot study [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 B06.

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.

High-purity capture of CTCs based on micro-beads enhanced isolation by size of epithelial tumor cells (ISET) method

In this paper, we develop a low-cost size-based microfluidic chip using conventional polycarbonate membrane to isolate CTCs from blood, and propose a strategy to increase the capture efficiency before cell filtration by a size enlargement method utilizing modified microbeads specifically binding to CTCs. Up to 91% of target cells were isolated from whole blood samples using our microfluidic capture system at a flow rate of 1mL/min. Moreover, a WBC depletion process is introduced which greatly decreases the WBC retaining on the filter membrane. The tests of immunofluorescence analysis of cells captured on the membrane were performed, which demonstrates that the device could provide a dependable CTC identification and CTC count in whole blood samples. Finally, the device was further validated in the detection of CTCs from blood samples of cancer patients, and it indicates a promising capability to detect CTC response to treatment.

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.

Detection of KRAS Exon 2 Mutations in Circulating Tumor Cells Isolated by the ISET System from Patients with RAS Wild Type Metastatic Colorectal Cancer

INTRODUCTION: The presence of KRAS mutations in patients with metastatic colorectal cancer (mCRC) predicts poor response to agents targeting the EGFR. Even in patients with RAS wild type (WT) tumors, resistance eventually develops due to multiple mechanisms, including the expansion of previously undetected KRAS mutated clones. In this feasibility study, we aimed to detect KRAS exon 2 mutations in serial samples of circulating tumor cells (CTCs) of RAS WT patients with mCRC captured by the Isolation by Size of Epithelial Tumor cells (ISET) system. METHODS: CTC isolation using the ISET system was performed from prospectively collected blood samples obtained from patients with RAS and BRAF WT mCRC prior to first-line therapy initiation, at first imaging assessment and on disease progression. CTCs were enumerated using hematoxylin & eosin and CD45 double stain on a single membrane spot. DNA was extracted from 5 spots and KRAS exon 2 mutations were detected using a custom quantitative Polymerase Chain Reaction (qPCR) assay. RESULTS: Fifteen patients were enrolled and 28 blood samples were analyzed. In 9 (60%) patients, at least one sample was positive for the presence of a KRAS exon 2 mutation. In 11 out of 28 samples (39.2%) with detectable CTCs a KRAS mutation was detected; the corresponding percentages for baseline and on progression samples were 27% and 37.5%, respectively. The most commonly detected mutations were G13D and G12C (n=3). The presence of KRAS mutated CTCs at baseline was not prognostic for either PFS (P=.950) or OS (P=.383). CTC kinetics did not follow tumor response patterns. CONCLUSION: The results demonstrate that using a qPCR-based assay, KRAS exon 2 mutations could be detected in CTCs captured by the ISET system from patients with RAS WT primary tumors. However, the clinical relevance of these CTCs remains to be determined in future studies.

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

Detection of FAM172A expressed in circulating tumor cells is a feasible method to predict high-risk subgroups of colorectal cancer.

Previous studies used to enumerate circulating tumor cells to predict prognosis and therapeutic effect of colorectal cancer. However, increasing studies have shown that only circulating tumor cells enumeration was not enough to reflect the heterogeneous condition of tumor. In this study, we classified different metastatic-potential circulating tumor cells from colorectal cancer patients and measured FAM172A expression in circulating tumor cells to improve accuracy of clinical diagnosis and treatment of colorectal cancer. Blood samples were collected from 45 primary colorectal cancer patients. Circulating tumor cells were enriched by blood filtration using isolation by size of epithelial tumor cells, and in situ hybridization with RNA method was used to identify and discriminate subgroups of circulating tumor cells. Afterwards, FAM172A expression in individual circulating tumor cells was measured. Three circulating tumor cell subgroups (epithelial/biophenotypic/mesenchymal circulating tumor cells) were identified using epithelial-mesenchymal transition markers. In our research, mesenchymal circulating tumor cells significantly increased along with tumor progression, development of distant metastasis, and vascular invasion. Furthermore, FAM172A expression rate in mesenchymal circulating tumor cells was significantly higher than that in epithelial circulating tumor cells, which suggested that FAM172A may correlate with malignant degree of tumor. This hypothesis was further verified by FAM172A expression in mesenchymal circulating tumor cells, which was strictly related to tumor aggressiveness factors. Mesenchymal circulating tumor cells and FAM172A detection may predict highrisk stage II colorectal cancer. Our research proved that circulating tumor cells were feasible surrogate samples to detect gene expression and could serve as a predictive biomarker for tumor evaluation.

Detection of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer

PURPOSE: Gastric cancer studies indicated a potential correlation between circulating tumor cells (CTCs) in peripheral blood and tumor relapse/metastasis. The prevalence and significance of circulating tumor microemboli (CTM) in gastric cancer remain unknown. We investigated the prevalence and prognostic value of CTCs and CTM for progression-free survival (PFS) and overall survival (OS) in gastric cancer patients. METHODS:Eighty-one gastric cancer patients consented to provide 5ml of peripheral blood before systematic therapy. CTCs and CTM were isolated using isolation by size of epithelial tumor cells and characterized by cytopathologists. For 41 stage IV gastric cancer patients, CTM was investigated as a potential biomarker to predict prognosis. RESULTS:CTCs were detected in 51 patients; the average count was 1.81. In clinical stage I, II, III, and IV patients, the average CTC counts were 1.40, 0.67, 1.24, and 2.71, respectively. CTM were detected in 3 of 33 clinical stage I to IIIb patients, at an average of 0.12 (0-2). CTM were detected in 13 of 53 clinical stage IIIc to IV patients, at an average of 1.26 (0-22). In stage IV patients, CTM positivity correlated with the CA125 level. PFS and OS in CTM-positive patients were significantly lower than in CTM-negative patients (P<.001). CTM positivity was an independent factor for determining the PFS (P=.016) and OS (P=.003) of stage IV patients in multivariate analysis. Using markers of the epithelial-mesenchymal transition, single CTCs were divided into three phenotypes including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. For CTM, CK−/Vimentin+/CD45− and CK+/Vimentin+/CD45− phenotypes were observed, but the CK+/Vimentin−/CD45− CTM phenotype was not. CA125 was detected in gastric cancer cell lines BGC823 and MGC803. CONCLUSIONS: In stage IV patients, CTM positivity was correlated with serum CA125 level. CTM were an independent predictor of shorter PFS and OS in stage IV patients. Thus, CTM detection may be a useful tool to predict prognosis in stage IV patients.

Inflammation-based indexes and clinicopathologic features are strong predictive values of preoperative circulating tumor cell detection in gastric cancer patients.

Circulating tumor cell (CTC) count and the host inflammatory response are two independent predictors for patients with various malignant disease. Several inflammation-based indicators have been demonstrated to have prognostic value in many malignant solid tumors, including systemic immune-inflammation index (SII), neutrophil lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), and prognostic nutrition index (PNI). The aim of this study was to evaluate the predictive value of the inflammation-based indexes including SII, NLR PLR, and PNI for CTC detection of gastric cancer patients before surgery. CTCs were measured using the isolation method by size of epithelial tumor cells and Wright staining for 60 patients with gastric cancer who underwent surgery. The indicators of SII, NLR, PLR, and PNI were calculated based on clinical laboratory testing. The detected CTC number was correlated with extension of tumor invasion (p=0.037), lymph node metastasis (p<0.001), and TNM stage (p<0.001). The CTC detection ratio was significantly correlated with T stage (p=0.041), lymph node metastasis (p=0.001), nerve fiber invasion of tumor outside the lymph nodes (p=0.017), and TNM stage (p<0.001). Statistical analysis showed that SII (p<0.001), NLR (p<0.001), PLR (p<0.001), and PNI (p<0.001) were significantly associated with positive CTC count and CTC detection rate. This study provides evidence that preoperative indicators consisting of SII, NLR, PLR, and PNI are robust predictors for CTC detection in gastric cancer patients undergoing tumor resection.