Measurement Of Circulating Tumor Cells Research Articles

Measurable morphological features of single circulating tumor cells in selected solid tumors-A pilot study.

Liquid biopsies developed into a range of sensitive technologies aiming to analyze for example, circulating tumor cells (CTCs) in peripheral blood, which significantly deepens understanding of the metastatic process. Nevertheless, examination of CTCs is mostly limited to their enumeration and usually only 2-3 markers-based phenotyping, not offering yet sufficient insight into their biology. In contrast, quantitative analysis of their morphological details might extend our knowledge about dissemination and even improve CTC isolation or label-free identification methods dependent on their physical features such as size, and deformability. Current study was conducted to describe CTCs' and their size, shape, presence of protrusions, and micronuclei across various types of cancers (lung, n = 29; ovarian, n = 24, breast, n = 54; and prostate, n = 33). Epithelial (pan-keratins), mesenchymal (vimentin), and two exclusion markers were used to identify CTCs and classify them into four epithelial and epithelial-mesenchymal transition-related phenotypes using standardized and throughput method, imaging flow cytometry. The morphological characteristics of CTCs, including their nuclei, such as circularity, the maximum, and minimum diagonal values were determined using an open-source software QuPath. On average, detected CTCs (n = 1156) were larger, and more irregular in shape compared to leukocytes/endothelial cells (n = 400). Epithelial and mesenchymal CTCs had the largest (median = 18.2 μm) and the smallest diameter (median = 10.4 μm), respectively. In terms of cancer-specific variations, the largest CTCs were identified in lung cancer, whereas the smallest-in prostate and breast cancers. Epithelial CTCs and those negative for both epithelial and mesenchymal markers exhibited the highest degree of elongation, whereas mesenchymal CTCs were the most irregular in shape. Protrusions and micronuclei were observed extremely rarely within CTCs of breast and prostate cancer (0.6%-0.8% of CTCs). Micronuclei were observed only in epithelial and epithelial-mesenchymal CTCs. This study underscores the significant variability in the morphological features of CTCs in relation to their phenotypic classification or even the particular organ of origin, potentially influencing for example, size-dependent CTC isolation methods. It demonstrates for the first time the morphological measurements of CTCs undergoing epithelial-mesenchymal transition, and some specific morphological details (i.e., protrusions, micronuclei) within CTCs in general.

Clinical significance of circulating tumor cells in colorectal cancer with peritoneal metastases: a prospective cohort study using a novel method for monitoring treatment response, and assessing minimal residual disease.

The treatment for patients with colorectal cancer with metastases to the peritoneum is complex and may involve both surgery and chemotherapy. Circulating tumor cells (CTCs) have been poorly investigated in peritoneal metastatic colorectal cancer. The aim of the study is to examine the role of circulating tumor cells (CTCs) as a biomarker for monitoring disease progression, treatment response and residual disease using CellMate® - a new promising in vitro diagnostic platform technology. We prospectively followed clinical outcomes of 46 patients treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) for colorectal cancer with peritoneal metastases and examined whether CTCs were present the week of surgery. The CTC measurements were made with the CellMate® technology, which is a platform technology to detect CTCs based on the difference in biomechanical properties compared to blood resident cells. The study was registered online (ClinicalTrials.gov). CTCs were detected in 17 (37%) patients. The presence of CTCs was associated with shorter recurrence-free survival and overall survival after CRS and HIPEC. Both recurrence free survival (HR 4.00, 95%CI 1.15-13.9; P=0.029) and overall survival (HR 5.91; 95% CI 1.18-29.7; P=0.03) were significantly worse if CTCs were detected after neoadjuvant treatment. In the subgroup of patients with CTCs detected, adjuvant therapy tended to improve the prognosis while in CTC negative patients it did not. Pending a prospective multi-center trial to validate these findings, CTCs may in the future be used as a dynamic personalized biomarker for prognostication, predicting response to therapy, and for monitoring disease progression in colorectal cancer with metastases to the peritoneum.

Controllable Assembly of a Quantum Dot-Based Aptasensor Guided by CRISPR/Cas12a for Direct Measurement of Circulating Tumor Cells in Human Blood.

Accurate and sensitive analysis of circulating tumor cells (CTCs) in human blood provides a non-invasive approach for the evaluation of cancer metastasis and early cancer diagnosis. Herein, we demonstrate the controllable assembly of a quantum dot (QD)-based aptasensor guided by CRISPR/Cas12a for direct measurement of CTCs in human blood. We introduce a magnetic bead@activator/recognizer duplex core-shell structure to construct a multifunctional platform for the capture and direct detection of CTCs in human blood, without the need for additional CTC release and re-identification steps. Notably, the introduction of magnetic separation ensures that only a target-induced free activator can initiate the downstream catalysis, efficiently avoiding the undesired catalysis triggered by inappropriate recognition of the activator/recognizer duplex structure by crRNAs. This aptasensor achieves high CTC-capture efficiency (82.72%) and sensitive detection of CTCs with a limit of detection of 2 cells mL-1 in human blood, holding great promise for the liquid biopsy of cancers.

A Paradigm Shift in Cancer Staging - Seeing the Unseen with Circulating Tumor Cell Measurement

A Paradigm Shift in Cancer Staging - Seeing the Unseen with Circulating Tumor Cell Measurement

Abstract P6-01-17: A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy

Abstract Background The presence of circulating tumor cells (CTCs) among women before and/or after completion of neoadjuvant chemotherapy (NAC) for breast cancer may be associated with an increased risk of recurrence, but limited data is available. Objectives To use the Epic Sciences platform to detect and enumerate CTCs in blood samples from women with a new diagnosis of non-metastatic breast cancer of any subtype both i) prior to commencing NAC, and ii) after completion of NAC and surgery. Methods Inclusion criteria included women of any age with non-metastatic breast cancer of any subtype who have not yet commenced NAC. Those diagnosed with prior invasive cancer at any site (apart from non-melanoma skin cancer diagnosed more than five years prior to enrollment) were excluded. Blood samples were obtained to measure CTCs prior to NAC and after NAC and surgery, respectively. CTC identification was based on immunofluorescence analysis using Epic Sciences platform as previously described (Ueno et al 2017). The presence of CTCs was correlated with clinical/pathological data and treatment response, which were abstracted from patients’ medical records. The association between the presence of CTCs and clinical/pathologic characteristics was tested using Fisher’s exact test for categorical variables and t-test or Wilcoxon rank sum tests for numerical variables. All analyses were performed using the R software package. An ad-hoc preliminary analysis was conducted among the first 34 of 50 participants. Results 41 patients (out of an intended 50) have been recruited to-date. 34 participants have a pre- and/or post-treatment CTC measurement available, but 1 was excluded because it was identified to have metastatic disease shortly after enrollment. Among 33 evaluable patients without metastatic disease, 6 (19%) had triple negative breast cancer (TNBC), 13 (39%) had HER2+ and 13 (39%) had hormone receptor (HR)+/HER2- breast cancer. Most (94%) received anthracycline and taxane-based NAC. The median age of breast cancer diagnosis was 50 (29-75). A total of 53 samples were tested for CTC enumeration (5 mL per sample) including 33 pre-treatment and 20 post-treatment samples. CTCs were detected in 32 samples (n=32/53, 60%), including 24 pre-NAC (n=24/33, 73%) with a median of 0.9 CTCs per mL (0.2-19) and 8 post-NAC and surgery (n= 8/20, 40%) with a median of 0.6 CTCs per mL (0.3-3.3). Among the 24 patients (73%) who had detectable CTCs pre-NAC, 10 had HR+/HER2- (41.7%), 9 had HER2+ (37.5%) and 4 (16.7%) had triple negative disease. Among the 20 patients for whom matched pre- and post-treatment CTC results were available, 16 (80%) had detectable CTCs pre-treatment and 8 (40%) had detectable levels post-NAC and surgery. Among the 8 patients (40%) for whom CTCs were detectable post NAC and surgery, 4 (50%) had HR+/HER2-, 2 had HER2+ (25%) and 2 (25%) had triple negative disease. 3 of these patients had numerically higher CTC levels after completion of NAC and surgery compared to pre-NAC levels, 2 of whom had HR+/HER2- breast cancer and one of whom had TNBC. A total of 7 of 33 patients achieved a pathological complete response (PCR) to NAC, among whom 3 had matched pre- and post-treatment CTC results available; none of these 3 patients had detectable CTCs post-treatment. Conclusions Approximately 3 in 4 women with non-metastatic breast cancer who undergo NAC have detectable CTCs pre-treatment using the Epic Sciences Platform. Of 20 patients with matched pre-/post-treatment results, a high proportion (40%) have persistently detectable CTCs. Hence, CTCs may represent an additional measure of minimal residual disease for patients undergoing NAC for breast cancer. Citation Format: Rania chehade, Arushi Jain, Veronika Moravan, Giuseppe Di Caro, Amanda Anderson, Megan M. Slade, Rick Wenstrup, Ana Elisa Lohmann, William Tran, Katarzyna Jerzak. A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-17.

Circulating tumor cells as diagnostic markers of early gastric cancer and gastric precancerous lesions.

Circulating tumor cells (CTCs) may be potential diagnostic biomarkers of various malignancies including gastric cancer. This study aimed to evaluate whether CTCs could be used to facilitate the diagnosis of early gastric cancer (EGC) or precancerous gastric lesions. The diagnostic study included consecutive patients with EGC, gastric precancerous lesions or fundic gland polyps admitted to the Gastroenterology Department, Beijing Friendship Hospital Affiliated to Capital Medical University (National Center for Digestive Diseases) between October 2016 and January 2018. A total of 92 patients were enrolled, including 57 patients with EGC, 14 patients with gastric precancerous lesions and 21 patients with fundic gland polyps (control group). CTCs were detected in 47.89% (34/71) of patients with EGC/gastric precancerous lesions and 4.76% (1/21) of patients with fundic gland polyps (P<0.001). CTC detection distinguished EGC/precancerous lesions from fundic gland polyps with an area under the ROC curve of 0.740 (95% confidence interval, 0.640-0.840; P=0.001), a sensitivity of 49.10%, a specificity of 95.00%, a positive predictive value of 97.00% and a negative predictive value of 64.90%. Peripheral blood CTCs are more common in patients with EGC or gastric precancerous lesions than in patients with fundic gland polyps. Measurement of CTCs may be a useful tool to aid the diagnosis of EGC and precancerous lesions.

SERS Multiplex Profiling of Melanoma Circulating Tumor Cells for Predicting the Response to Immune Checkpoint Blockade Therapy.

Immune checkpoint blockade (ICB) therapy has achieved remarkable success in many cancers including melanoma. However, ICB therapy benefits only a small proportion of patients and produces severe side effects for some patients. Thus, there is an urgent need to identify patients who are more likely to respond to ICB therapy to improve outcomes and minimize side effects. To predict ICB therapy responses, we design a surface-enhanced Raman scattering (SERS) assay for multiplex profiling of circulating tumor cells (CTCs) under basal and interferon-γ (IFN-γ) stimulation. Through simultaneous ensemble and single-cell measurements of CTCs, the SERS assay can reveal tumor heterogeneity and offer a comprehensive CTC phenotype for decision-making. Anisotropic gold-silver alloy nanoboxes are utilized as SERS plasmonic substrates for improved signal readouts of CTC surface biomarkers. By generating a unique CTC signature with four surface biomarkers, the developed assay enables the differentiation of CTCs from three different patient-derived melanoma cell lines. Significantly, in a cohort of 14 melanoma patients who received programmed cell death-1 blockade therapy, the changes of CTC signature induced by IFN-γ stimulation to CTCs show the potential to predict responders. We expect that the SERS assay can help select patients for receiving ICB therapy in other cancers.

Circulating tumor cells as a biomarker for monitoring: Disease progression, treatment response, and minimal residual disease.

e15021 Background: To analyze the role of Circulating tumor cells (CTC) as a confirmatory personalized biomarker for monitoring the disease progression, disease burden, and minimal residual disease in epithelial origin cancers. Methods: In this retrospective study, 127 patients with colorectal, breast, and ovarian cancer at stage III and IV were analyzed. The patients were at various stages of intensive chemo and radiotherapy while the CTCs were isolated and enumerated from 1.5 ml of blood. The decision to continue chemotherapy or change to oral metronomic therapy was based on the presence of circulating tumor cells in Stage III. While in stage IV, serial measurement of CTC guided therapy. CTCs were isolated using OncoDiscover platform possessing EpCAM antibody based immunomagnetic targeting of magnetic nanoparticles after RBC lysis. CTCs were imaged and identified as CK18+ and CD45- cells showing a well-defined nucleus using a motorized fluorescence microscope operational with a monochrome camera. CTCs were enumerated using automated image analysis software and counts were expressed as number per 1.5 ml of blood. Results: In this retrospective study we analyzed blood sample from 127 patients with the advanced stage epithelial cancers (breast- 50 %, ovarian -27 %, colorectal- 23 %) for the presence of CTCs. Amongst those, 52 % showed the presence of CTCs (breast- 52 %, ovarian -46 %, colorectal- 58 %). The CTC count ranged between 1-5 / 1.5 ml of blood with mean and median value of 2 and 1. Among the CTC positive population, majority had CTC count of 1 (44.4 %), while more than 2 CTCs were observed in 11 % of population. CTC clusters were detected in 13 % of population which predominantly were stage IV patients. 67 % among the follow up patients showed decrease in CTC count from the baseline due to the prescribed treatment, while 22 % patients showed increase in CTC count from the baseline. 11 % patients did not show change in CTC count from the baseline. When CTCs count was investigated as an independent variable to monitor the therapeutic response, it correlated well with the positive or negative outcome. In few representative cases, the reduction of CTC number from the basal value was indicative of an effective treatment. Exceptionally, in a representative colorectal cancer case, PET showed no primary as well secondary tumor burden, but the presence of CTCs in blood led further investigating an abdominal MRI that indicated multiple liver lesions suggesting micro-metastasis. Subsequent to SIRT treatment, the patient showed complete tumor regression and absence of CTCs in peripheral blood. Conclusions: Our data suggest that CTC can serve as a dynamic intermittent biomarker for monitoring the disease progression in advanced stages and assess the therapeutic response, thus emphasizing the role of CTCs in personalized cancer management.

Sensitive and dynamic CTCs measurement for prediction and monitoring of PD-1 therapy in GC/EGJC patients: Results from a pilot phase 2 study.

e16094 Background: Programmed death-ligand 1 (PD-L1) expression is currently being explored as a predictive biomarker for anti-PD-1antibody treatment in patients with advanced gastric or esophagogastric junction cancer (GC/EGJC) patients. Circulating tumor cells (CTCs) can be collected non-invasively and sequentially, and may allow real-time monitoring of immune activation in tumor. The aim of this study was to investigate whether PD-L1 expression on CTCs can accurately represent tumor PD-L1 expression and the potential of CTCs as a companion diagnosis (CDx) biomarker. Methods: We evaluated the CTCs counts and PD-L1 status on CTCs during treatment in a phase 2 clinical trial, where patients with previously treated gastric cancer received JS001 in combination with Apatinib. The multi-center trial was registered with ClinicalTrials.gov, number NCT04190745. Blood samples were collected from patients. Sixteen patients with GC were analyzed to investigate the association between clinical outcome and changes of CTC counts pre and post JS001 plus Apatinib. The correlation between the binary clinical outcome (SD/PR/CR vs. PD) and the change in CTCs counts pre and post treatment was analyzed using a logistic regression adjusting for baseline CTCs counts. PD-L1 expression on CTCs were analyzed to determine whether they were in accordance with tissue biopsy. Results: CTCs were detected in all the patients (16/16，100%) ranging from 1 to 33 CTCs/ml. PD-L1+ CTCs were detected in 12 of 16 patients (75%) ranging from 1 to 5 PD-L1 + CTCs/ml. PD-L1 expression in tumor tissue was positive in 11 of 13 patients (84.6%, positive means CPS &gt; = 1), the sensitivity of PD-L1+ CTCs is 90.9% (10/11) if using biopsy as gold standard. The only false negative patient though with CPS 40%, got no benefit from immunotherapy. The specificity of PD-L1+ CTCs detection is 100%, and the accuracy is 90.9%. Overall response rate of this regimen is 26.6% (4/15). Disease control rate is 53.3% (8/15). Progression free survival (PFS) was 2.43 months in all the evaluable patients. However, patients with PD-L1+ CTCs had a longer PFS compared to patients without PD-L1+ CTC (3.30months vs 1.37months, p = 0.001). Overall Survival is not reached. Change of CTCs counts after treatment was significantly correlated with clinical response (p = 0.024). The logistic model McFadden goodness of fit score was 0.435, which is a strong correlation value. Notably, one of the patients who had pseudo-progression shows remarked decreased CTCs count. Conclusions: Our results reveal the potential of CTCs as a noninvasive CDx biomarker in patients with advanced GC/EGJC.

Abstract 4604: Can breast cancer cells be distinguished from blood cells by mechanical parameters? A label-free CTC detection approach

Abstract Background: Even years after successful treatment of the primary tumor about one third of breast cancer patients are suffering from metastatic relapse. A possible reason might be hematogenous spread. Therefore circulating tumor cells (CTCs) in the blood might be interesting and easy accessible surrogate markers for disease monitoring. Usually, detection methods are based on cellular parameters like cell surface markers, size, density or flexibility. Due to phenotypical, mechanical and functional changes during cancer progression and treatment response, isolation of CTC subpopulations remains very challenging. Here we focused on the characterization of CTC mechanics to evaluate the utility of mechanical parameters for CTC separation from blood. Methods: For the first time, we investigated the active and passive mechanical CTC properties such as elasticity, viscosity and contractile force exertion. In an optical rheometer, cells are deformed non-invasively by a dual beam trap. Thus, the relative deformation of a single cell can be measured while phase contrast and fluorescent images are taken. For proof of premise we used peripheral blood mononuclear cells (PBMC) isolated from a healthy donor and human GFP-expressing MDA-MB 231 breast cancer cells to mimic a CTC model system and to measure cell type specific mechanical deformation profiles. For translational experiments blood samples were collected from breast cancer patients. Hematopoietic cells were depleted using CD45. The cell suspensions were applied to the optical stretcher and rheological parameters were measured in the same manner. Phase contrast images of potentially interesting cells were analyzed manually to eliminate dead cells and to identify possible CTC candidates. Results: We were able to reveal distinct mechanical profiles from hematopoietic cells and breast cancer cells, respectively. The optical deformability at the end of the step stress creep experiment (EOS) was significantly different in both, the model and the translational, systems comparing healthy PPMC to MDA-MB 231 cells respective possible CTC from breast cancer patients. Evaluation of secondary parameters like viscosity and activity using a machine learning algorithm allowed for a clear distinction between hematopoietic and cancer cells in the model system. Preliminary results of the translational system suggest that identification of CTC candidates exclusively based on mechanical parameters might be possible. Conclusion: Together with morphology and cell size, the cellular deformation pattern might be a proper tool for marker-free CTC detection in the peripheral blood of breast cancer patients. The optical stretcher device is not suitable for high throughput CTC measurement. However, it allows recommendations for pore size or flow rates which play an important role in the development of label-free isolation approaches including filtration by size or separation by cellular compressibility. Citation Format: Ivonne Nel, Erik W. Morawetz, Josef A. Kaes, Bahriye Aktas. Can breast cancer cells be distinguished from blood cells by mechanical parameters? A label-free CTC detection approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4604.

Serial Analysis of Circulating Tumor Cells in Metastatic Breast Cancer Receiving First-Line Chemotherapy.

We examined the prognostic significance of circulating tumor cell (CTC) dynamics during treatment in metastatic breast cancer (MBC) patients receiving first-line chemotherapy. Serial CTC data from 469 patients (2202 samples) were used to build a novel latent mixture model to identify groups with similar CTC trajectory (tCTC) patterns during the course of treatment. Cox regression was used to estimate hazard ratios for progression-free survival (PFS) and overall survival (OS) in groups based on baseline CTCs, combined CTC status at baseline to the end of cycle 1, and tCTC. Akaike information criterion was used to select the model that best predicted PFS and OS. Latent mixture modeling revealed 4 distinct tCTC patterns: undetectable CTCs (56.9% ), low (23.7%), intermediate (14.5%), or high (4.9%). Patients with low, intermediate, and high tCTC patterns had statistically significant inferior PFS and OS compared with those with undetectable CTCs (P < .001). Akaike Information Criterion indicated that the tCTC model best predicted PFS and OS compared with baseline CTCs and combined CTC status at baseline to the end of cycle 1 models. Validation studies in an independent cohort of 1856 MBC patients confirmed these findings. Further validation using only a single pretreatment CTC measurement confirmed prognostic performance of the tCTC model. We identified 4 novel prognostic groups in MBC based on similarities in tCTC patterns during chemotherapy. Prognostic groups included patients with very poor outcome (intermediate + high CTCs, 19.4%) who could benefit from more effective treatment. Our novel prognostic classification approach may be used for fine-tuning of CTC-based risk stratification strategies to guide future prospective clinical trials in MBC.

Baseline circulating tumor cell (CTC) count as a prognostic marker of PSA response and progression in metastatic castrate sensitive prostate cancer (mCSPC): Results from SWOG S1216, a phase III randomized trial of androgen deprivation plus orteronel (cyp17 inhibitor) or bicalutamide.

5506 Background: In mCSPC, androgen deprivation therapy (ADT) combined with chemotherapy or androgen receptor signaling inhibition (ARSI) is the new standard of care. Biomarkers that predict clinical outcomes with these therapies are needed. We hypothesized that CellSearch CTC count, an FDA-cleared biomarker in metastatic castrate resistant PC (mCRPC), may be a valuable biomarker in mCSPC. Methods: In S1216, peripheral blood was drawn with informed consent at registration (baseline), and CTCs were enumerated on the FDA-cleared CellSearch platform (Menarini) per standard manufacturer protocol. CTC counts were analyzed centrally for associations with 2 pre-specified trial intermediate endpoints: 7-month PSA (7mPSA) ≤ 0.2 ng/ml vs. 0.2–4.0 vs. &gt; 4.0, (intermediate endpoint for overall survival, OS); and progression-free survival (PFS) &lt; vs. &gt; 2 years. Because OS data have not matured, analysis was pooled and equal numbers of samples were analyzed from each treatment arm and outcome measure (7mPSA and PFS) as stipulated by the Data Safety Monitoring Committee. Results: From 2014 to 2017, 523 baseline samples were collected. In the 7mPSA analysis (n = 264), CTCs were detected in 38% of men, with a median of 4 CTCs in those with detectable CTCs. In the PFS analysis (n = 336), CTCs were detected in 37% of men, with a median of 3 CTCs in those with detectable CTCs. Adjusting for disease burden (minimal vs. extensive) and ADT status (already initiated or not) at the time of CTC measurement, men with undetectable CTCs were 6.1-fold more likely to attain 7mPSA ≤ 0.2 (OR 6.1, 95% CI 2.1-17.2, p &lt; 0.001) and 3.7-fold more likely to achieve &gt; 2 years PFS (OR 3.7, 95% CI 1.7-8.1, p &lt; 0.001) compared to men with baseline CTCs ≥ 5. Other cutpoints previously validated in mCRPC studies (CTC &lt; 5 vs. ≥5 and CTCs 0 vs. ≥1) also strongly discriminated 7mPSA and PFS with statistical significance in this mCSPC cohort. Conclusions: CTC count at the start of treatment for mCSPC was highly prognostic of 7-month PSA response (intermediate endpoint for OS) and of PFS at 2 years. To our knowledge, this is the first such strong evidence from a prospective phase 3 trial of this magnitude. Additional analyses are planned when the trial is fully reported. Baseline CTC count may serve as a valuable prognostic marker to discriminate men likely to respond favorably to hormonal therapies from those who may benefit from early alternate interventions.

Liquid Biopsy as Effective Predictor of Stage III Melanoma Relapse

Liquid Biopsy as Effective Predictor of Stage III Melanoma Relapse

Circulating Tumor Cells in Gastrointestinal Cancers: Current Status and Future Perspectives.

Circulating tumor cells (CTCs), which are now defined as the “break away” cancer cells that derive from primary- or metastatic-tumor sites and present in the bloodstream, are considered to be the precursors of metastases. Considering the key role of CTCs in cancer progression, researchers are committed to analyze them in the past decades and many technologies have been proposed for achieving CTCs isolation and characterization with highly sensitivity and specificity until now. On this basis, clinicians gradually realize the clinical values of CTCs' detection through various clinical studies. As a “liquid biopsy,” CTCs' detection and measurement can supply important information for predicting patient's survival, monitoring of response/resistance, assessment of minimal residual disease, evaluating distant metastasis, and sometimes, customizing therapy choices. Nowadays, eliminating CTCs of the blood circulation has been regarded as a promising method to prevent tumor metastasis. However, research on CTCs still faces many challenges. Herein, we present an overview to discuss the current concept of CTCs, summarize the available techniques for CTCs detection, and provide an update on the clinical significance of CTCs in gastrointestinal malignancies, especially focus on gastric and colorectal cancer.

Prognostic significance of circulating tumor cells in patients with gastric cancer: Epithelial mesenchymal transition and perioperative kinetics.

59 Background: Circulating tumor cells (CTCs) have been shown to be heterogeneous. This study aimed to identify the prognostic significance of CTCs in patients with gastric cancer, focusing on epithelial mesenchymal transition and perioperative kinetics. Methods: Peripheral blood (7.5 ml) was taken from patients (n = 54) before curative resection, and at 7 days, 1 and 6 months postoperatively. CTCs were enriched using density gradient centrifugation and magnetic-activated cell sorting (negative selection). Cell suspensions were characterized by multi-immunofluorescence staining against cytokeratin (CK) and N-cadherin, and by DAPI staining. CTCs were defined as nucleated cells expressing CK or N-cadherin. Threshold analysis identified 1 CTC/7.5 ml as an optimal cut-off value. The median observation period was 735 days. Results: CTCs were detected in seven patients (24%) with early cancer and 14 patients (56%) with advanced cancer (p &lt; 0.05). Cells were identified as either N-cadherin+/CK−/CD45− or N-cadherin+/CK+/CD45−, but no N-cadherin−/CK+/CD45− cells were observed. The median follow-up period was 24.5 months. After 2 years, postoperative recurrence was detected in nine patients, all of whom had advanced gastric cancer and N-cadherin+/CK−/CD45− CTCs preoperatively. In terms of perioperative kinetics (just before, 7 days and 1 month after surgery), we divided patients with advanced cancer into three risk groups: A, preoperative CTCs ≥1 and increased postoperatively; B, preoperative CTCs ≥1 and decreased postoperatively; C; no preoperative CTCs. The recurrence rates in the above groups were 80% (4/5), 44% (4/9), and 0% (0/11), respectively. Conclusions: Numerous CTCs expressed N-cadherin but not CK. Perioperative measurement of CTCs may be a useful surrogate marker for recurrence risk.

Abstract 4583: Combined analysis of circulating tumor cells, fecal occult blood test and serum carcinoembryonic antigen for early detection and monitoring recurrence of patients with colorectal cancer

Abstract Current methods for clinical early detection and disease monitoring of patients with colorectal cancer (CRC) are immunochemical fecal occult blood test (iFOBT) and serum levels of carcinoembryonic antigen (CEA), which are known with low sensitivity. There is an unmet need to seek for additional molecular tools for early detection and disease monitoring of patients with CRC. Whether enumeration of circulating tumor cells (CTCs) supplements current clinical tests and facilitates early detection and monitoring disease progression was investigated. A total of 109 pre-operative patients with CRC (including early and advanced stages) and 65 non-cancerous controls were enrolled in this study. CTCs were enriched from the peripheral blood of these individuals by using the PowerMag negative selection system. Immunofluorescence staining of epithelial cell adhesion molecule (EpCAM), and podoplanin (PDPN, a marker related to poor cancer progression) was performed to define the two distinct subtypes of CTCs. Heterogeneous CTCs expressing PDPN or EpCAM are defined in the peripheral blood of patients with CRC. Regardless clinical stages in patients, the number of both EpCAM+-CTCs and PDPN+-CTCs explicitly differentiated non-cancerous controls and patients with CRC, and were also correlated with clinicopathological diagnosis. Receiver operating characteristic analysis demonstrated that the sensitivity and specificity of EpCAM+-CTCs was 86.32% and 78.46%, respectively, when the cutoff value was 23 EpCAM+-CTCs/mL. Using 7 PDPN+-CTCs/mL as the cutoff value, the sensitivity and specificity of PDPN+-CTCs was 77.98% and 75.38%, respectively. 84.2% and 80.3% of patients with serum CEA &amp;lt; 5 ng/mL were found to have higher EpCAM+-CTCs and PDPN+-CTCs counts, respectively. In addition, simultaneous measurements of iFOBT and CTCs could compromise the false-positive rate of iFOBT, while measurements of CEA and CTCs are applicable to monitor cancer recurrence after surgery. This study implicates that CTC testing supplements the current clinical methods to facilitate early detection and disease monitoring in CRC. Citation Format: Wei-Shan Hung, Wen-Sy Tsai, Tzu-Min Wang, Hsueh-Ling Hsu, Hsuan Liu, Chia-Yu Yang, Ching-Ping Tseng, Ju-Chien Cheng. Combined analysis of circulating tumor cells, fecal occult blood test and serum carcinoembryonic antigen for early detection and monitoring recurrence of patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4583.

Circulating Tumor Cells in Minimally Invasive Follicular Thyroid Carcinoma and Benign Thyroid Tumors with a Follicular Pattern: Pilot Experience

Minimally invasive follicular thyroid carcinomas (MIFCs) are uncommon; literature offers limited guidance on their natural history and management. Starting January 2015 we measured circulating tumor cells (CTCs) in patients with MIFC (n=22) or benign thyroid tumors with follicular features (n=4). In a retrospective analysis, we assessed detectability of and serial changes in CTC, compared demographic/clinical differences between CTC-positive versus CTC-negative subgroups using Student's t-test, and examined correlations between CTC status and serum thyroglobulin using Spearman's test. CTCs were quantitated via immunomagnetic separation/microscopic inspection. Thirteen patients (50%: 12/22 MIFC, 1/4 benign tumor) were initially CTC-positive; 3 remained CTC-positive in ≥1 subsequent measurement. CTC-positive patients had larger tumors and more frequent multifocality and vascular invasion versus CTC-negative patients (n=13). However, no tested variable differed significantly between the subgroups. After 17.2±10.5 months, neither subgroup showed evidence of disease. Significant correlation was absent (p ≥ 0.263) between CTC and Tg negativity (r = 0.243; n=13 evaluable) or initial CTC positivity and Tg positivity (r = -0.418; n=9 evaluable). In the studied settings, CTC measurement is feasible, has unclear clinical/outcome implications, but may provide different information versus thyroglobulin testing. Lengthier assessment is warranted in larger series.

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.

Clinical significance of circulating tumor cells in squamous cell lung cancer patients.

Squamous cell carcinoma (SCC) of the lung represents 20-30% of non-small cell lung cancers (NSCLC) and is associated with a poor prognosis. This study aims to investigate the presence of circulating tumor cells (CTCs) in squamous cell lung cancer patients and what its role might be in providing prognostic information. Serial blood samples from 100 patients both before and after initiation of one cycle of standard chemotherapy were analyzed using CellSearch system. Of 105 patients enrolled, 100 were evaluable. ≥ 2 CTCs per 7.5 mL of blood were present in 29% of patients at baseline before chemotherapy, and 9% patients have more than 5 CTCs. Based on the current literature, the CTC measurements were dichotomized as 2-4 versus ≥ 5 CTCs. In the univariate analysis, CTC count ≥ 5 at baseline and CTC count ≥ 5 at both time points (before and after one cycle of chemotherapy) were significantly associated with a poor PFS and OS outcome. Both factors remained independent poor prognostic markers in the stepwise multivariate analysis. Our study indicate that the CTC count is a prognostic factor for PFS and OS outcomes in Chinese patients with locally advanced SCC of the lung.

A nonrandomized, prospective, clinical study on the impact of circulating tumor cells on outcomes of urothelial carcinoma of the bladder patients treated with radical cystectomy with or without adjuvant chemotherapy.

To investigate outcomes of urothelial carcinoma of the bladder (UCB) patients treated with radical cystectomy (RC) according to the presence of circulating tumor cells (CTC) and the administration of adjuvant chemotherapy (AC). We prospectively enrolled 226 UCB patients treated with RC without neoadjuvant chemotherapy at our institution between 2007 and 2013. Blood samples were obtained from all patients preoperatively and analyzed for CTC using the CellSearch® system. Platinum-based AC was administered in 50 patients (27.0%). Cox regression models evaluated the association of CTC with disease recurrence, cancer-specific and overall mortality according to AC administration. 185 patients were available for analyses. CTC were present in 41 patients (22.2%). Patients with presence of CTC received AC more frequently, compared to patients without CTC (p = 0.027). At a median follow-up of 31months, the presence of CTC was associated with disease recurrence, cancer-specific and overall mortality (p-values < 0.001) in patients without AC administration. In patients who received AC, there was no difference in either endpoint between patients with or without presence of CTC. In multivariable analysis of patients without AC administration, the presence of CTC was an independent predictor for disease recurrence (HR: 4.9; p < 0.001), cancer-specific (HR: 4.2; p = 0.003) and overall mortality (HR: 4.2; p = 0.001). The CTC status may be implemented in decision-making regarding AC administration in UCB patients following RC. CTC measurement should be implemented in future UCB studies on systemic chemotherapy to validate our findings.