Increase In Circulating Tumor Cells Research Articles

Flow-based immunomagnetic enrichment of circulating tumor cells from diagnostic leukapheresis product.

The clinical utility of circulating tumor cells (CTCs) is hampered by the low number of cells detected. Diagnostic leukapheresis (DLA) offers a solution but, due to the observed non-specific binding and clumping, processing of DLA samples using the CellSearch system only allows for the processing of aliquots consisting of ~ 2% of the total DLA sample per test. Here, we introduce a flow enrichment target capture Halbach-array (FETCH)-based separation method in combination with a DNase preprocessing step to capture CTCs from larger fractions of DLA products without clumping. To evaluate the FETCH method, we processed peripheral blood samples from 19 metastatic castration-naïve prostate cancer (mCNPC) patients with CellSearch, and processed 2% aliquots of leukapheresis samples from the same patients with CellSearch as well as FETCH with or without DNase preprocessing. Using 2% aliquots from six patients, the use of FETCH with fewer immunomagnetic epithelial cellular adhesion molecule (EpCAM) conjugated ferrofluids was tested, whereas 20% aliquots from four patients were used to evaluate the processing of 10-fold larger DLA samples using FETCH. Results show that the cell clumping normally seen after immunomagnetic enrichment of DLA material was greatly reduced with the use of DNase pretreatment, while the number of CTCs detected was not affected. The number of CTCs detected in 2% aliquots of DLA using FETCH was unchanged compared to CellSearch and did not decrease when using down to 10% of the volume of immunomagnetic anti-EpCAM ferrofluids normally used in a CellSearch test, whereas the number of co-enriched white blood cells reduced a median 3.2-fold. Processing of a 20% aliquot of DLA with FETCH resulted in a 14-fold increase in CTCs compared to the processing of 2% aliquots of DLA using CellSearch and a total 42-fold median increase in CTCs compared to peripheral-blood CellSearch.

A clinically feasible circulating tumor cell sorting system for monitoring the progression of advanced hepatocellular carcinoma

BackgroundHematogenous metastasis is essential for the progression of advanced hepatocellular carcinoma (HCC) and can occur even after patients receive multidisciplinary therapies, including immunotherapy and hepatectomy; circulating tumor cells (CTCs) are one of the dominant components of the metastatic cascade. However, the CTC capture efficiency for HCC is low due to the low sensitivity of the detection method. In this study, epithelial cell adhesion molecule (EpCAM)/vimentin/Glypican-3 (GPC3) antibody-modified lipid magnetic spheres (LMS) were used to capture tumor cells with epithelial phenotype, mesenchymal phenotype and GPC3 phenotype, respectively, in order to capture more CTCs with a more comprehensive phenotype for monitoring tumor metastasis.ResultsThe novel CTC detection system of Ep-LMS/Vi-LMS/GPC3-LMS was characterized by low toxicity, strong specificity (96.94%), high sensitivity (98.12%) and high capture efficiency (98.64%) in vitro. A sudden increase in CTC counts accompanied by the occurrence of lung metastasis was found in vivo, which was further validated by a clinical study. During follow-up, the rapid increase in CTCs predicted tumor progression in HCC patients. Additionally, genetic testing results showed common genetic alterations in primary tumors, CTCs and metastatic tissues. The proportion of patients predicted to benefit from immunotherapy with the CTC detection method was higher than that for the tissue detection method (76.47% vs. 41.18%, P = 0.037), guiding the application of clinical individualized therapy.ConclusionsThe Ep-LMS/Vi-LMS/GPC3-LMS sequential CTC capture system is convenient and feasible for the clinical prediction of HCC progression. CTCs captured by this system could be used as a suitable alternative to HCC tissue detection in guiding immunotherapy, supporting the clinical application of CTC liquid biopsy.

The Impact of Surgery on Circulating Malignant Tumour Cells in Oral Squamous Cell Carcinoma.

The extent to which surgical management of oral squamous cell carcinoma (OSCC) disseminates cancer is currently unknown. To determine changes in numbers of malignant cells released into systemic circulation immediately following tumour removal and over the first seven post-operative days. An observational study from March 2019 to February 2021. This study was undertaken at Queen Mary University Hospital, Hong Kong. Patients with biopsy-proven oral SCC were considered for eligibility. Patients under 18 years of age, pregnant or lactating women and those unable to understand the study details or unable to sign the consent form were excluded. Twenty-two patients were enrolled (12 male and 10 female) with mean age of 65.5 years. Primary tumour management was performed in accord with multi-disciplinary team agreement. Anaesthesia and post-operative care were unaltered and provided in accord with accepted clinical practice. Three types of malignant cells detected in peripheral blood samples were enumerated and sub-typed based on the presence of chromosomal aneuploidy and immunohistochemical characteristics. To test the hypothesis that malignant cells are released by surgery, the numbers of single circulating tumour cells (CTCs), circulating tumour microemboli (CTM) and circulating endothelial cells (CTECs) were recorded pre-operatively, upon tumour removal and the second and seventh post-operative days. Of a potential 88 data collection points, specimens were not obtainable in 12 instances. Tumour removal resulted in a statistically significant increase in CTCs and a non-statistically significant rise in CTMs. CTCs, CTMs and CTECs were detected in the majority of patients up to the seventh post-operative day. Individual patients demonstrated striking increases in post-operative CTCs and CTECs numbers. Surgical management of OSCC has a significant impact on the systemic distribution of cancer cells. Malignant cells persisted post-operatively in a manner independent of recognised staging methods suggesting differences in tumour biology between individuals. Further investigation is warranted to determine whether circulating malignant cell enumeration can be used to refine risk stratification for patients with OSCC.

Circulating tumor cells detected in follow-up predict survival outcomes in tri-modality management of advanced non-metastatic esophageal cancer: a secondary analysis of the QUINTETT randomized trial

BackgroundOur aim was to establish if presence of circulating tumor cells (CTCs) predicted worse outcome in patients with non-metastatic esophageal cancer undergoing tri-modality therapy.MethodsWe prospectively collected CTC data from patients with operable non-metastatic esophageal cancer from April 2009 to November 2016 enrolled in our QUINTETT esophageal cancer randomized trial (NCT00907543). Patients were randomized to receive either neoadjuvant cisplatin and 5-fluorouracil (5-FU) plus radiotherapy followed by surgical resection (Neoadjuvant) or adjuvant cisplatin, 5-FU, and epirubicin chemotherapy with concurrent extended volume radiotherapy following surgical resection (Adjuvant). CTCs were identified with the CellSearch® system before the initiation of any treatment (surgery or chemoradiotherapy) as well as at 6-, 12-, and 24-months post-treatment. The threshold for CTC positivity was one and the findings were correlated with patient prognosis.ResultsCTC data were available for 74 of 96 patients and identified in 27 patients (36.5%) at a median follow-up of 13.1months (interquartile range:6.8-24.1 months). Detection of CTCs at any follow-up visit was significantly predictive of worse disease-free survival (DFS;hazard ratio [HR]: 2.44; 95% confidence interval [CI]: 1.41-4.24; p=0.002), regional control (HR: 6.18; 95% CI: 1.18-32.35; p=0.031), distant control (HR: 2.93; 95% CI: 1.52-5.65;p=0.001) and overall survival (OS;HR: 2.02; 95% CI: 1.16-3.51; p=0.013). After adjusting for receiving neoadjuvant vs. adjuvant chemoradiotherapy, the presence of CTCs at any follow-up visit remained significantly predictive of worse OS ([HR]:2.02;95% [Cl]:1.16-3.51; p=0.013) and DFS (HR: 2.49;95% Cl: 1.43-4.33; p=0.001). Similarly, any observed increase in CTCs was significantly predictive of worse OS (HR: 3.14; 95% CI: 1.56-6.34; p=0.001) and DFS (HR: 3.34; 95% CI: 1.67-6.69; p<0.001).ConclusionThe presence of CTCs in patients during follow-up after tri-modality therapy was associated with significantly poorer DFS and OS regardless of timing of chemoradiotherapy.

Circulating Tumor Cells and Biomarker Modulation with Olaratumab Monotherapy Followed by Olaratumab plus Doxorubicin: Phase Ib Study in Patients with Soft-Tissue Sarcoma

This phase Ib study enumerated whole blood circulating tumor cells (CTC) and evaluated biomarkers in patients with potentially resectable soft-tissue sarcoma (STS) treated with olaratumab monotherapy (20 mg/kg) for one cycle followed by up to six cycles of olaratumab (20 mg/kg, cycles 1-2; 15 mg/kg, cycles 3-7) plus doxorubicin (75 mg/m2 on day 1). CTCs, platelet-derived growth factor receptors (PDGFR), and PDGF ligand expression in tumor tissue pre- and post-olaratumab monotherapy were evaluated. Antitumor activity, safety, pharmacokinetics, and PET/biomarker association with clinical outcome were assessed. Of 51 treated patients, 35, 43, and 37 were evaluable for CTC enumeration, PDGFRs, and PDGF ligand expression, respectively. An increase in CTCs at cycle 1 day 8 was observed, followed by a significant reduction by cycle 3 day 1 or 30-day follow-up. Decrease in CTC counts after olaratumab monotherapy was higher in patients with disease control than without disease control (57.9% vs. 31.2%). Baseline IHC expression was positive in most patients for PDGFRα [n = 31 (72.1%)] and PDGFRβ [n = 36 (83.7%)]. Similar rates were observed post-olaratumab monotherapy [PDGFRα, n = 30 (69.8%); PDGFRβ, n = 33 (76.7%)]. Eleven patients (29.7%) showed a 30% reduction by RT-PCR in PDGFRα at cycle 2. PDGFR expression and PET response showed no correlation with clinical outcome. Safety and pharmacokinetic profiles were consistent with previous reports. This study, the first to use a validated method for CTC detection, confirms that CTC enumeration in STS is feasible. However, no correlation was observed between PDGFRα expression and clinical outcome.

NCMP-13. LEPTOMENINGEAL DISEASE FOLLOWING RESECTION OF INTRAVENTRICULAR MELANOMA METASTASIS DIAGNOSED THROUGH CIRCULATING TUMOR CELLS

Abstract Leptomeningeal disease in the setting of malignant melanoma metastatic to the brain provides a dismal prognosis. The relationship between intraventricular metastatic tumor seeding following surgical resection and development of leptomeningeal disease (LMD) is not completely clear, although there appears to be correlation. While the mechanisms that drive the development of LMD is not well understood, monitoring of cerebrospinal fluid (CSF) for circulating tumor cells (CTCs) in high risk patients may allow for early intervention for LMD prior to radiographical diagnosis. This report describes a patient with metastatic melanoma who developed ventricular trapping from an intraventricular melanoma metastasis. The patient underwent endoscopic assisted resection of the tumor. Due to concern for leptomeningeal seeding given the location of the tumor and use of surgical resection, CSF analysis was performed. CTC count was increased shortly after surgical resection along with cytology that was suspicious for malignancy. Due to the increase in CTCs, the patient was treated for LMD with whole brain radiation therapy and intrathecal pembrolizumab. Subsequent CSF analysis revealed clearing of malignant cells in the CSF. The patient developed symptoms consistent with LMD approximately 9 months after the surgery and died 21 months after resection of his brain metastasis. This case illustrates a rare occurrence of an intraventricular melanoma metastasis, and the use of CTC presence within the CSF to diagnose LMD for early intervention. This emphasizes that the risk of developing LMD must be considered with intraventricular metastasis or ventricular exposure during tumor resection, and that CTCs may be an effective factor to monitor for early development of disease with possible prolonged survival benefit.

Abstract B19: The impact of circulating tumor cells on treatment response in early breast cancer patients with hormone receptor-positive and HER2 positive who underwent the neoadjuvant therapy

Abstract Introduction: Accumulating evidence has described that circulating tumor cells (CTCs) imply a poor prognosis in patients with early breast cancer. In this study, we evaluated the association between CTCs and response of neoadjuvant therapy in patients with hormone receptor (HR)-positive and HER2-positive breast cancer. Method: The HERAKLES study was a prospective single-arm, multicenter, phase II trial designed to evaluate the efficacy and safety of letrozole and trastuzumab for neoadjuvant therapy in postmenopausal patients with HR-positive and HER2-positive breast cancer (NCT02214004). All patients received trastuzumab every 3 weeks for eight cycles and daily letrozole for 24 weeks, followed by the surgery. After neoadjuvant therapy, the clinical response was assessed by the radiologic modalities. The CTCs were investigated by using a microfabricated porous filter in which the size of the square pores was optimized as 8.5 × 8.5 μm for the nickel electroformed membrane filter from 10mL of whole blood. Then, immunofluorescence staining was conducted with antibodies against CD45, EpCAM, cytokeratins, and then counterstained with DAPI. The CTCs were determined as DAPI-positive, EpCAM-positive, CK-positive, and CD45-negative cells. The CTCs analysis before and after neoadjuvant therapy was performed in 21 patients. In addition, we classified the patients into 4 groups: Group A, CTC-negative at both times (n=4); Group B, presence of CTC at pretreatment and CTC-negative at post-treatment (n=4); Group C, CTC-negative at pretreatment and presence of CTC at post-treatment (n=6); Group D, presence of CTC at both times (n=7). Results: The CTCs were detected in 11 (52.4%; median CTCs count, 2; range, 1-26) patients at pre-treatment and 13 (61.9%; median CTCs count, 4; range, 1-11) patients at post-treatment. CTC detection and CTC changes after neoadjuvant therapy were not associated with primary tumor characteristics. There was no patient with disease progression after neoadjuvant therapy in group A and B. However, the disease progression was observed in one (16.7%) patient in group C and two (28.6%) patients in group D (p=0.470). When the CTC was analyzed as continuous value, the CTCs were increased in all (the average change of CTCs, +6.0) patients with disease progression after neoadjuvant therapy, although the CTCs were increased in 7 (38.9%; the average change of CTCs, -1.3) of 18 patients without progression disease (p=0.09). Conclusions: The increase in CTCs after neoadjuvant treatment, including aromatase inhibitor and trastuzumab, tended to be correlated with poor treatment response in HR+/HER2+ breast cancer patients, although it remains underpowered. Our results supported that the monitoring of CTCs may reflect the response to neoadjuvant aromatase inhibitor and trastuzumab therapy in HR+/HER2+ breast cancer. Citation Format: Soong June Bae, Soeun Park, Chi Hwan Cha, Dooreh Kim, Janghee Lee, Sung Gwe Ahn, Joon Jeong. The impact of circulating tumor cells on treatment response in early breast cancer patients with hormone receptor-positive and HER2 positive who underwent the neoadjuvant therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B19.

Circulating tumour cell enumeration does not correlate with Miller\u2013Payne grade in a cohort of breast cancer patients undergoing neoadjuvant chemotherapy

PurposeThe association between pathological complete response (pCR) in patients receiving neoadjuvant chemotherapy (NAC) for breast cancer and Circulating Tumour Cells (CTCs) is not clear. The aim of this study was to assess whether CTC enumeration could be used to predict pathological response to NAC in breast cancer as measured by the Miller–Payne grading system.MethodsTwenty-six patients were recruited, and blood samples were taken pre- and post-NAC. CTCs were isolated using the ScreenCell device and stained using a modified Giemsa stain. CTCs were enumerated by 2 pathologists and classified as single CTCs, doublets, clusters/microemboli and correlated with the pathological response as measured by the Miller–Payne grading system. χ2 or ANOVA was performed in SPSS 24.0 statistics software for associations.Results89% of patients had invasive ductal carcinoma (IDC) and 11% invasive lobular carcinoma (ILC). At baseline 85% of patients had CTCs present, median 7 (0–161) CTCs per 3 ml of whole blood. Post-chemotherapy, 58% had an increase in CTCs. This did not correlate with the Miller–Payne grade of response. No significant association was identified between the number of CTCs and clinical characteristics; however, we did observe a correlation between pre-treatment CTC counts and body mass index, p < 0.05.ConclusionsPatients with a complete response to NAC still had CTCs present, suggesting enumeration is not sufficient to aid surgery stratification. Additional characterisation and larger studies are needed to further characterise CTCs isolated pre- and post-chemotherapy. Long-term follow-up of these patients will determine the significance of CTCs in NAC breast cancer patients.

Circulating Tumor Cells Predict Outcome in Trimodality Management of Advanced Non-Metastatic Esophageal Cancer: A Pre-planned Correlative Study from a Randomized Trial

Circulating tumor cells (CTC) correlate with poor prognosis in metastatic esophageal cancer, but there are few data describing the importance of CTCs in patients with non-metastatic disease. Our aim was to establish if CTCs predicted worse outcome in patients with non-metastatic esophageal cancer. We prospectively collected CTC data from patients with operable non-metastatic esophageal cancer from April 2009 to November 2016 enrolled in our QUINTETT esophageal cancer clinical trial (NCT00907543). Patients were randomized to receive either adjuvant or neoadjuvant chemoradiotherapy plus surgery. CTCs were enumerated with the CellSearch® system (Menarini Silicon Biosystem, Philadelphia, PA) before the initiation of any treatment (surgery, or chemoradiotherapy) as well as at 6, 12 , and 24 months post-treatment. CTC findings were correlate with standard tumor characteristics, including tumor size, and grade. Oncological outcomes were examined using Kaplan Meier estimates and Cox proportional hazards regression. CTCs were identified in 26 of 74 patients (35%) at a median follow up of 12.7months (interquartile range: 6.8-23.9 months). Detection of CTCs at any follow-up visit was significantly predictive of worse disease free survival (p=0.004), regional control (p=0.042), distant control (p<0.001) and overall survival (p=0.034). After adjusting for receiving neoadjuvant vs. adjuvant chemoradiotherapy, the presence of CTCs at any follow-up visit remained significantly predictive of worse overall survival (hazard ratio:1.82[HR];95% confidence interval [Cl]:1.03-3.22; p=0.04) and disease free survival (HR: 2.26 ;95% Cl: 1.29-3.97; p=0.005). Similarly, any observed increase in CTCs was significantly predictive of worse overall survival (HR: 2.70; 95% CI: 1.30-5.62; p=0.008) and disease free survival (HR: 2.92; 95% CI: 1.43-5.95; p=0.003). Patients with positive CTCs post trimodality therapy was associated with significantly poorer disease free and overall survival regardless of timing of chemoradiotherapy. Further investigation is warranted for the role of CTCs as a prognostic indicator in management of advanced non-metastatic esophageal cancer.

Nano-Based Quantification of Circulating Tumor Cells as a Biomarker of Disease Status in Oligometastatic Patients Following Metastases-Directed Radiation Therapy

Circulating tumor cells (CTCs) have been demonstrated as a biomarker in certain advanced cancers. Additionally, there has been increasing recognition that definitive treatment, including radiotherapy (RT), may lead to cure or significantly increased time to progression in oligometastatic solid malignancies. However, differentiating between patients with truly oligometastatic disease and those who will develop systemic disease is difficult. In most cases, secondary to a lack of reliable biomarker, surveillance often relies heavily on imaging. We hypothesized that CTCs are a sensitive biomarker in post-treatment surveillance after metastasis-directed definitive RT in oligometastatic patients. Patients with oligometastatic solid-tumor malignancies with up to 3 metastases undergoing definitive RT to all known metastatic sites were enrolled on a prospective study. Peripheral blood (7 mL) was drawn prior to initiation of RT, 1-3 time points during RT, and at subsequent follow up appointments. CTCs were quantified using CapioCyte, a nanotechnology based CTC capture system that takes advantage of biomimetic cell rolling and dendrimer-mediated strong multivalent binding. A total of 33 patients were enrolled with a median follow-up of 12.4 months. All patients had detectable CTCs on enrollment with a median of 24 CTCs/mL (range 3-120). Pre-treatment CTCs did not predict subsequent recurrence (p=0.94). 20 patients (67%) subsequently had progression of disease, with an overall median PFS of 4.6 months. Of those who recurred, 10 patients had CTCs quantified at or near the time of progression, with 8 of these patients showing a corresponding increase in CTCs (2.5-28.7 fold increase). Out of 6 patients who were started on systemic therapy after progression, CTCs declined or were stable in all 5 patients with a partial response or stable disease, and increased in 1 patient who progressed. Our data suggests CTCs as quantified by the CapioCyte technology correspond with disease status and are a reliable biomarker for post-treatment surveillance in oligometastatic solid-tumor malignancies.

Abstract 3606: Circulating tumor cells and tumor-derived extracellular vesicles as a possible marker for tumor response and survival in patients with non-small cell lung cancer treated with immunotherapy

Abstract Background: Lung cancer remains the number one cause of cancer related death worldwide. Immune modulating therapy have shown long lasting survival, however, they are only effective in about 20% of patients. Although PD-L1 expression predicts tumor response, it is not a robust predictor. Circulating tumor cells (CTCs) or tumor derived extracellular vesicles (tdEV) may be another tumor response predictor. CTCs have been associated with survival in NSCLC. Our hypothesis is that CTC or tdEV changes predict early tumor response and it is an early marker of treatment efficacy. Methods: Patients with proven advanced NSCLC referred to our center for immunotherapy by PD-L1 or PD-1 antagonists were eligible for inclusion. CellSave tubes were drawn at start of therapy (T0) and after 4 weeks of treatment (T1). EpCAM expressing CTCs were detected by the CellSearch system. EpCAM expressing tdEV which are present in the blood during CTC enumeration, were counted in the images from the CellTracks Analyzer, which were analyzed with the open source program ACCEPT. Response to therapy was measured by the response evaluation criteria in solid tumors (RECIST) and according to clinical evaluation of the treating physician. Patients were divided in groups based on response to therapy and presence of CTCs. Cox regression analyses were used to study the association between CTCs and tdEVS with both OS and PFS. Results: 114 patients have been included (104 T0 samples, 71 T1 samples, 52 both a T0 and T1 sample). 53 patients have progressive disease (PD), 24 stable disease (SD), 23 partial response (PR) and 2 a complete response; 12 could not be evaluated yet. CTCs were present in 33 T0 samples (32%), and 20 T1 samples (28%). When CTCs were present at T0, patients had no response to therapy in 83% of cases, while patients without CTCs had no response in 68% of cases. Patients with increased CTC counts at T1 responded in 9% of the cases, versus 33% in patients with decreased CTC counts. CTCs were significantly correlated to survival at both time points independently (T0, HR=1.06, p=0.042; T1, HR=1.04, p=0.009). Comparing patients with a decrease of CTCs with those with an increase, Cox regressions showed a HR of 8.9 (p=0.041), while using the difference of CTCs between T1 and T0 as a continuous variable in the cox regression gave a HR of 1.41 (p=0.02).tdEVS were present in 90% of cases (median 7, range 0-1752)at T0 and in 94% at T1 (median 5, range 0-1975). Cox regression showed a significant correlation with survival of tdEVs at T0 (HR=1.0, p=0.004) and T1 (HR=1.0, p=0.009) and the change in tdEVs counts between T0 and T1 (HR=1.01, p=0.04) Conclusion: CTC and tdEV decrease in the first weeks of immunotherapy was associated with tumor response, whereas an increase in CTC was associated with no response to immunotherapy, indicating a possible use as an early marker. Citation Format: Menno Tamminga, Sanne de Wit, Joost F. Swennenhuis, T. Jeroen N. Hiltermann, Ed M. Schuuring, Leon W.M. Terstappen, Harry J. Groen. Circulating tumor cells and tumor-derived extracellular vesicles as a possible marker for tumor response and survival in patients with non-small cell lung cancer treated with immunotherapy [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 3606.

Impact of liver tumor percutaneous radiofrequency ablation on circulating tumor cells.

Radiofrequency ablation has become an increasingly common therapeutic technique for patients with hepatocellular carcinoma or metastatic liver tumors. However, reports on the effect of percutaneous radiofrequency ablation (PRFA) on circulating tumor cells (CTCs) are limited. The present study aimed to further investigate the impacts of PRFA on the numbers and phenotypes of CTCs in patients with hepatocellular carcinoma or metastatic liver tumors. A total of 43 patients with hepatocellular carcinoma or 7 types of metastatic liver tumors were treated with PRFA. A total of 5 ml blood per sample were collected from the peripheral circulation 30 min before and 3 days after PRFA. The total number of CTCs significantly increased 3 days after PRFA, and the mesenchymal phenotype CTCs, which also increased significantly, significantly contributed to the overall increase in CTCs. Furthermore, the lymphocyte levels were significantly decreased following PRFA, and the CTC level was significantly higher in patients with decreased lymphocyte levels compared with those with increased lymphocyte levels. Liver tumor PRFA may increase the level of mesenchymal phenotype CTCs, which is significantly associated with the lymphocyte count. Factors pertaining to the performance of PRFA were also investigated in the present research, but no significant results were identified.

Perioperative detection of circulating tumour cells in patients with lung cancer.

Lung cancer is a leading cause of mortality and despite surgical resection a proportion of patients may develop metastatic spread. The detection of circulating tumour cells (CTCs) may allow for improved prediction of metastatic spread and survival. The current study evaluates the efficacy of the ScreenCell® filtration device, to capture, isolate and propagate CTCs in patients with primary lung cancer. Prior to assessment of CTCs, the present study detected cancer cells in a proof-of-principle- experiment using A549 human lung carcinoma cells as a model. Ten patients (five males and five females) with pathologically diagnosed primary non-small cell lung cancer undergoing surgical resection, had their blood tested for CTCs. Samples were taken from a peripheral vessel at the baseline, from the pulmonary vein draining the lobe containing the tumour immediately prior to division, a further central sample was taken following completion of the resection, and a final peripheral sample was taken three days post-resection. A significant increase in CTCs was observed from baseline levels following lung manipulation. No association was able to be made between increased levels of circulating tumour cells and survival or the development of metastatic deposits. Manipulation of the lung during surgical resection for non-small cell lung carcinoma results in a temporarily increased level of CTCs; however, no clinical impact for this increase was observed. Overall, the study suggests the ScreenCell® device has the potential to be used as a CTC isolation tool, following further work, adaptations and improvements to the technology and validation of results.

Screening for Circulating Tumour Cells Allows Early Detection of Cancer and Monitoring of Treatment Effectiveness: An Observational Study

Background:Circulating-Tumour-Cells (CTC) provide a blood biomarker for early carcinogenesis, cancer progression and treatment effectiveness. An increase in CTCs is associated with cancer progression, a CTC decrease with cancer containment or remission. Several technologies have been developed to identify CTC, including the validated Isolation-by-Size-of-Epithelial-Tumour (ISET, Rarecells) technology, combining blood filtration and microscopy using standard histo-pathological criteria.Methods:This observational study compared CTC count to cancer status and cancer risk, by monitoring treatment effectiveness in cancer patients and by screening for CTC in asymptomatic patients with risk factors, including family history of cancer.Results:Between Sept-2014 and Dec-2016 we undertook 600 CTC tests (542 patients), including 50% screening requests of patients without cancer diagnosis but with risk factors. CTC were detected in all cancer patients (n=277, 100%), and in half of the asymptomatic patients screened (50%, 132 out-of 265 patients). Follow-up tests including scans, scheduled within 1-10 months of positive CTC tests, found early cancerous lesions in 20% of screened patients. In 50% of male patients with CTC and normal PSA (prostate-specific-antigen) levels, PSMA-PET scans revealed increased uptake in the prostate, indicative of early prostate cancer. Other types of cancers detected by CTC screening and subsequent scans included early breast, ovarian, lung, or renal cancer. Patients with CTC were advised on integrative approaches including immune-stimulating and anti-carcinogenic nutritional therapies. CTC repeat tests were available in 10% of patients with detected CTC (40 out-of 409 patients, n=98 CTC tests) to assess treatment effectiveness, suggesting nutritional therapies to be beneficial in reducing CTC count.Conclusions:CTC screening provided a highly sensitive biomarker for the early detection of cancer, with higher CTC counts being associated with higher risk of malignancy. CTC monitoring over time indicated treatment effectiveness. Nutrients with anti-carcinogenic properties could reduce CTC count, and included curcumin, garlic, green tea, grape seed, modified citrus pectin, and medicinal mushroom extract.

Screening for Circulating Tumour Cells Allows Early Detection of Cancer and Monitoring of Treatment Effectiveness: An Observational Study

Background: Circulating-Tumour-Cells (CTC) provide a blood biomarker for early carcinogenesis, cancer progression and treatment effectiveness. An increase in CTCs is associated with cancer progression, a CTC decrease with cancer containment or remission. Several technologies have been developed to identify CTC, including the validated Isolation-by-Size-of-Epithelial-Tumour (ISET, Rarecells) technology, combining blood filtration and microscopy using standard histo-pathological criteria. Methods: This study compared CTC count to cancer status and cancer risk, by monitoring treatment effectiveness in cancer patients and by screening for CTC in asymptomatic patients with risk factors, including family history of cancer. Results: Between Sept-2014 and Dec-2016 we undertook 600 CTC tests (542 patients), including 50% screening requests of patients without cancer diagnosis but with risk factors. CTC were detected in all cancer patients (n=277, 100%), and in half of the asymptomatic patients screened (50%, 132 out of 265 patients). Follow-up tests including scans were scheduled within 1-6 months of CTC tests. In up to 50% of male patients with normal PSA (prostatespecific- antigen) levels but detected CTC, PET scans using PSMA (Ga-68-prostate-specific-membrane-antigens) revealed increased uptake in the prostate, indicative of early prostate cancer. Other types of cancer, including early breast, ovarian, lung, or renal cancer were detected in a small number of asymptomatic men or women with a positive CTC count. A subgroup of patients with detected CTC underwent interventions, including nutritional therapy with immunestimulating and anti-carcinogenic nutrients. CTC repeat tests were available in 10% of patients with detected CTC (40 out of 409 patients, n=98 CTC tests) to assess treatment effectiveness. Conclusion: CTC screening provided a highly sensitive biomarker for the early detection of cancer, with higher CTC counts being associated with higher risk of malignancy. CTC monitoring over time indicated treatment effectiveness. Nutrients with anti-carcinogenic properties could reduce CTC count, and included curcumin, garlic, green tea, grape seed, modified-citrus-pectin, and medicinal mushroom-extract.

Abstract 1539: Live circulating tumor cells as a predictive biomarker of response to neoadjuvant chemoradiotherapy for resectable pancreatic cancer

Abstract The detection of increase in the number of circulating tumor cells (CTCs) during a patient's clinical course may be a harbinger of forthcoming overt metastasis. To detect live CTCs (l-CTCs) in blood samples of cancer patients (pts), we employed a new genetically modified telomerase-specific replication-selective adenovirous, expressing GFP (TelomeScanF35), containing both type 35 fiber that induce broad infectivity and miR-142-3p target sequence that can prevent a false positive toward blood cells. Recently, we indicated that preoperative chemoradiation therapy comprising gemcitabine and S-1 administration concurrent with full-dose radiation (NACRT) lead to encouraging survival rate in pts with resectable pancreatic cancer. But, NACRT may have disadvantages. NACRT requires several months, potentially delaying surgical resection, and can be waste of time and may result in the pts missing the chance for surgery. We assessed the l-CTCs burden in pts treated with NACRT. This study was approved by the Osaka Police Hospital IRB. Pts with resectable cytologically or histologically proven ductal adenocarcinoma of the pancreas were enrolled. Treatment consisted of an intervenous infusion of Gem 800 mg/m2 on day 1, 8, 22, and 29; and S-1 80 mg/m2 orally on day 1-5, 8-12, 22-26, and 29-33 given concurrently with IMRT to 60 Gy. Surgical exploration was scheduled 4-7 weeks after the final radiation fraction. 7.5 ml of blood samples were obtained from the pts included in this clinical study before NACRT, after 1 month of NACRT, and after 2 months of surgical resection. To distinguish between leucocyte and cells with epithelial origin, cells were stained with anti-CD45 and anti-Cytokeratin Abs. To distinguish cells with mesenchymal origin, cells were labeled with anti-Vimentin Ab. GFP-positive and CD45-negative cells were counted as l-CTC. 16 pts aged 44-78 years (5 males and 11 females) were enrolled. No treatment-related deaths occurred. CA19-9 was reduced to &amp;lt;50% of baseline values in 10 of 12 measureable pts. 13 of 16 enrolled pts successfully underwent surgical resection. 5 of 16 enrolled pts had l-CTCs (1∼4CTCs,Vimentin[+]) detected before NACRT. An increase in CTCs was seen in 4 of 5 pts after NACRT, but no detectable l-CTCs were observed in only 1 pt after NACRT. These 4 CTC-positive pts continuously had CTCs detected after surgery. 2 of 4 CTC-positive pts early developed liver metastasis and died, despite R0 resection. On the contrary, small increase (1∼2CTCs) of CTCs was seen after NACRT in 4 of 11pts, who had no CTCs detected before NACRT. But, no l-CTCs were detected in these 11 pts after surgery and they survived without recurrence. CTC-positive pts before NACRT presented a higher rate of liver metastasis after surgery than CTC-negative pts (50% vs. o%). We may consider surgery first for the CTC-positive pts before NACRT. The capture of l-CTCs may be useful biomarker for prognosis assessment or stratification. Citation Format: Masahiro Tanemura, Kenta Furukawa, Masaki Wakasugi, Kentaro Kishi, Yasuo Urata, Kiyomi Taniyama, Hiroki Akamatsu. Live circulating tumor cells as a predictive biomarker of response to neoadjuvant chemoradiotherapy for resectable pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1539.

Interleukin-17A Modulates Circulating Tumor Cells in Tumor Draining Vein of Colorectal Cancers and Affects Metastases

Metastasis is the major cause of death in patients with colorectal cancer (CRC). Circulating tumor cells (CTC) are believed to cause metastasis and serve as a prognostic marker for mortality in clinical stage IV patients. However, most studies are conducted in late-stage cases when distant metastases have already occurred; thus, such results provide limited clinical use. This study focused on whether CTCs can predict the risk of metastasis after treatment of the primary tumor in early-stage patients with CRC. CTCs were quantified using EpCAM-positive/CD45-negative immunoselection and flow cytometry in patients with CRC. A mouse model was used to investigate the mechanistic roles of CTCs and interleukin (IL)-17A in metastasis. The number of mesenteric CTCs obtained from stage II patients was higher than that obtained from patients in stages I, III, and IV. In addition, following invasion of orthotopically implanted tumors in our mouse model, we found that CTCs exhibited an increase-then-decrease pattern, accompanied by corresponding changes in serum IL-17A levels and opposing changes in serum granulocyte macrophage colony-stimulating factor (GM-CSF) levels. Ablation of IL-17A and administration of rGM-CSF effectively suppressed the increase in CTCs and prevented metastasis in mice. Moreover, IL-17A promoted cancer cell motility, matrix digestion, and angiogenesis, whereas GM-CSF stimulated the elimination of CTCs by boosting host immunity. Notably, serum levels of IL-17A were also correlated with disease-free survival in patients with CRC. Our results showed that CTCs and IL-17A could serve as prognostic markers and therapeutic targets for CRC metastasis. Clin Cancer Res; 20(11); 2885-97. ©2014 AACR.

Reduced Dissemination of Circulating Tumor Cells With No-Touch Isolation Surgical Technique in Patients With Pancreatic Cancer

Circulating tumor cells (CTCs) disseminate from the primary tumor and travel through the bloodstream and lymphatic system. The detection of and/or increase in the number of CTCs during a patient’s clinical course may be a harbinger of forthcoming overt metastasis. We aimed to examine the impact of 2 different surgical techniques, standard (ST) pancreaticoduodenectomy (PD) and no-touch isolation (NT) PD, on tumor behavior and outcome in patients with pancreatic cancer by using CTCs as biomarkers. In this pilot study, patients were randomized to either ST-PD (n = 6) or NT-PD (n = 6). Intraoperatively, blood samples were taken from the portal vein for measurement of CTCs before and immediately after removal of the tumor. An increase in CTCs was seen in 5 of 6 patients (83%) with ST-PD but no patients with NT-PD (P = .003). In the ST-PD and NT-PD groups, median overall survival was 13.0 and 16.7 months, respectively (P = .33); there was no difference in disease-free survival (P = .42). The use of NT-PD significantly reduced the number of CTCs in the portal vein with no benefit in survival outcomes compared with ST-PD, although more extensive studies are required.

090 * SIGNIFICANT INCREASE IN CIRCULATING TUMOUR CELLS IN PULMONARY VENOUS BLOOD DURING SURGICAL MANIPULATION IN PATIENTS WITH PRIMARY LUNG CANCER

Objectives: Circulating tumour cells (CTCs) are tumour cells shed from primary tumour which circulate in peripheral blood after passing through the drainage vein. This prospective study was conducted to assess changes in CTCs in the drainage pulmonary vein (PV) during lung cancer surgery. Methods: A total of 30 consecutive peripheral-type primary lung cancer patients who underwent lobectomy via thoracotomy (adenocarcinoma 73.3%; pathologic stage I 60%) were included. For each patient, 2.5 ml blood was sampled from the lobar PV of the primary tumour site before and after surgical manipulation for lobectomy. CTCs were quantitatively examined with the CellSearch system. Results: Before surgical manipulation, CTCs were detected in PV blood in the majority of patients (22/30, 73%), whereas CTCs were detected in peripheral blood in only two patients. The median number of CTCs in PV blood before manipulation (prePV-CTCs) was 4.0 (cells/2.5 ml), and there was no significant difference in prePV-CTCs according to any clinico-pathological characteristics including tumour size, progression, or histologic type. After manipulation, that is at the time of completion of lobectomy, PV-CTCs significantly increased (median postPV-CTCs 60.0, P < 0.001). Increase in postPV-CTCs was associated with microscopic lymphatic invasion. PV-CTCs significantly increased in tumours with lymphatic invasion (pre and postPV-CTCs, 4.0 and 90.5, respectively; P = 0.006), but not in tumours without lymphatic invasion (pre and postPV-CTCs, 3.5 and 7.0, respectively). Conclusions: We documented a significant increase in CTCs in drainage PV after surgical manipulation, especially in tumours with lymphatic invasion. We are awaiting survival data with 5-year follow-up, which may provide insight into the clinical significance of PV-CTCs.

Circulating tumor cells as emerging tumor biomarkers in lung cancer.

Enumeration of circulating tumor cells (CTCs) has been shown to correlate with poor progression free survival (PFS) and overall survival (OS) in breast, colorectal, and prostate cancers. In lung cancer, there is a large need for more and multiple tumor biomarkers that will allow monitoring of patient progress over the course of therapy. Studies have suggested that the primary treatment for lung cancer, surgery, can lead to an increase of CTCs (1,2). This may lead to a different sort of residual disease even in successful cases of full tumor resection. In CTC studies involving lung cancer, CTC enumeration as a method to monitor patient progress has not been as convincing because CTCs were not found in many study patients, and those patients with CTCs had numbers far lower than other cancer types (3-5). In order for CTCs to become a useful biomarker for lung cancer, there is a need to look beyond enumeration and an understanding of the molecular characteristics of the CTCs and their similarities and differences from their tumor of origin. The ability to quickly define a patient’s disease based on molecular signatures as well as identify whether a patient is responding to therapy is potentially a powerful clinical tool for a fast-acting disease.