Circulating Tumor Cells Levels Research Articles

P3.01-008 Association between Icotinib Efficacy and Circulating Tumor Cell Levels in Advanced Non-Small Cell Lung Cancer

Advanced non-small cell lung cancer (NSCLC) studies indicated a potential association between chemotherapy efficacy and circulating tumor cells (CTC) counts in peripheral blood. The icotinib efficacy and circulating tumor cells (CTC) counts in non-small cell lung cancer remain unknown. The aim is to investigate association between the icotinib efficacy and CTC counts in advanced NSCLC patients. A total of 74 advanced NSCLC patients consented to provide 5ml of peripheral blood before systematic therapy, and divided into two groups (those with high CTC counts and those with low CTC counts) based on the patients′ median CTC counts. All the patients were treated with icotinib, and the icotinib efficacy and prognoses were compared. The treatment efficacies were 46.88% (15/32) and 23.81% (10/42) for the low CTC group and the high CTC group, respectively. The median overall survival was 22.0 months (95%CI: 19.6-28.7 months) for the low CTC group and 18.3 months (95% CI: 15.3-25.4 months) for the high CTC group. The median progression-free survival was 11.6 months (95% CI: 8.7-15.6 months) and 7.2 months (95% CI: 3.4-8.7 months) for the low group and the high CTC group, respectively. The CTC counts can be used as a important biomarker for therapy monitoring the icotinib effect on patients with advanced NSCLC. Efficacy and prognosis of icotinib and CTC counts were considered important, and the CTC counts could be used to predict the efficacy of icotinib and prognosis of advanced NSCLC. The change in CTC count levels can be used as a biomarker for evaluating the prognosis of patients with advanced NSCLC.

Circulating tumor cell count and thrombosis in metastatic breast cancer

Background Circulating tumor cell (CTC) count is a major prognostic factor in metastatic breast cancer (MBC) and has been reported to be associated with thrombosis in short-term studies on MBC patients. Objective To assess whether CTC detection (CellSearch® ) before first-line chemotherapy impacts the risk of thrombosis throughout the course of MBC. Patients/Methods Among patients included before first-line chemotherapy for MBC in the prospective IC2006-04 CTC detection study (NCT00898014), the electronic medical files of those patients treated at Institut Curie (Paris, France) were searched in silico and manually checked for incident venous or arterial thrombotic events (TE) in the course of MBC. Univariate and multivariate analyses were performed using Cox and Fine-Gray models, adjusted for age and Khorana score. Results/Conclusions With a median follow-up of 64months (25-81months), among the 142 patients included, 34 (24%) experienced a TE (incidence rate, 8 TE/100 patient-years). The TE incidence rate was 13 TE/100 patient-years for the 80 patients with ≥1 CTC/7.5mL of blood before initiating first-line chemotherapy, vs. only 4 TE/100 patient-years for the 62 CTC-negative patients. Fine-Gray multivariate analysis (with death as competing event) included age, Khorana score and baseline lactate dehydrogenase and CTC levels: detectable CTC was the only factor significantly associated with an increased risk of TE (sub-distribution hazard ratio[SHR] for patients with [1-4] CTC=3.1, 95% CI [1.1; 8.6], SHR for patients with ≥5 CTC=1.4, 95% CI [0.5; 4.6]). This study shows that CTC detection before starting first-line chemotherapy is an independent risk factor for TE in MBC patients.

NanoVelcro-captured CTC number concomitant with enhanced serum levels of MMP7 and MMP9 enables accurate prediction of metastasis and poor prognosis in patients with lung adenocarcinoma.

Lung adenocarcinoma (LADC) is among the most malignant cancers that frequently develops micrometastases even in early stages of the disease. Circulating tumor cell (CTC) number, matrix metalloproteinase (MMP) 7, and MMP9 show great prospects as predictive biomarkers in many tumors. However, the interactions between these biomarkers and the molecular basis of their roles in the metastasis and prognosis of LADC remain unclear. The present study revealed that an elevated CTC count and overexpression of MMP7 and MMP9 correlate with metastasis and clinical progression in LADC patients (n=143). Furthermore, MMP7 and MMP9 upregulation facilitates LADC cell migration in vitro and enhances serum CTC levels in a xenograft mouse model. More importantly, receiver operating characteristic (ROC) curves and Kaplan–Meier analysis confirmed more accurate prediction of metastasis and overall survival (OS) with a combination panel of CTC, MMP7, and MMP9. Taken together, our data show, for the first time, the involvement of MMP7 and MMP9 in the release of CTCs into the peripheral blood, and our data reveal that CTC count and expression of MMP7 and MMP9 can be used together as an effective clinical prediction panel for LADC metastasis and prognosis.

Circulating tumor cells in peripheral and pulmonary venous blood predict poor long-term survival in resected non-small cell lung cancer patients

We tested the hypothesis that circulating tumor cells (CTCs) in preoperative peripheral blood (PPB) and intraoperative pulmonary venous blood (IPVB) could predict poor long-term survival in resected non-small cell lung cancer (NSCLC) patients. CTCs were separated from blood using magnetic beads coated with antibodies against epithelial-cell adhesion molecule (EpCAM) via magnetic-activated cell sorting (MACS). CTCs were quantified with fluorescence-labeled antibodies against pan-cytokeratin through flow cytometry. CTCs were quantified in PPB and IPVB in 23 consecutive stage I-IIIA patients with resected NSCLC. The association between CTCs and prognosis in these patients was evaluated after a 5-year follow-up. In NSCLC patients, outcomes were assessed according to CTC levels at surgery. NSCLC patients identified as high-risk groups exhibited >5 CTCs/15 mL in PPB and >50 CTCs/15 mL in IPVB. Univariate Cox proportional-hazards regression analysis showed that the CTC count in PPB or IPVB was an independent risk factor for tumor-free surivival (TFS) and overall survival (OS). The high-risk group of patients had a shorter median TFS (22 months vs. >60.0 months, p < 0.0012) and shorter OS (27 months vs. >60 months, p < 0.0015). The number of CTCs counted in PPB and IPVB was an independent risk factor for TFS and OS in resected NSCLC patients.

Serum CA19-9 as a marker of circulating tumor cells in first reflux blood of colorectal cancer patients.

Circulating tumor cells (CTCs) are used for metastasis surveillance in cancer patients, but low detection rates limit their use in colorectal cancer (CRC). We investigated the distribution of CTCs in peripheral and portal blood of CRC patients, and analyzed the relationship between serum tumor CEA/CA19-9 markers and CTCs blood levels. CTC levels detected in first reflux/portal vein blood were higher than in peripheral blood, and liver reduced CTCs amount. CTCs-positive patients had increased serum CEA and CA 19-9 levels, and the CEA and CA 19-9 levels correlated with the CTCs levels. Even in non-metastatic CRC patients with barely detectable CTCs in peripheral blood, serum CA 19-9 levels correlated with the CTC levels in first reflux/portal vein blood. These results demonstrate that CTC detection in the first reflux vein/portal vein blood is more sensitive than in peripheral blood, suggesting that clinical diagnosis using the CellSearch System should be based on the CTC detection in first reflux vein blood due to the high detection rates. In addition, our results indicate that serum CA 19-9 levels may serve as a diagnostic marker for further evaluation of CTC levels in portal blood.

Abstract 4953: Single cell signaling analysis reveals circulating tumor cell markers of drug susceptibility and tumor heterogeneity

Abstract Complexity of cancer requires advancements of tools for intervention and diagnostics. Rare circulating tumor cells (CTCs) from patient blood could be used for real-time monitoring of solid cancer. We recently developed a microfluidic device, the CTC-iChip, which enriches well-preserved CTCs in solution through removal of the cellular components (red and white blood cells) and platelets of whole blood. Protein expression analysis in CTCs has been limited to immunofluorescent staining for a few well-characterized tumor markers (including EpCAM, HER2, PSA and Keratins) due to the limitations inherent to immunofluorescence labels and their precise resolution through microscopy. The scarcity of cells precludes large-scale proteomic analysis such as mass spectrometry of CTCs to effectively evaluate changes in total as well as phospho-proteins, which serve as excellent surrogate markers to monitor in real time the rapid alterations in signaling pathways within cells. This approach would be particularly important since the PI3K, mTOR, Akt, MAPK signaling pathways are aberrantly activated in many cancers and are promising therapeutic targets. In this work, we applied inductively-coupled plasma mass spectrometry (ICP-MS) measurement of cells (mass cytometry) that can measure up to ~40 proteins in millions of single cells due to quantitative, non-overlapping mass signals acquired from antibodies tagged with purified lanthanides. Sample preparation for mass cytometry was modified to minimize the loss of cells using our microfluidic platform in lieu of washing via centrifugation. Mass cytometry allowed identification of tumor cells simultaneous analysis of markers that are associated with proliferation, response or resistance to pathway inhibition due to administered therapeutic agent. We quantified the expression of twelve phospho-proteins to monitor changes multiple signaling nodes within single tumor cells. This approach allowed assay and compare the target proteome within single CTCs as well as individual tumor cells derived from primary and metastatic tumors. We found that some protein levels of CTCs represent primary tumor protein levels well. When targeted therapeutic was administered two of these epitopes were found to be significantly changed in both CTCs and primary tumor cells. We also found a portion of cancer cells in primary tumor were more susceptible to the targeted therapeutic than the rest of the tumor, showing a heterogeneity in response within the tumor. Overall, single cell multiplexed molecular analysis revealed tumor molecular heterogeneity and circulating markers of drug susceptibility. Citation Format: N Murat Karabacak, Yu Zheng, Erin Emmons, Michael Koulopoulos, Daniel A. Haber, Mehmet Toner, Shyamala Maheswaran. Single cell signaling analysis reveals circulating tumor cell markers of drug susceptibility and tumor heterogeneity [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 4953. doi:10.1158/1538-7445.AM2017-4953

Circulating tumor cells correlate with patterns of recurrence in patients with hormone-sensitive prostate cancer.

The aim of this study was to evaluate the correlation between circulating tumor cells (CTCs) and patterns of recurrence in patients with hormone-sensitive prostate cancer. The study involved patients with histologically confirmed, advanced prostatic adenocarcinoma, who were tested for CTCs (Veridex®) when they developed recurrence after radical prostatectomy or external beam radiation between 2008 and 2014. Forty-two prostate cancer patients were evaluated. CTCs were detected in 14 out of 42 (33.3%) patients (Group A), while the remaining 28 (66.7%) showed undetectable levels of CTCs (Group B). The mean prostate-specific antigen value was higher in Group A in comparison to Group B (6.2 vs 3.3 ng/dL) (P=0.48). Presence of bone metastases alone or along with nodal metastases was more common in Group A (57.1%) in comparison to Group B (25%) (P=0.04). In a univariate analysis, the presence of CTCs at diagnosis correlated with the development of bone recurrence (OR: 4; 95% CI: 1.0–15.9; P=0.05). Even if the study enrolled only a small number of patients, the detection of CTCs in the blood appears to correlate with the pattern of progression in patients with hormone-sensitive prostate cancer, suggesting a possible role in anticipating recurrence at the bone in men with higher tumor load. Further prospective studies are warranted in this setting.

Circulating tumor cells detected by lab-on-a-disc: Role in early diagnosis of gastric cancer.

BackgroundThe use of circulating tumor cells (CTCs) as an early diagnostic biomarker and prognostic indicator after surgery or chemotherapy has been suggested for various cancers. This study aimed to evaluate CTCs in patients who underwent gastrectomy for gastric cancer and to explore their clinical usefulness in the early diagnosis of gastric cancer.MethodsA total of 116 patients with gastric cancer who underwent gastrectomy and 31 healthy volunteers were prospectively included between 2014 and 2015. Peripheral blood samples were collected before gastrectomy, and CTCs were examined using a centrifugal microfluidic system with a new fluid-assisted separation technique.ResultsAfter creating a receiver operating characteristic curve to identify the discriminative CTC value needed differentiate patients with gastric cancer from healthy volunteers, sensitivity and specificity were nearly optimized at a CTC threshold of 2 per 7.5 mL of blood. Of the 102 persons with a CTC level ≥2 per 7.5 mL of blood, 99 (97.1%) had gastric cancer, and of the 45 persons with a CTC level <2 per 7.5 mL of blood, 28 (62.2%) were healthy controls. Accordingly, the sensitivity and specificity for the differentiation of patients with gastric cancer from healthy controls were 85.3% and 90.3%, respectively. However, the presence of CTCs was not associated with any clinicopathologic features such as staging, histologic type, or mucin phenotype.ConclusionAlthough we could not prove the clinical feasibility of CTCs for gastric cancer staging, our results suggest a potential role of CTCs as an early diagnostic biomarker of gastric cancer.

Role of Circulating Tumor Cell (CTC) Monitoring in Evaluating Prognosis of Triple-Negative Breast Cancer Patients in China.

BackgroundBreast cancer (BC) is the most common malignant tumor in females. This study investigated the role and utility of CTC monitoring in evaluating the prognosis of triple-negative breast cancer patients.Material/MethodsWe enrolled 286 female triple-negative breast cancer patients who were diagnosed at and received radical resection surgery in our hospital. Peripheral venous blood samples were collected preoperatively and at 3 and 7 days postoperative, and the Cell Search system was used to detect CTC in peripheral blood. We analyzed the relationship between preoperative CTC level and clinical pathological characteristics of patients. Kaplan-Meier method was used to establish progression-free survival curves and overall survival curves, we used the log-rank test to compare the survival rate, and we explored the effects of preoperative and postoperative CTC levels on patient survival.ResultsCompared with preoperative levels, the average CTC content in peripheral blood of breast cancer patients was significantly increased at 3 days after surgery, and then decreased to the preoperative baseline level by 7 days after surgery. The 3-year overall survival rate and progression-free survival rate in patients with CTC >5/7.5 mL peripheral blood were significantly lower than in patients with CTC <5/7.5 mL peripheral blood detected preoperatively and at 3 and 7 days postoperatively.ConclusionsDynamic monitoring of preoperative and postoperative CTC levels can accurately predict recurrence and progression of disease, and is important in postoperative monitoring and prognosis evaluation.

Immune status parameters in lung cancer patients with various levels of circulating tumor cells.

e20018 Background: The purpose of the study was to evaluate parameters of cellular immunity in lung cancer patients with various levels of circulating tumor cells (CTCs) and different tumor histogenesis. Methods: Blood samples of 28 patients (24 men, 4 women, mean age 62±1.3 years) were studied. 18 (64.3%) patients had neuroendocrine tumors (small cell lung cancer, SCLC), and 10 (35.7%) patients had adenocarcinoma (AC). 11 (39.3%) patients were diagnosed with stage III disease and 17 (60.7%) – stage IV. CTCs were detected in the blood before treatment using the CellSearch System (Janssen Diagnostics, LLC) with iron microparticles coated with antibodies to EpCAM, CD45 and cytokeratins 8, 18 and 19; subsets of lymphocytes, including minor ones, were studied on FACSCantoII flow cytometer (BD). Results: CTCs were found in 25 patients (89.3%); their number ranged 1-6888. 15 (83.3%) patients with SCLC had 0-6888 CTCs, with an average of 655.7, median 38.5. All 10 patients with AC had 1-486 CTCs, with an average of 57.2, median 5.0. The number of CTCs in CTC-positive patients with SCLC was, despite high individual variability, significantly higher than in patients with AC (786.9±474 vs. 57.2±47.9, respectively; p &lt; 0.05). Such differences were accompanied by higher levels of Th-lymphocytes with activation/apoptosis markers (CD3+CD4+CD95+): 84.2±2.7 vs. 72.2±3.7%, and cytotoxic CD16bright/56dim NKcells: 91.4±1.8 vs. 83.4±3.7%. Patients with CTC+ AC had higher levels of IFNγ-producing NK-cells, compared to SCLC patients (15.3±3.8 vs. 7.2±1.6%), as well as levels of Th with the early activation marker (CD4+CD38+) (27.8±3.5 vs. 17.4±2.3%). Presence of CTCs in SCLC patients was associated with lower levels of activated lymphocytes of various subsets: T-cells (CD4+CD38+, CD8+CD38+, CD3+CD8+HLA-DR+) and NК-cells (Granzyme B+ and CD335+). Conclusions: Levels of CTCs were significantly higher in patients with SCLC than in those with AC; immune status parameters had a number of differences in CTC+ tumors with various histogenesis. The presence of CTCs in SCLC was accompanied by reduced blood levels of activated T- and NK-cells. The reported study was partially supported by RFBR, research project No. 16-34-00244mol_a

Enumeration of circulating tumor cells and assessment of immunotherapy biomarker PD-L1 in patients with colorectal cancer.

e23029 Background: Circulating tumor cells (CTC) play a prognostic role in patients with metastatic colorectal cancer (mCRC). Studies investigating detection of CTC in peripheral blood (PB) showed limited clinical sensitivity. This study compares clinical sensitivity of CTC analysis in mesenteric venous blood (MVB) vs PB. PD-L1 expression on isolated CTCs was also characterized in various stages of CRC patients. Methods: 8mL PB and MVB samples were collected into K2EDTA-containing vacationer tubes and subsequently performed CTC enumeration and PD-L1 analysis by MiSelect R system. CTC is defined as EpCAM+, cytokeratin+ and CD45- with intact nuclei. CTC and PD-L1+ CTC number were counted and correlated to the patients’ clinical manifestations. Results: Blood samples were collected from 75 CRC patients with various stages. In MVB, CTC detection rate was 20%, 33%, 44% and 58% for stage I, II, III, and IV respectively, and the overall detection rate is 41%. In PB, CTC only detected in sporadic cases (7%). Also, the amount of CTC detected in MVB was more abundant than that in PB ( P &lt; 0.001) In addition, preoperative serum CEA levels were significantly associated with presence of CTC in MVB ( P= 0.011). PD-L1 expression was detected in 32% of the isolated CTCs, and was more abundant in those from late stage patients. The expression level of PD-L1 showed heterogeneity among CTCs. Conclusions: CTC enumeration in MVB could potentially increase the clinical sensitivity for CTC analysis in CRC. The CTC level in MVB significantly correlated with CEA levels which could serve as an important biomarker information for post-operative management. In additions, using MiSelect R System to determine PD-L1 expression on CTC could provide a promising approach for both patient selection or therapeutic monitoring of immunotherapies for CRC.

Arginine methylation of EGFR in circulating tumor cells: A new biomarker for predicting resistance to anti-EGFR agents.

3590 Background: Arginine methylation of the epidermal growth factor receptor (meEGFR) increases binding affinity of EGF and other EGFR ligands, reduces the efficacy of anti-EGFR agents in vivo, and is reported to have a role in predicting response to anti-EGFR agents. This study aimed to investigate the predictive impact of meEGFR in metastatic colorectal cancer (mCRC) patients (pts) treated with anti-EGFR agents using blood-based testing. Methods: 15 mL of blood were collected from mCRC pts with documented disease progression following anti-EGFR treatment (Rx). Circulating tumor cells (CTCs) were isolated using antibody (ab)-independent micro-fluidic cassette-based technology (Parsortix system), which separates CTCs on the basis of size and deformability. CTCs were identified based on negative staining for CD45ab and positive staining for EpCAMab. meEGFR was identified based on positive staining for me-R198/200ab on CTCs. Associations between meEGFR-CTCs and total CTCs with progression free survival (PFS) were determined by Kaplan-Meier method and compared by the log-rank test. Results: A total of 47 mCRC pts were prospectively included in this study. CTCs were identified in 30 out of 47 cases (64%). Of those 30, meEGFR-CTCs were identified in 19 cases (63%). Mean total CTCs and meEGFR-CTCs counts were 3.6 (range 0-52) and 2.3 (range 0-30) cells per 7.5ml, respectively. There was no association between meEGFR-CTCs and clinic-pathological features (age, sex, tumor site &amp; grade), line of anti-EGFR Rx, previous irinotecan used, or NRAS, BRAF, PIK3CA, and MSI status. However, in RASwt/BRAFwtmCRC pts, high levels of meEGFR ratio (defined as &gt; 0.25 meEGFR-CTCs per total CTCs) was associated with significantly inferior PFS with anti-EGFR Rx (median PFS 5.4 mo vs. 8 mo, HR 3.4, 95% CI of 1.5-7.9, P = 0.004). By contrast, high levels of total CTCs ( &gt; 3 cells/per 7.5 ml) had no impact on PFS with anti-EGFR Rx. Conclusions: We have successfully isolated CTCs from mCRC pts’ blood using Parsortix system. Elevated levels of arginine methylated EGFR is associated with a shorter PFS with anti-EGFR-based Rx. Assessment of meEGFR-CTCs may provide a “liquid biopsy” biomarker for reduced efficacy from anti-EGFR Rx.

Improving circulating tumor cells enumeration and characterization to predict outcome in first line chemotherapy mCRPC patients.

IntroductionThere is a critical need of new surrogate markers for improving the therapeutic selection and monitoring of metastatic prostate cancer patients. Nowadays clinical management of these patients is been driven by biochemical and clinical parameters without enough accuracy to allow a real personalized medicine. The present study was conducted to go insight the molecular profile of circulating tumor cells (CTCs) isolated from advanced metastatic castration-resistant prostate cancer (mCRPC) with the aim of identifying prognostic marker with potential utility for therapy selection and monitoring.Materials and MethodsCTCs isolation was carried out in peripheral blood samples from 29 mCRPC patients that undergo systemic chemotherapy based on taxanes (docetaxel/cabazitaxel) and 19 healthy controls using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize the CTC population. A panel of 17 genes related with prostate biology, hormone regulation, stem properties, tumor aggressiveness and taxanes responsiveness was analysed to identify an expression signature characterizing the CTCs.ResultsPatients with ≥ 5 CTCs/7.5ml of peripheral blood at baseline and during the treatment showed lower progression free survival (PFS) and overall survival (OS). Changes of CTCs levels during the treatment were also associated with the patient's outcome. These results confirmed previous data obtained using CellSearch in mCRPC. In addition, we found a CTC profile mainly characterized by the expression of relevant genes for the hormone dependent regulation of PCa such as AR and CYP19 together with genes strongly implicated in PCa progression and resistance development such as BIRC5, TUB1A, GDF15, RAB7 and SPINK1. Our gene-expression profiling also permitted the identification of valuable prognostic biomarkers. Thus, high levels of AR, CYP19 and GDF15 were associated with poor PFS rates while AR, GDF15 and BIRC5 were also found as reliable predictors of OS. Besides, a logistic model using KLK3 and BIRC5 showed a high specificity and sensitivity compared to CellSearch to discriminate patients with a more aggressive evolution.ConclusionsThe molecular characterization of CTCs from advanced mCRPC patients provided with a panel of specific biomarkers, including genes related to taxanes resistance, with a promising applicability as “liquid biopsy” for the management of these patients.

Characteristics of circulating tumor cells in organ metastases, prognosis, and T lymphocyte mediated immune response.

Circulating tumor cells (CTCs) possess profound influence on tumor metastases and disease progression. This study aimed to investigate the correlation of CTCs with clinical characteristics and T-cell immunity, and to explore whether CTCs and the subpopulations can serve as an independent prognostic factor in advanced non-small cell lung cancer (NSCLC). A prospective study was conducted in late stages of NSCLC patients. The levels of overall CTCs and the three subpopulation CTCs were enumerated using the CanPatrol™ CTC enrichment system. The information about the patients which included the clinical characteristics, survival status at the 200th day postdiagnosis, and the levels of T cells was collected. Mann–Whitney U test, Kruskal–Wallis H test, Cox regression, and Spearman’s rank correlation coefficient were the statistical methods used in this study. We detected CTCs in 27 of the 31 eligible patients; the level of epithelial–mesenchymal circulating tumor cells (EMCTCs) was higher than that of epithelial circulating tumor cells and that of mesenchymal circulating tumor cells (MCTCs) in the majority of NSCLC patients. Organ metastases were positively associated with the levels of overall CTCs, EMCTCs, and MCTCs (P<0.05). EMCTCs and MCTCs were associated with worse clinical outcomes. Additionally, the levels of EMCTCs were negatively associated with the levels of CD3+ T cells (P=0.01) and CD8+ T cells (P=0.04). In conclusion, the levels of CTCs were positively associated with organ metastases, particularly bone metastases, but were negatively associated with T-cell levels. The levels of EMCTCs and MCTCs had negative prognostic value.

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.

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

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

Effect of NK cell immunotherapy on immune function in patients with hepatic carcinoma: A preliminary clinical study

ABSTRACTWe investigated the effectiveness of adoptive transfer of KIR ligand-mismatched highly activated nature killer (HANK) cells in patients with hepatic carcinoma. Peripheral blood mononuclear cells were obtained and cultured in vitro to induce expansion and activation of HANK cells. After 12 d of culture, the cells were divided into 3 parts and infused intravenously on days 13 to 15. The patients (n = 16) were given one to 6 courses of immunotherapy. No side effects were observed. The lymphocyte subsets and cytokine, thymidine kinase 1 (TK1) and circulating tumor cell (CTC) levels were measured 1 day before treatment and 1 month after the final infusion: the absolute number of total T cells and NK cells and the IL-2 and TNF-β levels were significantly higher, and the TK1 and CTC levels were significantly lower at 1 month after treatment. The percentage of patients who experienced partial response, disease stabilization, and disease progression at 3 months after treatment was 18.8%, 50.0% and 31.2%, respectively. The total follow-up period was 2–12 months. The median progression-free survival from treatment was 7.5 months. This is the first study on the benefits of HANK cell immunotherapy for hepatic carcinoma These encouraging preliminary observations imply that HANK cell immunotherapy is safe, can improve the immune function of patients with liver cancer, and may even reduce the rate of tumor metastasis and recurrence. However, further studies on larger samples of patients with a longer follow-up period are required to confirm these findings.

Abstract P1-01-02: Circulating tumor cells (CTC) and endothelial cells (CEC) prognostic value in HER2 negative metastatic breast cancer patients treated with first line weekly paclitaxel and bevacizumab: First results of a prospective cohort from the French Breast Cancer InterGroup Unicancer (UCBG): COMET study

Abstract Background: increased levels of circulating tumor cells (CTC) are associated with worse progression-free survival (PFS) and overall survival (OS) in patients (pts) with metastatic breast cancer (MBC). It has been hypothesized that bevacizumab could modify CTC prognostic value. CEC variations to predict benefit of anti-angiogenic treatment is still controversial. Predictive markers for response to bevacizumab combined to chemotherapy in MBC remains a clinical unmet need. Patients &amp; methods: The French cohort COMET is a prospective study including first line HER2 negative patients (pts) receiving weekly paclitaxel and bevacizumab according to EMA approved combination. The aim of this cohort is to evaluate clinical, biological and radiological parameters associated with pts outcome (CTC, CEC, serum markers, ctDNA, pharmacogenomic polymorphisms, metabolomic parameters, visceral fat assessed by initial CTscan, serum estradiol level, and quality of life). We present here the first planned analysis on 203 pts evaluated for CTC and CEC using the FDA cleared CellSearch method. Results: For CTC substudy, 211 patients were included from 09/2012 to 5/2014. Median follow-up is 24 months. Median PFS was 10 months (CI95 9-12) and response rate was 57%. Median OS was not reached. 203 patients were evaluable for both CTC and CEC at baseline and first day of second cycle of CT (D1C2). At baseline, 97/203 (48%) pts had ≥ 5 CTC (median 4 (range 0-30,000). Median number CEC was 21 (0-2231) at baseline and 22 (1-881) at D1C2. LDH, CEA, CA15.3 and CYFRA 21 were above normal at baseline in 44%, 46%, 73% and 71% of the cases respectively. CTC level was not correlated with any patients' characteristics except a number of metastatic site &amp;gt;3. After one cycle of chemotherapy (D1C2) 37 pts (22%) had still ≥ 5 CTC: 36 pts with initial high level and only one patient with low CTC at baseline had increased CTC above 5. Prognostic factors for PFS at univariate analysis were visceral disease, number of metastatic sites (&amp;gt; 3), triple negative status, LDH, CTC level at baseline and CTC level after one cycle of chemotherapy (D1C2). None of serum marker nor CEC level at baseline or any variations had prognostic value. In multivariate analysis for PFS, CTC level after one cyle of chemotherapy predicts poor outcome Table 1 Multivariate analysis for PFSPts' characteristicsnRRCI 95%p-valueNumber of metastatic sites &amp;lt;3 sites971 &amp;gt;=3 sites1001.65[1.13 ; 2.41]0.010CTC D1C2 &amp;lt; 51321 &amp;gt;= 5372.17[1.43 ; 3.29]&amp;lt;0.001Hormonal status Luminal (HR+)1531 Triple negative372.86[1.85 ; 4.54]&amp;lt;0.001 Conclusion: We confirm in a large prospective series the lack of clinical validity of CEC to predict response to an antiangiogenic based treatment in MBC. A persistent elevated level of CTC after just one cycle of chemotherapy is a very strong and independent marker of poor outcome in a homogeneously bevacizumab-treated cohort of MBC patients. This marker could be used to stopped earlier an inefficient and costly treatment. Citation Format: Pierga J-Y, Tredant O, Chevrier M, Dubot C, Lorgis V, Romieu G, Goncalves A, Debled M, Levy C, Ferrero J-M, Jouannaud C, Luporsi E, Mouret-Reynier M-A, Dalenc F, Berger F, Lemonnier J, Proudhon C, Bidard F-C. Circulating tumor cells (CTC) and endothelial cells (CEC) prognostic value in HER2 negative metastatic breast cancer patients treated with first line weekly paclitaxel and bevacizumab: First results of a prospective cohort from the French Breast Cancer InterGroup Unicancer (UCBG): COMET study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-02.

Abstract P6-07-21: High reduction of circulating tumour cells in HER2-negative locally advanced or metastatic breast cancer (LAMBC) patients treated with eribulin as third line chemotherapy (ONSITE study)

Abstract Background Circulating tumour cells (CTC) may be a good biomarker to guide the management of patients with LAMBC (Cristofanilli, NEJM 2004). Detection of CTCs in peripheral blood is a simple procedure. Eribulin monotherapy has shown improved survival in patients with LAMBC that has progressed after 2 or 3 chemotherapy regimens (EMBRACE study). The aims of this study were to evaluate the kinetics of CTCs before and after eribulin treatment, and its correlation with clinical outcomes. Methods Patients received eribulin (1.23 mg/m2 on days 1 and 8 of every 21-day cycle) as third-line therapy until progression, unacceptable toxicity or withdrawal. CTCs were measured in 7.5ml of blood at baseline and after the second cycle of treatment. Cell counting was performed using the CellSearch System, Veridex. Cox proportional hazards regression modeling was used to identify independent prognostic factors for survival endpoints. Results Out of 59 eligible women (mean age 57.7 years), 58 (98.3%) had received previous taxanes and/or anthracyclines. Nearly all (98.3%) had HER2-negative tumors, 72% were positive for estrogens, 21% were triple-negative; 64.4% had liver metastasis and 57.6% bone metastasis. The mean number of administered cycles was 6.9 ± 5.4. Follow-up was performed in 54 patients, with 18.5% (n=10) partial response, 42.6% (n=23) stable disease, and 38.9% (n=21) progressive disease. Clinical benefit was achieved in 33 patients (61.1%). Median progression-free survival (PFS) was 5.13 months (95% CI 3.23, 8.90) and median overall survival (OS) was 13.6 months (95% CI 11.8, not reached). CTC levels were measured in 50 patients. The mean number of CTCs at baseline was 16.8 (IQR 0-21) and on cycle 2, 5.4 (IQR 0-8.5), p&amp;lt;0.001. In patients with CTC ≥ 5 at baseline, 52.4% reduced their levels to CTC &amp;lt; 5 on cycle 2 (p=0.043). No significant differences (p=0.066) were found in PFS when baseline CTC levels were &amp;lt; 5 or ≥ 5 cells/ml. Statistical differences were, however, found in OS (p= 0.0083). On cycle 2, significant differences were observed for both PFS (p=0.045) and OS (p=0.0129) with CTC levels &amp;lt;5 or ≥ 5 cells/ml. No correlation could be found between CTC levels and the objective response at baseline or on cycle 2. Conclusions Our study suggests there is a significant correlation between levels of CTCs and disease prognosis. Eribulin monotherapy was related to a significant reduction in CTCs. (EudraCT number 2013-001416-30). Keywords eribulin, metastatic, breast cancer, circulating tumour cells. Citation Format: Manso Sánchez L, Moreno Antón F, Izarzugaza Perón Y, Delgado Mingorance I, Borrega García P, Echarri González MJ, Martínez Jáñez N, López González A, Olier Garate C, Ballesteros García A, Chacón López-Muñiz I, Ciruelos Gil EM, García Sáenz JA, Paz-Ares Rodríguez L. High reduction of circulating tumour cells in HER2-negative locally advanced or metastatic breast cancer (LAMBC) patients treated with eribulin as third line chemotherapy (ONSITE study) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-21.

Abstract P1-01-01: Circulating tumor cell number and CTC-endocrine therapy index predict clinical outcomes in ER positive metastatic breast cancer patients: Results of the COMETI Phase 2 trial

Abstract Introduction: Only half of hormone receptor positive (HR+) metastatic breast cancer (MBC) patients (pts) benefit from endocrine therapy (ET). Circulating tumor cells (CTC) are prognostic in pts with MBC using CellSearch® technology. The CTC-endocrine therapy index (CTC-ETI) provides semi-quantitative analyses of CTC-ER (estrogen receptor), BCL2, HER2, and Ki67 expression. We hypothesized that CTC-ETI high (elevated CTC number and/or low expression of ER and BCL2, and high expression of HER2 and Ki-67) might predict resistance to ET in a prospective, multi-institutional clinical trial: COMETI-P2-2012.0 (NCT01701050). Methods: 121 pts with ER+, HER2 negative (-), and progressive MBC after one or more lines of ET or within 12 months (mos) of completing adjuvant ET, who were initiating a new ET, were enrolled after informed consent. CTC and CTC-ETI were determined as previously reported (Paoletti C et al, CCR 2015) at baseline (BL), 1, 2, 3, and 12 mos, and/or at the time of progression. Imaging was performed every 3 mos. Association of CTC levels and CTC-ETI with patient outcomes (progression free survival (PFS); rapid progression (RP) defined as progression within 3 mos) was assessed using logrank and Fisher's exact tests. Trial design estimated 85 PFS and 51 RP events, providing &amp;gt;90% power (2-sided a=0.05); pts with unsuccessful BL CTC-ETI or ineligible were unevaluable. Only baseline (BL) data are reported in this abstract. Results: 32% of enrolled pts had progression within 12 mos of completing adjuvant ET, whereas 40%, 20%, and 8% had 1, 2, ≥3 lines of ET for MBC. CTC-ETI was successfully determined in 93% of pts (90% CI, 88% to 97%). CTC were ≥5 CTC/7.5 ml whole blood in 37/108 (34%) pts evaluable for clinical validity. Elevated CTC was associated with worse PFS (median (m) PFS: 3.3 vs. 5.9 mos; P&amp;lt;0.01). Low, intermediate, and high CTC-ETI were observed in 75 (69%), 6 (6%), and 27 (25%) pts, respectively. CTC-ETI was associated with PFS (logrank P&amp;lt;0.01): pts with low, intermediate, and high CTC-ETI had mPFS of 5.7, 8.5, and 2.8 mos, respectively. In the 96 pts eligible for determination, elevated CTC was associated with RP, (65.6% vs. 42.2%; P=0.05) as was CTC-ETI (P=0.003): 79.2% (95% CI, 57.8% to 92.9%) of pts with high CTC-ETI had RP versus 41.2% (95% CI, 29.4% to 53.8%) with low CTC-ETI; in the small group with intermediate CTC-ETI 1 of 4 pts (25%) had RP. Conclusions: In this multi-institutional, prospective study, CTC-ETI was accurately determined, confirming the previously established analytical validity of the assay, meeting the primary objective of the trial. Elevated CTC and CTC-ETI high compared to low were associated with poor outcomes to ET. CTC-ETI distribution resulted in a small number of patients assigned to the intermediate group, restricting our ability to associate this group with outcomes. These results suggest that CTC-biomarker phenotype and enumeration have clinical validity. CTC-ETI may identify ER+ HER2– MBC pts who are unlikely to benefit from ET and might be better treated with ET in combination with other therapies or proceed to chemotherapy. Further analyses including CTC-ETI at serial time points during ET are planned. Citation Format: Paoletti C, Regan MM, Liu MC, Marcom PK, Hart LL, Smith II JW, Tedesco KL, Amir E, Krop IE, DeMichele AM, Goodwin PJ, Block M, Aung K, Cannell EM, Darga EP, Baratta PJ, Brown ME, McCormack RT, Hayes DF. Circulating tumor cell number and CTC-endocrine therapy index predict clinical outcomes in ER positive metastatic breast cancer patients: Results of the COMETI Phase 2 trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-01.