CellSearch System Research Articles

Epidermal Growth Factor Receptor Status in Circulating Tumor Cells as a Predictive Biomarker of Sensitivity in Castration-Resistant Prostate Cancer Patients Treated with Docetaxel Chemotherapy.

Objective: We examined whether epidermal growth factor receptor (EGFR) expression in circulating tumor cells (CTCs) can be used to predict survival in a population of bone-metastatic castration-resistant prostate cancer (mCRPC) patients treated with docetaxel chemotherapy. Methods: All patients with mCRPC who had experienced treatment failure with androgen-deprivation therapy and had received docetaxel chemotherapy were eligible. CTCs and EGFR expression in CTCs were enumerated with the CellSearch System in whole blood. This system is a semi-automated system that detects and enriches epithelial cells from whole blood using an EpCAM antibody-based immunomagnetic capture. In addition, the EGFR-positive CTCs were assessed using CellSearch® Tumor Phenotyping Reagent EGFR. Results: The median CTC count at baseline before starting trial treatment was eight CTCs per 7.5 mL of blood (range 0–184). There were 37 patients (61.7%) who had ≥5 CTCs, with median overall survival of 11.5 months compared with 20.0 months for 23 patients (38.3%) with <5 CTCs (p < 0.001). A total of 15 patients (40.5%, 15/37) with five or more CTCs were subjected to automated immunofluorescence staining and cell sorting for EGFR protein. Patients with EGFR-positive CTCs had a shorter overall survival (OS) (5.5 months) than patients with EGFR-negative CTCs (20.0 months). CTCs, EGFR-positive CTCs, and alkaline phosphatase (ALP) were independent predictors of overall survival time (p = 0.002, p < 0.001, and p = 0.017, respectively). Conclusion: CTCs may be an independent predictor of OS in CRPC treated with docetaxel chemotherapy. The EGFR expression detected in CTCs was important for assessing the response to chemotherapy and predicting disease outcome.

Vertical Magnetic Separation of Circulating Tumor Cells for Somatic Genomic-Alteration Analysis in Lung Cancer Patients.

Efficient isolation and genetic analysis of circulating tumor cells (CTCs) from cancer patients’ blood is a critical step for clinical applications using CTCs. Here, we report a novel CTC-isolation method and subsequent genetic analysis. CTCs from the blood were complexed with magnetic beads coated with antibodies against the epithelial cell adhesion molecule (EpCAM) and separated vertically on a density-gradient medium in a modified well-plate. The recovery rate of model CTCs was reasonable and the cell purity was enhanced dramatically when compared to those parameters obtained using a conventional magnetic isolation method. CTCs were recovered from an increased number of patient samples using our magnetic system vs. the FDA-approved CellSearch system (100% vs. 33%, respectively). In 8 of 13 cases, targeted deep sequencing analysis of CTCs revealed private point mutations present in CTCs but not in matched tumor samples and white blood cells (WBCs), which was also validated by droplet digital PCR. Copy-number alterations in CTCs were also observed in the corresponding tumor tissues for some patients. In this report, we showed that CTCs isolated by the EpCAM-based method had complex and diverse genetic features that were similar to those of tumor samples in some, but not all, cases.

A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation.

IntroductionAmplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples.MethodsUsing healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4′,6-diamidino-2′-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs.ResultsA method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC HER-2 amplification during treatment against metastatic disease.ConclusionHER-2 amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of HER-2 status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift in HER-2 status who may benefit from treatment adjustments.

Prognostic Impact of HER2 and ER Status of Circulating Tumor Cells in Metastatic Breast Cancer Patients with a HER2-Negative Primary Tumor

BACKGROUND: Preclinical and clinical studies have reported that human epidermal growth factor receptor 2 (HER2) overexpression yields resistance to endocrine therapies. Here the prevalence and prognostic impact of HER2-positive circulating tumor cells (CTCs) were investigated retrospectively in metastatic breast cancer (MBC) patients with a HER2-negative primary tumor receiving endocrine therapy. Additionally, the prevalence and prognostic significance of HER2-positive CTCs were explored in a chemotherapy cohort, as well as the prognostic impact of the estrogen receptor (ER) CTC status in both cohorts. METHODS: Included were MBC patients with a HER2-negative primary tumor, with ≥1 detectable CTC, starting a new line of treatment. CTCs were enumerated using the CellSearch system, characterized for HER2 with the CellSearch anti-HER2 phenotyping reagent, and characterized for ER mRNA expression. Primary end point was progression-free rate after 6 months (PFR6months) of endocrine treatment in HER2-positive versus HER2-negative CTC patients. RESULTS: HER2-positive CTCs were present in 29% of all patients. In the endocrine cohort (n=72), the PFR6months was 53% for HER2-positive versus 68% for HER2-negative CTC patients (P=.23). In the chemotherapy cohort (n=82), no prognostic value of HER2-positive CTCs on PFR6months was observed either. Discordances in ER status between the primary tumor and CTCs occurred in 25% of all patients but had no prognostic value in exploratory survival analyses. CONCLUSION: Discordances regarding HER2 status and ER status between CTCs and the primary tumor occurred frequently but had no prognostic impact in our MBC patient cohorts.

Influence of Circulating Tumour Cells on Production of IL-1α, IL-1β and IL-12 in Sera of Patients with Primary Diagnosis of Breast Cancer Before Treatment.

Circulating tumour cells (CTCs) have been found to be a prognostic marker for reduced disease-free survival (DFS), distant DFS, breast cancer-specific survival, and overall survival (OS) before the start of systemic treatment. Determination of CTCs with the CellSearch System (Veridex, Raritan, NJ, USA) is a valuable but time-consuming and costly method. Therefore, the aim of this study was to evaluate cytokine profiles as a marker for CTC involvement. Patients chosen for this study were defined as women with breast cancer who agreed to participate in the phase I SUCCESS study. CTC analysis, the blood sampling time points, and the methodology were prospectively designed, and the prognostic value of the CTCs was defined as a scientific objective of the study protocol. A total of 100 patients positive for CTCs and an additional 100 patients negative for CTCs were matched into pairs. Matching criteria were histopathological grading, lymph node status, hormone receptor type, TNM classification and survival vs. tumour associated death. Commercial enzyme-linked immunosorbent assay (ELISA) was used to screen the blood serum samples for the Th1 cytokines: interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), IL-1α, IL-1β, IL-2 and IL-18. The correlation of cytokine levels to the matching criteria listed above were analyzed with the Spearman correlation coefficient and the Mann-Whitney-U rank-sum test. The IL-1α level was significantly lower in the CTC-positive patient group (p=0.043) but significantly higher in the CTC-negative progesterone receptor-positive collective (p=0.029). Furthermore, in patients who survived, significantly higher IL-12p40 levels were found in those with lymph node involvement (p=0.041) and those with triple-negative breast cancer (p=0.043). Of patients who died, those with oestrogen receptor-negative disease had higher IL-1α (p=0.050) and higher IL-1β (p=0.034) levels. Moreover, of those who died, those with triple-negative breast cancer had significantly higher IL-1α levels (p=0.033). In patients with grade 2 tumour, patients with HER2/neu expression had significantly higher IFN-γ levels (p=0.031) and those with no lymph node involvement had significantly higher IL-1α levels (p=0.014). In the collective with grade 3 tumour, patients with progesterone receptor-negative disease had significantly higher IL12p70 concentrations (p=0.048), while those with triple-negative breast cancer had lower IL12p40 levels (p=0.033). Regarding CTC involvement, we speculate that IL-1α might be a marker for the release of tumour cells into the circulation and not into the lymphatic system. In addition, IL-1α like IL-1β appears to be related to CTC release in patients with breast cancer.

Detection of circulating tumour cells may add value in endometrial cancer management

ObjectiveTo evaluate the role of circulating tumour cells (CTCs) in patients with endometrial cancer (EC). Study designThis study included 40 patients with a pre-operative diagnosis of high-risk EC between April 2015 and May 2016. Patients were further divided into high-risk (grade 3, non-endometrioid, myometrial invasion ≥1/2 and stage III–IV) and high-intermediate-risk (grade 2–3, endometrioid, myometrial invasion <1/2 and stage I–II) groups according to postoperative pathological results. CTCs were detected using the CellSearch system, and CTC results were correlated with standard clinicopathological characteristics and serum tumour marker CA125/HE4 status using Chi-squared test, continuity correction or Fisher’s exact test. The pharmacodynamic effect was detected after the first cycle of adjuvant therapy. Patients were followed up for 13 months to assess outcomes. ResultsFifteen percent of patients had one or more CTCs. The presence of CTCs was found to be significantly associated with cervical involvement (83.33% vs 11.76%, p=0.00). No significant difference in CTC-positive rates was detected between the high-risk and high-intermediate-risk groups, and no significant correlation was found between CTCs and serum CA125/HE4, either by positive rates or exact serum levels of the conventional tumour markers. No more CTCs were detected after the first cycle of standard chemotherapy in this study, and no distant metastases or recurrence were found in the CTC-positive patients during the follow-up period. ConclusionThe presence of CTCs was correlated with cervical involvement. Early-stage EC patients with CTCs may benefit from additional adjuvant therapies. Assessment of CTCs may be useful in the management of high-risk EC patients.

886P - Circulating tumor cell number predicts time to progression (TTP) in patients with heavily pretreated gynecological cancers treated with selinexor (SEL)

SEL, an oral, first-in-class selective inhibitor of XPO1-mediated nuclear export (SINE), induces nuclear retention and activation of tumour suppressor proteins including p53, BRCA1/2, CDKN2A and pRB. SEL has anti-cancer activity in preclinical models of cervical cancer (CC) & ovarian cancer (OC) and in a phase I clinical study. In an effort to identify markers predictive of disease control with SEL, circulating tumour cells (CTCs) were enumerated during the phase 2 trial from patients (pts) with heavily pretreated OC, CC & endometrial cancer (EC). NCT02025985. Patients with ≥ 2 lines of prior therapy, ECOG PS 0-1, were treated with single agent SEL. At predose C1D1, 7.5 mL of blood was collected in CellSave tubes and CTCs were identified using the Janssen Diagnostics CellSearch System. Intact cells that measured at least 4 microns in size and stained positive for DAPI, EpCAM, and cytokeratin while negative for CD45 were counted as CTCs. CTC were enumerated at predose C1D1 in 47 (33 OC, 8 EC, 6 CC) of 114 patients. To date, 31 pts had <2 CTCs at predose C1D1 with a median days on study of 108 days while 16 pts had >2 CTCs with a median days on study of 56 days (p = 0.01). Ten patients with <2 CTCs at predose showed stable disease for at least 12 weeks and 4 demonstrated partial response by RECIST 1.1. This study demonstrates that the presence of CTCs in pts prior to SEL treatment may correlate with a shorter TTP. Detection of CTC in the peripheral blood of cancer patients has proven feasible and of prognostic value in different neoplasms, including in patients with OC. These results suggest that low CTC count (<2) may also be used to identify pts with heavily pre-treated OC, CC & EC that may benefit from prolonged disease control with single agent oral single agent SEL. Additional studies are planned.

Detection and clinical significance of circulating tumor cells in gastric cancer

The death of patients with gastric cancer is mainly due to its recurrence and metastasis, and circulating tumor cell (CTC) is the necessary condition of metastasis. As liquid biopsy, CTC detection has its certain clinical significance. The detection is required after enrichment because circulating tumor cells are rare. Many enrichment methods have been developed: methods based on physical characteristics of TCT, like density, size and dielectric properties and so on; immunogenicity, like Cell Search System; and microfluidic chip technology. The immunofluorescence is commonly used to identify CTC in gastric cancer and the isolated CTC can also be used for the following analysis on the level of nucleic acid, protein and gene regulation. Detection of CTC in gastric cancer is helpful to judge the prognosis, assess staging, monitor the curative effect and guide the development of drug. There are many challenges for clinical transformation of CTC: the lower enrichment efficiency, the less specific surface markers, the uncertain diagnostic efficiency and so on, but it also has the good research prospect because it is non-invasive, repeatable and can real-time monitor the condition and guide the clinical treatment compared with pathological biopsy. In this paper, the detection and identification methods, and clinical value of CTC in gastric cancer patients are reviewed.

Phenotypic diversity of circulating tumour cells in patients with metastatic castration-resistant prostate cancer.

To use a non-biased assay for circulating tumour cells (CTCs) in patients with prostate cancer (PCa) in order to identify non-traditional CTC phenotypes potentially excluded by conventional detection methods that are reliant on antigen- and/or size-based enrichment. A total of 41 patients with metastatic castration-resistant PCa (mCRPC) and 20 healthy volunteers were analysed on the Epic CTC platform, via high-throughput imaging of DAPI expression and CD45/cytokeratin (CK) immunofluorescence (IF) on all circulating nucleated cells plated on glass slides. To confirm the PCa origin of CTCs, IF was used for androgen receptor (AR) expression and fluorescence in situ hybridization was used for PTEN and ERG assessment. Traditional CTCs (CD45- /CK+ /morphologically distinct) were identified in all patients with mCRPC and we also identified CTC clusters and non-traditional CTCs in patients with mCRPC, including CK- and apoptotic CTCs. Small CTCs (≤white blood cell size) were identified in 98% of patients with mCRPC. Total, traditional and non-traditional CTCs were significantly increased in patients who were deceased vs alive after 18 months; however, only non-traditional CTCs were associated with overall survival. Traditional and total CTC counts according to the Epic platform in the mCRPC cohort were also significantly correlated with CTC counts according to the CellSearch system. Heterogeneous non-traditional CTC populations are frequent in mCRPC and may provide additional prognostic or predictive information.

Association of CA27.29 and Circulating Tumor Cells Before and at Different Times After Adjuvant Chemotherapy in Patients with Early-stage Breast Cancer - The SUCCESS Trial.

Evidence for the prognostic value of circulating tumor cells (CTCs) in early-stage breast cancer is swiftly increasing. An alternative approach for identifying patients at risk for recurrence is based on the detection of the mucin-1 (MUC1)-based tumor marker CA27.29. Here we report the association of these two prognostic markers before and immediately after chemotherapy (CHT), as well as after 2 and 5 years of follow-up. The SUCCESS trial compared fluorouracil, epirubicin and cyclophosphamide followed by docetaxel vs. FEC followed by docetaxel plus gemcitabine, and 2 vs. 5 years of treatment with zoledronic acid in 3,754 patients with node-positive or high-risk node-negative early-stage breast cancer. CA27.29 was measured with the ST AIA-PACK CA27.29 reagent (Tosoh Bioscience, Belgium). The cutoff for CA27.29 positivity was >31 U/ml. CTCs were assessed with the CellSearch System (Veridex, USA). The cutoff for CTC positivity was ≥1 CTC/15 ml whole blood. The relationship between CTC positivity and CA27.29 positivity was assessed based on Chi-square statistics and Cramer's V, which varies from 0 (no association between the variables) to 1 (complete association). Samples for CA27.29 and CTC determinations during follow-up were only drawn from patients that had no relapse. Both CA27.29 and CTC data were available for 1,981, 1,602, 1,159 and 707 patients before, immediately after and at 2 and 5 years after CHT, respectively. Positivity rates for CTC were 21.3%, 22.8%, 18.6% and 8.5%, respectively. CA27.29 was positive in 7.9%, 21.0%, 2.8%and 7.5%, respectively. Positivity for both CA27.29 and CTC was found in 2.4%, 4.2%, 0.7% and 1.8% of patients, respectively. The association between CA27.29 and CTC was significant but weak before CHT (p=0.0015; Cramer's V=0.063) and 5 years after CHT (p<0.001; Cramer's V=0.164), and not significant immediately after CHT (p=0.162; Cramer's V=0.035) and 2 years after (p=0.349; Cramer's V=0.028). We showed that CTC and CA27.29 positivity were significantly, but only weakly associated before CHT and 5 years after CHT, while no significant association was found immediately or 2 years after CHT during the course of early-stage breast cancer. It, therefore, seems reasonable to further evaluate the prognostic value of CTCs and CA27.29 as a combined prognostic test of two potentially independent markers that might provide complementary prognostic information.

The preliminary role of circulating tumor cells obtained from the hepatic or portal veins in patients with hepatobiliary–pancreatic cancer

Metastases, which result from circulating tumor cells (CTC) that have detached from the primary cancer and survived in distant organs, cause the majority of cancer deaths. The present study aimed to assess the prognostic relevance of CTC in patients with hepatobiliary–pancreatic cancer. A total of 30 consenting patients were enrolled. Pre-resection blood samples were obtained from the hepatic or portal veins of patients, respectively. CTCs were analyzed using the CellSearch system and isolated using antibodies against the epithelial cell adhesion molecule and cytokeratin. Patients were separated into 2 groups based on their CTC counts. The CTC-positive group included patients with baseline CTC counts >10/7.5 ml. Of the 30 patients, 12 (40 %) were considered CTC positive. CTC-positive patients had a shorter 1‑year progression-free survival (PFS) than CTC-negative patients (71.1 % vs. 36.4 %, p = 0.0324); however, no difference was observed in the 3‑year overall survival (47.1 % vs. 45.5 %, p = 0.7576). In particular, CTC-positive pancreatic carcinoma patients had a shorter 1‑year PFS (22.2 % vs. 0.0 % for CTC-negative, p = 0.0018). Elevated CTC from hepatic or portal veins might predict a shorter PFS in patients with hepatobiliary–pancreatic cancer, particularly those with pancreatic carcinoma.

Real-time HER2 status detected on circulating tumor cells predicts different outcomes of anti-HER2 therapy in histologically HER2-positive metastatic breast cancer patients.

BackgroundThis study was initiated to investigate the difference in HER2 status between tumor tissue and circulating tumor cells (CTCs), as well as the predictive value of CTC HER2 status for predicting the outcomes of anti-HER2 therapy in histologically HER2-positive metastatic breast cancer (MBC) patients.MethodsHER2 expression on CTCs was detected using a CellSearch system within 7 days before a new line of anti-HER2 therapy was begun. According to the criterion proposed in our previous report, patients were defined as CTC HER2-positive or -negative. After close follow-up, the correlation between CTC HER2 status and the outcome of the treatment was evaluated by statistical analysis.ResultsCTCs were detected in 57.4 % (58/101) of the patients. Notably, 62.1 % (36/58) of these patients had an inconsistent HER2 status between their tissue and CTCs. The discordant rate may correlate with the time interval between histological and CTC HER2 testing and is more likely to occur in the subgroup of patients with an interval of > 1 year than in those with an interval < 1 year (70.7 % vs. 41.2 %, P = 0.043). For PFS, positive HER2 status on CTCs was shown to be a valuable predictor, both in univariate (HR = 0.321, 95%CI, 0.156–0.62, P = 0.0011) and multivariate (HR = 0.383, 95%CI, 0.166–0.831, P = 0.019) Cox regression analysis. Meanwhile, Kaplan-Meier survival curves revealed that the median PFS of CTC HER2-positive patients was significantly longer than CTC HER2-negative ones (8.5 vs. 3.5 months, P < 0.001).ConclusionsHER2 status on CTCs was different from that of tumor tissues and predicted a different outcome of the patients’ anti-HER2 therapy. This difference may be correlated with the time interval between tissue and CTC HER2 testing, indicating the necessity of real-time HER2 analysis for histologically HER2-positive MBC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2578-5) contains supplementary material, which is available to authorized users.

Abstract 2243: Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts)

Abstract Background: NSCLC exhibits intratumor heterogeneity, with subpopulations of cells undergoing epithelial-mesenchymal transition. Such CTCs from NSCLC pts may be missed by the EpCAM-based CELLSEARCH® system (CS). The label-free ClearCell® FX inertial microfluidic system (FX) isolates CTCs based on size and inertia and may lead to more accurate CTC capture. PD-1/PD-L1 inhibitors have shown benefit in PD-L1+ NSCLC pts, but responses are still seen in PD-L1- pts, suggesting limitations in tumor PD-L1 testing. Also, PD-L1 testing on CTCs may be more practical than tumor rebiopsies and may provide insights into cancer heterogeneity. Methods: FX was validated with EpCAM-high and EpCAM-low cancer cell lines labeled with CellTracker dyes spiked into healthy volunteer (HV) blood for repeatability and reliability. Enriched cells were detected with the automated Bioview Duet imaging system for recovery (%). Identical spiking studies were conducted on CS for comparison. Antibodies for 5-color immunofluorescence (IF) (CK, CD45, DAPI, TTF1 [to detect lung adenocarcinoma cells], PD-L1) were optimized on EpCAM-high and EpCAM-low cell lines. 8ml of blood from NSCLC pts were enriched with FX for 5-color IF characterization. 7.5ml of blood from the same pts taken at identical timepoints were enumerated with CS for comparison. Blood was obtained from HV for CTC enumeration on FX and CS as controls. Results: Cell recovery of EpCAM-high cancer cell lines using FX produced similar counts to CS, e.g. lung NCI-H2066 cells: FX 64.9%±4.5 vs CS 74.4%±9.2 (p = 0.05); lung H1975 cells: FX 74.1%±11.9 vs CS 74.7%±10.7 (p = 0.91). In contrast, for EpCAM-low cells, a significant difference in cell recovery between FX and CS was seen, e.g. lung A549 cells: FX 59.3%±5.8 vs CS 26.1%±7.6 (p&amp;lt;0.0001); and prostate PC3 cells: FX 68.6%±7.4 vs CS 35.5%±6.9 (p&amp;lt;0.0001). CTCs were detected in 19/21 pts with progressing stage IV NSCLC (adenocarcinoma) (mean 57; range 4-304) using FX, vs 7/21 pts (mean 7; range 1-38) using CS. CTC counts were higher with FX vs CS in 19/21 (90%) NSCLC pts, p = 0.012. No CTCs were detected in HV with FX (n = 10) and CS (n = 5). After FX enrichment, CTCs from 17/21 NSCLC pts were available for 5-color IF testing. Of these 17 pts, 15 had TTF1+ CTCs. All 15 pts with TTF1+ CTCs had ≥1 PD-L1+ CTC. 6/15 (40%) pts only had PD-L1+ TTF1+ CTCs, with no PD-L1- TTF1+ CTCs. PD-L1 CTC heterogeneity was seen in the other 9/15 pts, with co-existing PD-L1+ and PD-L1- TTF1+ CTCs. 6 of these 9 pts had more PD-L1+ TTF1+ CTCs than PD-L1- TTF1+ CTCs. Conclusions: FX resulted in consistently high cell recovery rates regardless of EpCAM status. Higher CTC counts were isolated with FX vs CS in 90% of NSCLC pts. 40% of NSCLC adenocarcinoma pts had PD-L1+ TTF1+ CTCs, but PD-L1 CTC heterogeneity was seen in other pts, which may in part explain differences in responses to PD-1/PD-L1 inhibitors in NSCLC. Citation Format: Zai Ahmad, Jen Fraser-Fish, Rajiv Kumar, Bernadette Ebbs, Gemma Fowler, Penny Flohr, Mateus Crespo, Sanjay Popat, Jaishree Bhosle, Udai Banerji, Mary O’Brien, Johann S. de Bono, Timothy A. Yap. Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts). [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 2243.

Abstract 2244: Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients

Abstract Background and Purpose: Circulating tumor cells (CTCs) are relatively rare cells defined as tumor cells circulating in the peripheral blood of patients with solid tumors. Diagnosis utilizing CTCs is expected to help guide decision-making for precision cancer medicine. We developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Here we evaluated its performance using preclinical spike-in model and blood samples from non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients. Material and method: The automated MCA system consists of components such as chambered cartridge containing micro metal filter, reagent and waste reservoirs, and peristaltic pump. To evaluate the recovery of CTCs, preclinical experiments using NSCLC cells, NCI-H820, A549, NCI-H441 and NCI-H23 spiked into peripheral whole blood from healthy volunteers were performed. For clinical evaluation, 6 mL of peripheral whole blood was collected from 50 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Samples were collected in an EDTA-containing tube and were processed within 3 hours of blood draw. Recovered cells on the filter were then fixed, permeabilized, and stained automatically and high-resolution fluorescent images were obtained using fluorescence microscope. We defined CTC as DAPI-positive, cytokeratin-positive and CD45-negative cell. Results: Results of the preclinical study showed that up to 90% of spiked-in tumor cells were recovered, confirming that the detection sensitivity by this automated device is on par with that by previous manual detection procedure. Demographics of 50 lung cancer patients enrolled in clinical study were as follows: median age 72 (range, 48 to 85); male 66%; stage III/IV 12/88%; NSCLC/SCLC 78/22%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 / 6 mL, respectively. Three or more CTCs were detected in 71% of patients with advanced lung cancer (39 out of 50) and five or more CTCs were detected in 52% of patients (26 out of 50) (median CTC count 13.5). Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those with intrathoracic metasitasis (median CTC count, 8 versus 4, p = 0.058). A head-to-head comparison between CellSearch system and our system was conducted in NSCLC patients, showing the superiority of our system (median CTC count, 0 versus 11.25, p = 0.0001, n = 17). Conclusions Our results suggest that the automated MCA device has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This device also enables higher throughput owing to its automated procedure. Further clinical evaluation including the detection of PD-L1 expression will be performed in an expansion cohort. Citation Format: Satomi Yagi, Yasuhiro Koh, Hiroaki Akamatsu, Woong Kim, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients. [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 2244.

Abstract 1532: The isolation of CTC from diagnostic leukapheresis

Abstract Introduction At present, the CellSearch system is the only validated method for the detection of circulating tumor cells (CTC) that has been cleared by the U.S. Food and Drug Administration. This system, designed for the enumeration of CTC in 7.5 mL of blood, detects CTC based on their expression of EpCAM and cytokeratins and negativity for CD45. However, the number of CTC that are detected in patients with metastatic carcinomas is in most cases too small to reliably determine tumor heterogeneity and to be representative as a ‘liquid biopsy’. Our aim is to identify and isolate a sufficient number of circulating tumor cells in virtually all metastatic cancer patients to enable their characterization and to represent a real-time liquid biopsy. For this purpose we used Diagnostic LeukApheresis (DLA) to increase the blood volume to be analyzed. We developed several techniques to isolate CTC from DLA to enable a multicenter comparison of CTC detection in DLA products. Methods DLAs were performed for ∼1 hour to obtain 40 mL of product containing ∼4 x10⁁9 mononuclear cells representing ∼1 liter of blood. Using CellSearch a maximum of 2 mL of DLA could be processed for EpCAM+ CTC (Fisher et al. doi: 10.1073/pnas.1313594110) and EpCAM- CTC (de Wit et al doi: 10.1038/srep12270). Using filtration through microsieves with 5 μm pores a maximum of 1.0 mL of DLA could be processed. To process 18 mL DLA product protocols were developed for leukocyte depletion using RosetteSep™ (StemCell Technologies, USA) and for EpCAM selection using an anti-EpCAM coated column (Leukocare AG). All enriched cell fractions were stained using CD45 PerCP, Cytokeratins PE and the nuclear dye DAPI, followed by fluorescence microscopy scanning and analysis. Results Leukocyte depletion using the RosetteSep™ CTC Enrichment cocktail was first optimized using small sample volumes (1 mL) spiked with cells from cancer cell lines. Depletion of leukocytes ranged from 3.1 to 3.9 logs with an average recovery of spiked cancer cells of 50-60%. Isolation of CTC expressing EpCAM was pursued using anti-EpCAM coated columns and optimized for selection and release of EpCAM expressing cells by passage of cells from cancer cell lines through the column resulting in 34-100% recovery. Both procedures were scaled up to enable processing of 18 mL of DLA. Leukocytes were depleted using RosetteSepTM by 3.1 - 3.9 logs whereas with anti-EpCAM columns only 1.7 - 1.8 logs depletion were reached. Using RosetteSepTM 21% and with the anti-EpCAM coated columns 2% of the tumor cells spiked into 18ml DLA were recovered. Conclusion Standard operating procedures were developed to isolate CTC in DLA's from breast, prostate cancer and lung cancer patients for evaluation and comparison in the EU sponsored consortiums CTCTrap (www.utwente.nl/tnw/ctctrap/) and CANCER-ID (www.CANCER-ID.eu). Isolation of EpCAM expressing CTC using the anti-EpCAM coated columns will need further optimization before it can proceed to multicenter comparison. Citation Format: Kiki C. Andree, Anouk Mentink, Martin Scholz, Roland Kirchner, Rui P. Neves, Christiane Driemel, Rita Lampignano, Hans Neubauer, Dieter Niederacher, Tanja Fehm, Wolfram T. Knoefel, Johannes C. Fischer, Nikolas H. Stoecklein, Leon WMM Terstappen. The isolation of CTC from diagnostic leukapheresis. [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 1532.

Abstract 501: PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer

Abstract Background: Programmed cell Death receptor Ligand 1 (PD-L1) is a very promising biomarker for the selection of patients for cancer immunotherapy. We recently developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA. The aim of the present study was to study the expression of PD-L1 in CTCs from breast cancer patients with verified metastasis. Methods: We quantified the expression of PD-L1 mRNA transcripts in EpCAM-positive CTCs, by using our recently developed RT-qPCR assay, based on the following procedure: i) immunomagnetic enrichment of EpCAM-positive CTCs from 20mL of peripheral blood, ii) total RNA isolation iii) cDNA synthesis and iv) RT-qPCR for PD-L1. PD-L1 expression in respect to the expression of B2M as a reference gene, was normalized using the 2-ΔΔCt approach. Peripheral blood samples (20mL) were obtained from 22 breast cancer patients with verified metastasis and 14 healthy donors. Results: According to our results, 11/22 (50%) of metastasis-verified breast cancer patients were found to be positive for PD-L1 overexpression in CTCs. These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing. Our results are in concordance with a recent study by Mazel et al (Mol Oncol 2015), that by using the CellSearch(®) system found PD-L1((+)) CTCs in 11/16(68.8%) patients with metastatic breast cancer. Conclusion: This is the first time that a quantitative RT-qPCR molecular assay is used for the evaluation of PD-L1expression levels in EpCAM-positive CTCs in metastatic breast cancer patients. The assay is closed-tube, quantitative, highly specific and sensitive, and high-throughput. We are currently evaluating the assay in a large number of clinical samples. Citation Format: Martha Zavridou, Areti Strati, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. PD-L1 expressing circulating tumor cells (CTCs) in patients with breast 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 501.

Abstract 2254: Variations in the epithelial-mesenchymal transition (EMT) program by non-small cell lung cancer (NSCLC) circulating tumor cells (CTCs) do not influence survival

Abstract Background By definition, CTCs must undergo the EMT to enter the bloodstream where they can be isolated from cancer patients for translational and biological study. Here we examined survival patterns in relation to CTC EMT expression in different molecular subgroups of NSCLC. Methods 125 patients (pts) with advanced treatment-naïve stage IIb-IV NSCLC were prospectively included for CellSearch CTC analysis as part of the Gustave-Roussy MSN study. Patients signed an informed consent for one CellSave tube prior to chemotherapy. Anti-vimentin (vim) antibody was added to the free channel in the CellSearch system for examination of EMT. Association of CTC number with clinical characteristics were assessed using Fisher's exact, Mann-Whitney and chi-squared tests. Kaplan-Meier method and log-rank tests were used to analyse progression-free survival (PFS) and overall survival (OS) of vimentin-expression in molecular subgroups of NSCLC. Results 51/125 pts (40.8%) were CTC-positive by CellSearch (≥2 CTC), and 29/125 (23.2%) patient samples contained at least 1 vimentin-positive (+) CTC. 19/76 (25%) adenocarcinomas were KRAS mutated. In the KRAS subgroup, 0/19 patient samples (0%) from pts with mutated KRAS contained vim+ CTC, compared to 17/48 pts (35.4%) with wild-type (WT) KRAS (p = 0.0027). There was also a significantly higher overall number of vim+ CTCs in pts with KRAS WT cancer compared to KRAS mutated cancer (mean 0 vs 1.63, respectively, p = 0.0035). For KRAS WT pts, no survival difference was evident between vim+ and vim- subgroups in terms of PFS or OS. 21/89 adenocarcinomas were EGFR mutated (23.6%). In this subgroup, statistically higher numbers of EGFR mutated pts with both vim+ and total CTCs were observed compared to EGFR WT pts (vim+ CTC: 9/21 EGFR mutated vs 9/56 EGFR WT, p = 0.0134; total CTC: 12/21 EGFR mutated vs 18/56 EGFR WT, p = 0.0451). Similarly, there was a significantly higher overall number of vim+ CTCs in pts with EGFR mutated cancer compared to pts with EGFR WT cancer (mean 1.24 vs 0.91, respectively, p = 0.0189), but no PFS or OS difference was evident between vim+ and vim- subgroups in EGFR mutated pts. 14/71 (19.7%) adenocarcinomas were ALK rearranged, with further results pending. Conclusions At baseline stage IIIb-IV disease, there are statistically fewer vim+ CTCs (and pts with vim+ CTCs) in KRAS mutated NSCLC, while vim+ CTC (and vim+ CTC pts) are statistically higher in EGFR mutated NSCLC. Despite this differential CTC vimentin expression between molecular subgroups, no PFS or OS difference is evident between vim+ and vim- patients. This biological variation coupled to a lack of overall clinical impact favours the hypothesis that each individual CTC is a highly plastic cell that can cover a range of EMT expression. Citation Format: Colin R. Lindsay, Vincent Faugeroux, Emma Pailler, Maria-Virginia Bluthgen, Chloé Pannet, Maud Ngo-Camus, Guillaume Bescher, Fanny Billiot, Jordi Remon, Philippe Vielh, David Planchard, Jean-Charles Soria, Benjamin Besse, Françoise Farace. Variations in the epithelial-mesenchymal transition (EMT) program by non-small cell lung cancer (NSCLC) circulating tumor cells (CTCs) do not influence survival. [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 2254.

Prognostic impact of circulating tumor cell apoptosis and clusters in serial blood samples from patients with metastatic breast cancer in a prospective observational cohort.

BackgroundPresence of circulating tumor cells (CTCs) is a validated prognostic marker in metastatic breast cancer. Additional prognostic information may be obtained by morphologic characterization of CTCs. We explored whether apoptotic CTCs, CTC clusters and leukocytes attached to CTCs are associated with breast cancer subtype and prognosis at base-line (BL) and in follow-up (FU) blood samples in patients with metastatic breast cancer scheduled for first-line systemic treatment.MethodsPatients with a first metastatic breast cancer event were enrolled in a prospective observational study prior to therapy initiation and the CellSearch system (Janssen Diagnostics) was used for CTC enumeration and characterization. We enrolled patients (N = 52) with ≥5 CTC/7.5 ml blood at BL (median 45, range 5–668) and followed them with blood sampling for 6 months during therapy. CTCs were evaluated for apoptotic changes, CTC clusters (≥3 nuclei), and leukocytes associated with CTC (WBC-CTC, ≥1 CTC + ≥1 leukocytes) at all time-points by visual examination of the galleries generated by the CellTracks Analyzer.ResultsAt BL, patients with triple-negative and HER2-positive breast cancer had blood CTC clusters present more frequently than patients with hormone receptor-positive cancer (P = 0.010). No morphologic characteristics were associated with prognosis at BL, whereas patients with apoptotic CTCs or clusters in FU samples had worse prognosis compared to patients without these characteristics with respect to progression-free (PFS) and overall survival (OS) (log-rank test: P = 0.0012 or lower). Patients with apoptotic or clustered CTCs at any time-point had impaired prognosis in multivariable analyses adjusting for number of CTCs and other prognostic factors (apoptosis: HROS = 25, P < 0.001; cluster: HROS = 7.0, P = 0.006). The presence of WBC-CTCs was significantly associated with an inferior prognosis in terms of OS at 6 months in multivariable analysis.ConclusionsPatients with a continuous presence of apoptotic or clustered CTCs in FU samples after systemic therapy initiation had worse prognosis than patients without these CTC characteristics. In patients with ≥5 CTC/7.5 ml blood at BL, morphologic characterization of persistent CTCs could be an important prognostic marker during treatment, in addition to CTC enumeration alone.Clinical Trials (NCT01322893), registration date 21 March 2011Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2406-y) contains supplementary material, which is available to authorized users.

Molecular characteristics of circulating tumor cells resemble the liver metastasis more closely than the primary tumor in metastatic colorectal cancer

BackgroundCTCs are a promising alternative for metastatic tissue biopsies for use in precision medicine approaches. We investigated to what extent the molecular characteristics of circulating tumor cells (CTCs) resemble the liver metastasis and/or the primary tumor from patients with metastatic colorectal cancer (mCRC).ResultsThe CTC profiles were concordant with the liver metastasis in 17/23 patients (74%) and with the primary tumor in 13 patients (57%). The CTCs better resembled the liver metastasis in 13 patients (57%), and the primary tumor in five patients (22%). The strength of the correlations was not associated with clinical parameters. Nine genes (CDH1, CDH17, CDX1, CEACAM5, FABP1, FCGBP, IGFBP3, IGFBP4, and MAPT) displayed significant differential expressions, all of which were downregulated, in CTCs compared to the tissues in the 23 patients.Patients and MethodsPatients were retrospectively selected from a prospective study. Using the CellSearch System, CTCs were enumerated and isolated just prior to liver metastasectomy. A panel of 25 CTC-specific genes was measured by RT-qPCR in matching CTCs, primary tumors, and liver metastases. Spearman correlation coefficients were calculated and considered as continuous variables with r=1 representing absolute concordance and r= -1 representing absolute discordance. A cut-off of r>0.1 was applied in order to consider profiles to be concordant.ConclusionsIn the majority of the patients, CTCs reflected the molecular characteristics of metastatic cells better than the primary tumors. Genes involved in cell adhesion and epithelial-to-mesenchymal transition were downregulated in the CTCs. Our results support the use of CTC characterization as a liquid biopsy for precision medicine.

Serum HER2 extracellular domain levels and HER2 circulating tumor cell status in patients with metastatic breast cancer.

To shed light on the clinical role of HER2 status in serum as extracellular domain (ECD) and corresponding circulating tumor cells (CTCs) in metastatic breast cancer patients. 68 patients were analyzed. Serum HER2 was determined by ADVIA Centaur(®) Serum HER2 test. CellSearch System was performed for CTC quantification. HER2 was overexpressed in 21 primary tumors. In total, 19 patients had ECD >15 ng/ml (the cut-off used), 48 patients had at least one CTC. ECD positivity was associated with CTC number (p = 0.01), HER2-positive CTC (p = 0.01) and the ratio HER2-positive CTC/total CTC (p = 0.02). ECD was not associated with survival. ECD in combination with HER2 CTC status would deserve further investigation in larger series for addressing its putative prognostic relevance.