Apoptotic Circulating Tumor Cells Research Articles

Relationship of Ki-67 index in biopsies of metastatic breast cancer tissue and circulating tumor cells (CTCs) at the time of biopsy collection

BackgroundThe proliferation marker Ki-67 is a major pathological feature for the description of the state of disease in breast cancer. It helps to define the molecular subtype and to stratify between therapy regimens in early breast cancer and helps to assess the therapy response. Circulating tumor cells (CTCs) are a negative prognostic biomarker for progression free (PFS) and overall survival (OS) in patients with metastatic breast cancer. Therefore, the CTC count is often described as surrogate for the tumor burden. Both, decrease of Ki-67 and CTC count are considered as evidence for therapy response. The presented work analyzed the correlation between the Ki-67 indices of metastatic tissue biopsies and CTC counts in biopsy time-adjacent peripheral blood samples.Patients and methodsBlood samples from 70 metastatic breast cancer patients were obtained before the start of a new line of systemic therapy. CTCs were enumerated using CellSearch® (Menarini Silicon Biosystems, Bologna, Italy) whereas intact CTCs (iCTCs) and non-intact or apoptotic CTCs (aCTCs) were distinguished using morphologic criteria. The proportion of cells expressing Ki-67 was evaluated using immunohistochemistry on biopsies of metastases obtained concurrently with CTC sampling before the start of a new line of systemic therapy.Results65.7% of patients had a Ki-67 index of > 25%. 28.6% of patients had ≥ 5, 47.1% ≥ 1 iCTCs. 37.1% had ≥ 5, 51.4% ≥ 1 aCTCs. No correlation was shown between Ki-67 index and iCTC and aCTC count (r = 0.05 resp. r = 0.05, Spearman’s correlation index). High CTC-counts did not coincide with high Ki-67 index. High Ki-67, ≥ 5 iCTCs and aCTCs are associated with poor progression free (PFS) and overall survival (OS).ConclusionCTCs and Ki-67 are independent prognostic markers in metastatic breast cancer. High Ki-67 in metastatic tumor tissue is not correlated to high iCTC or aCTC counts in peripheral blood.

The Novel Association of Early Apoptotic Circulating Tumor Cells with Treatment Outcomes in Breast Cancer Patients.

Stemness and epithelial–mesenchymal plasticity are widely studied in the circulating tumor cells of breast cancer patients because the roles of both processes in tumor progression are well established. An important property that should be taken into account is the ability of CTCs to disseminate, particularly the viability and apoptotic states of circulating tumor cells (CTCs). Recent data demonstrate that apoptosis reversal promotes the formation of stem-like tumor cells with pronounced potential for dissemination. Our study focused on the association between different apoptotic states of CTCs with short- and long-term treatment outcomes. We evaluated the association of viable CTCs, CTCs with early features of apoptosis, and end-stage apoptosis/necrosis CTCs with clinicopathological parameters of breast cancer patients. We found that the proportion of circulating tumor cells with features of early apoptosis is a perspective prognosticator of metastasis-free survival, which also correlates with the neoadjuvant chemotherapy response in breast cancer patients. Moreover, we establish that apoptotic CTCs are associated with the poor response to neoadjuvant chemotherapy, and metastasis-free survival expressed at least two stemness markers, CD44 and CD133.

Cut-Off Analysis of CTC Change under Systemic Therapy for Defining Early Therapy Response in Metastatic Breast Cancer.

Detection of circulating tumor cells (CTC) can distinguish between aggressive and indolent metastatic disease in breast cancer patients and is thus considered an independent, negative prognostic factor. A clear decline in CTCs is observed in patients who respond to systemic therapy. Nevertheless, CTCs can decrease in patients experiencing disease progression during systemic therapy, too. This study aims to determine the differences between CTC decline in patients responding to therapy and those in whom disease is progressing. Therefore, CTC values were compared at the start and after one cycle of a new line of systemic therapy. In all, 108 initially CTC-positive patients (with ≥5 intact CTCs in 7.5 mL blood) were enrolled in this study and intact and apoptotic CTCs were measured via the CellSearch® system. A cut-off analysis was performed using Youden’s J statistics to differentiate between CTC change in the two groups. Here, 64 (59.3%) patients showed stable disease or partial response vs. 44 (40.7%) presenting disease progression. Median overall survival was 23 (range: 4–92) vs. 7 (2–43) months (p < 0.001). Median intact CTC count at enrollment was 15.0 (5–2760) vs. 30.5 (5–200000) cells (p = 0.39) and 2.5 (0–420) vs. 8.5 (0–15000) cells after one cycle of systemic therapy (p = 0.001). Median apoptotic CTC count at enrollment was 10.5 (0–1500) vs. 9 (0–800) cells (p = 0.475) and 1 (0–200) vs. 3 (0–250) cells after one cycle of systemic therapy (p = 0.01). A 50% reduction in baseline apoptotic CTC count represents the optimal cut-off to differentiate between therapy response and disease progression. An apoptotic CTC reduction of ≤10% is 74% specific for early disease progression.

Dynamics of Circulating Tumor Cells Early After Targeting Therapy to Human EGFR-mutated Lung Cancers and HER2 Gene-amplified Gastric Cancers in Mice.

Dynamics of circulating tumor cells (CTCs) after molecular targeting therapy remain unclear. We examined changes in CTC numbers and morphology early after targeting therapy in EGFR-mutated PC-9 human lung cancer and HER2-gene amplified GLM-1 gastric cancer mouse CTC models using a cytology-based semi-automated CTC detection platform. Erlotinib and T-DM1 inhibited cell growth mainly by induction of apoptosis in vitro. The number of CTCs detected 5-10 days after targeting therapy in mice was significantly increased compared to CTC numbers before therapy. The increased CTCs after therapy consisted of apoptotic CTCs and viable CTCs. This heterogeneous population of CTCs reflects well the cell population of the primary tumor disrupted by therapy. CTCs can be mobilized from the primary tumor due to tissue disruption in acute response to targeting therapy, suggesting potential usefulness of CTC monitoring as a predictor of therapeutic response in the clinical settings.

Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products.

Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients. DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture. Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture. The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

Molecular Subtype Conversion between Primary and Metastatic Breast Cancer Corresponding to the Dynamics of Apoptotic and Intact Circulating Tumor Cells.

The presence of circulating tumor cells (CTCs), detected as a form of liquid biopsy is associated with poor survival in both early and metastatic breast cancer. Monitoring tumor biology based on intrinsic subtypes delivers treatment-relevant information on the heterogeneity or biomarker conversion between primary and metastatic tumors. This study aimed to correlate the change of the apoptotic and intact CTC counts with mRNA-assessed intrinsic subtype change. Thirty-four breast cancer patients with available triplets of primary tumors, distant metastasis biopsies and data on intact and apoptotic CTC dynamics were included in the analysis. The intrinsic subtype was determined per RT-qPCR quantification of the gene expression ESR1, PGR, ERBB2 and MKI67. Both luminal (p = 0.038) and triple negative (p = 0.035) patients showed a significant downregulation of apoptotic CTCs. Repeated biopsies of distant metastatic sites, as well as determining a potential shift of the intrinsic subtype, combined with data on intact and apoptotic CTC dynamics from liquid biopsies might help personalize systemic therapy and generate additional surrogate markers for successful systemic therapy.

Detection of Apoptotic Circulating Tumor Cells Using in vivo Fluorescence Flow Cytometry.

Most cancer patients die from metastatic disease as a result of a circulating tumor cell (CTC) spreading from a primary tumor through the blood circulation to distant organs. Many studies have demonstrated the tremendous potential of using CTC counts as prognostic markers of metastatic development and therapeutic efficacy. However, it is only the viable CTCs capable of surviving in the blood circulation that can create distant metastasis. To date, little progress has been made in understanding what proportion of CTCs is viable and what proportion is in an apoptotic state. Here, we introduce a novel approach toward in situ characterization of CTC apoptosis status using a multicolor in vivo flow cytometry platform with fluorescent detection for the real-time identification and enumeration of such cells directly in blood flow. The proof of concept was demonstrated with two-color fluorescence flow cytometry (FFC) using breast cancer cells MDA-MB-231 expressing green fluorescein protein (GFP), staurosporine as an activator of apoptosis, Annexin-V apoptotic kit with orange dye color, and a mouse model. The future application of this new platform for real-time monitoring of antitumor drug efficiency is discussed. © 2018 International Society for Advancement of Cytometry.

Abstract 1727: Circulating Tumor Cells (CTCs) in patients with extensive stage small cell lung cancer and their association with clinical outcome

Abstract Background: The NOTCH pathway has been identified as a key therapeutic pathway in SCLC. Tarextumab (TRXT, anti-Notch2/3, OMP-59R5) is a fully human monoclonal antibody that targets the Notch2 and Notch3 receptors. PINNACLE is a Phase 1b/2 trial of TRXT in combination with etoposide and platinum therapy (EP) in patients with untreated extensive stage small cell lung cancer (ES-SCLC). Baseline CTCs and post treatment changes in CTCs have previously been shown to predict the response to chemotherapy in SCLC. CTCs may also serve as pharmacodynamic biomarkers. Here we describe a study of baseline and longitudinal CTCs in ES-SCLC patients from the PINNACLE phase 1b trial (clinicaltrials.gov:NCT01859741). Materials and methods: CTCs, CTC clusters, apoptotic CTCs and N-Cadherin+ CTCs were identified and enumerated from patient blood samples using Epic Sciences CTC technology. Baseline CTCs from 26 patients were correlated with clinical outcome: progression-free survival (PFS), overall survival (OS) and best overall response, as well as metastatic status. A mixed effects model was used to investigate the post treatment changes in CTCs among the dose groups. Association of CTCs with PFS/OS and best overall response, including CTCs at each time point, as well as temporal changes of CTC status, were studied. Multivariate analysis was performed to identify CTC numbers in a subset of time points to correlate with response to treatment. Results: CTCs were present in 81% of the patients (21/26). CTC clusters and apoptotic CTCs were detected in 38% and 77% of the patients, respectively. At baseline, CTC counts ≥ 5/mL were significantly associated with poor OS (p=0.04). There was a trend that the presence of CTC clusters was associated with worse OS. With a cut-off of 3.4/mL, apoptotic CTCs showed a trend in association with overall survival. CTC numbers in patients with liver metastasis were significantly higher than in patients without liver metastasis. CTCs were also found to be correlated significantly with the number of metastatic sites. When measuring at Day 7 post dosing, CTC numbers were significantly decreased. Conclusions: Our findings suggest that CTCs are frequently detectable in patients and are a prognostic factor in ES-SCLC. CTCs decrease with TRXT and platinum-based chemotherapy. Updated results will be presented. CTCs will be further evaluated in the phase 2 portion of the PINNACLE trial. Citation Format: Chun Zhang, Ryon Graf, Adam Jendrisak, Amanda K. Anderson, Priscilla Ontiveros, Sarah Orr, Anne Chiang, David Spigel, Charles Rudin, Eric Holmgren, Jakob Dupont, Gretchen Argast, Leonardo Faoro, Lei Zhou, John Lewicki, Ann M. Kapoun. Circulating Tumor Cells (CTCs) in patients with extensive stage small cell lung cancer and their association with clinical outcome [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 1727. doi:10.1158/1538-7445.AM2017-1727

Midkine promotes hepatocellular carcinoma metastasis by elevating anoikis resistance of circulating tumor cells.

Midkine is overexpressed in hepatocellular carcinoma (HCC) and plays a role in tumor progression, but less is known about its role in resistance of circulating tumor cells (CTCs) to anoikis which leading to recurrence and metastasis. The aim of the present study was to analyze whether midkine was associated with HCC progression with anoikis resistance. We found that cultured HCC cells were more resistant to anoikis, which paralleled midkine expression, and midkine treatment significantly inhibited anoikis in a dose-dependent manner. Furthermore, in in vitro and in vivo assays, knockdown of midkine resulted in significant sensitivity to anoikis, decreased cell survival and significantly decreased tumor occurrence rate. Patients with midkine-elevated HCC had higher CTC counts and less apoptotic CTCs, as well as significantly higher recurrence rate and shorter recurrence-free interval. To understand the molecular mechanism underlying the midkine with HCC progression, we performed in vitro and in vivo studies. We found that midkine plays an important role in enhancement of HCC cell resistance to anoikis, thereby promoting subsequent metastasis. Activation of PI3K/Akt/NF-κB/TrkB signaling by midkine-activated anaplastic lymphomakinase (ALK) is responsible for anoikis resistance.

Impact of apoptotic circulating tumor cells (aCTC) in metastatic breast cancer.

While intact circulating tumor cells (iCTC) have independent negative prognostic impact on patients with metastatic breast cancer (MBC), the prognostic relevance of apoptotic CTC (aCTC) has not been validated in larger patient cohorts. This study assessed aCTC and iCTC statuses at baseline (CTCBL) and CTC kinetics (CTCKIN) as changes from CTCBL to one completed treatment cycle for their utility in predicting response, progression-free survival (PFS), and overall survival (OS) in MBC. Status of iCTC and aCTC was prospectively assessed in 442 patients using the CellSearch™ system. Different cutoffs were analyzed both for iCTC and aCTC (≥5, ≥10, ≥25 and ≥50 CTC/7.5ml). CTCKIN were characterized by ≥25% changes in CTC counts. Numbers of iCTC and aCTC at baseline correlated strongly (r=0.7). For iCTCBL positive patients, additional detection of aCTCBL had a significant prognostic impact on OS (aCTCBL positive 10.3 vs. aCTCBL negative 16.4months, p=0.012). Worst prognosis for OS was observed in patients with ≥50 iCTC/7.5ml and simultaneously detected aCTC. Determination of aCTCKIN showed stronger discriminating power than iCTCKIN, with higher PFS and OS for the group with decreasing CTCs (PFS 7.7 vs. 6.1; OS 22.2 vs. 16.4). Intact and aCTC are predictive of outcome in MBC. Apoptotic CTC counts ≥ 5/7.5ml in conjunction with iCTC at baseline have an independent unfavorable prognostic impact on OS. Decreasing aCTCKIN at ≥ 5/7.5ml in serial enumeration is associated with favorable outcome. Therefore, separate enumeration of iCTC and aCTC is useful in tailoring systemic treatment.

Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) in muscle invasive and metastatic bladder cancer patients.

BackgroundWhile programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes.MethodsBlood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)+ and (CK)−CTCs (CD45−, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis.ResultsCTCs were detected in 20/25 (80 %) patients, inclusive of CK+ CTCs (13/25, 52 %), CK−CTCs (14/25, 56 %), CK+ CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1+ CTCs; 4 of these patients had exclusively CK−/CD45−/PD-L1+ CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1+/CD45−CTC burden and low burden of apoptotic CTCs had worse overall survival.ConclusionsCTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK+ and CK−CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2758-3) contains supplementary material, which is available to authorized users.

Abstract 496: Androgen receptor expression on circulating tumor cells in metastatic breast cancer

Abstract Background: Androgen receptor (AR) is a nuclear transcription factor and a member of the steroid hormone receptor superfamily that can be detected by immunohistochemical staining in all three subtypes of invasive breast cancer with frequency of &amp;gt;70% of estrogen receptor-positive tumors (ER+), &amp;gt;60% of HER2-positive tumors (HER2+), and 40-50% of triple-negative breast cancers (TNBC) respectively. AR-target therapies are showing early activity in AR+ breast cancer. Given early evidence of activity with agents targeting AR, it is desirable to improve our understanding of the AR biology in breast cancer. Although circulating tumor cells (CTCs) have been proposed as a liquid biopsy for biomarker detection in multiple cancer types, the rate and heterogeneity of AR expression in CTCs in breast cancer is still unclear. In this study, we determine the rate and heterogeneity of AR expression in CTCs of patients with metastatic breast cancer. Methods: Blood samples from patients with metastatic breast cancer were smeared onto glass slides and subjected to nuclear staining with DAPI and reacted with fluorescent-labeled antibodies to detect CD45 on leukocytes and cytokeratin and AR on epithelial cells; the slides were scanned on the Epic Sciences CTC platform and the data analyzed using established algorithms. Results: Among the 59 patients analyzed, 25 (42.4%) had ER+/HER2-, 12 (20.3%) had ER+/HER2+, 6 (10.2%) had ER-/HER2+, and 16 (27.1%) had TNBC based on the most recent staining. CTCs were detected in 50 of 59 (84.7%) patients. Characterization of CTCs identified CK+ CTCs (45 of 59, 76.3%), CK-negative CTCs (16 of 59, 27.1%), CK+ CTC clusters (16 of 59, 27.1%), and apoptotic CTCs (45 of 59, 76.3%), respectively. Number of CTCs per patient ranged from 0 to 282.1 per mL (median = 4.4). Six of the 50 patients (12%) with CTCs had AR+ CTCs. The number of AR+ CTCs per patient ranged from 0.3 to 52.6 per mL (median = 1.15). Among the patients with AR+ CTCs, the percentage of CTCs that were AR+ ranged from 5% to 50% (median = 21%). Among the six patients with AR+ CTCs, five patients had ER+ breast cancer and one had TNBC. Conclusions: We demonstrated that the Epic Sciences non-enriching or selecting platform can detect AR expression in CTCs in breast cancer. These preliminary results suggest the need for clinical validation of CTC AR expression as a potential test to stratify and identify patients who might benefit from AR therapy. The heterogeneity of intra-patient CTC AR expression leads us to another hypothesis that patients with AR+ CTCs might have heterogeneous disease with multiple drivers. Further studies are warranted to allow serial monitoring of changes in AR and to investigate the clinical applicability of AR+ CTCs and their heterogeneity. Citation Format: Takeo Fujii, Yipeng Wang, James M. Reuben, Rachel Krupa, Ryon Graf, Lyndsey Dugan, Jessica Kouw, Dena Marrinucci, Bora Lim, Carlos H. Barcenas, Angela N. Marx, Debu Tripathy, Ryan Dittamore, Naoto T. Ueno. Androgen receptor expression on circulating tumor cells in metastatic 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 496.

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.

Abstract P2-02-06: Impact of apoptotic circulating tumor cells in metastatic breast cancer

Abstract Background Circulating tumor cells (CTC) are a heterogeneous cell population and an independent negative prognostic factor for progression-free (PFS) and overall survival (OS) in patients with metastatic breast cancer (MBC). The study aimed to prospectively assess CTC status for the subtypes apoptotic CTC (aCTC) and intact CTC (iCTC) at baseline (CTCBL) and after one cycle of a new line of systemic therapy (CTC1C). Changes from CTCBL to CTC1C (CTC kinetics, CTCKIN) were evaluated for their utility in predicting response, progression-free (PFS) and overall survival (OS). Methods 423 MBC patients were included in a prospective trial prior to a new regimen of treatment. Intact and apoptotic CTC were analyzed at baseline (CTCBL) and after one cycle of systemic therapy (CTC1C) using CellSearch™ (Veridex) and morphologic criteria. Samples with ≥5 CTC/7.5ml blood were regarded as positive. Therapy response was assessed using the RECIST-criteria on three-monthly radiological controls. CTCKIN were characterized by ≥25% change from CTCBL to CTC1C to differentiate stable, increased and decreased CTC kinetic. Results 35% of patients were iCTCBL-positive and 28% aCTCBL-positive at baseline (CTCBL). PFS and OS differ significantly between the iCTCBL-positive and the iCTCBL-negative group (PFS 4.5 vs. 8.0; OS 12.5 vs. 27.2 (months)). Positive aCTC in conjunction with positive iCTCBL at baseline has worst prognostic impact (PFS 6.3; OS 8.7). Regarding the CTCKIN (BL to 1C), aCTC-decrease (≥25%) is a positive prognostic factor compared to aCTC-stable and aCTC-increase (PFS 7.6 vs. 3.7; 3.3 and OS 21.0 vs. 4.8; 5.7). Decreasing aCTCKIN shows favorable prognostic impact versus decreasing iCTCKIN (PFS 7.6 vs. 5.9 and OS 21.0 vs 16.4). Conclusion Elevated aCTC levels at baseline have an unfavorable prognostic impact on both OS and PFS in conjunction with elevated iCTC. Additionally, the decrease of aCTC is a relevant prognostic value for systemic therapy response. aCTCKIN allows better differentiation for therapy response in patients with positive CTC-status at baseline. Differentiated enumeration of intact and apoptotic CTC should be considered in clinical application. Citation Format: Wallwiener M, Deutsch TM, Riethdorf S, Hartkopf AD, Taran F-A, Trumpp A, Brucker S, Schütz F, Rom J, Pantel K, Schneeweiss A. Impact of apoptotic circulating tumor cells in metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-06.

Are circulating tumor cells (CTCs) a feasible tool for predicting disease recurrence and survival in nonmetastatic (M0) colorectal cancer (CRC)?

650 Background: Based on the correlation between CTCs and clinical outcome of metastatic breast, prostatic and CRC cancer, CTCs might be an ideal model to investigate the individual risk to develop metastasis. On the other way, data on their significance in M0 CRC are still limited. In a previous study we investigated CRC cell death in the circulating compartment, finding high value of apoptotic CTCs (M30-positive) independently from the disease stage. Our aim was to evaluate the prognostic role of total and M30-positive CTCs in M0 CRC patients (pts). Methods: From January 2009 to December 2011, we prospectively enrolled 192 stage 0-III CRC pts (stage 0 identified rectal cancer pts with Tis or complete pathological response after neoadjuvant therapy). We used CellSearch assay to quantify total and M30-positive CTCs in peripheral blood of CRC pts candidate to curative resection at diagnosis, time of surgery and four months later. Multivariate survival analyses were conducted using a Cox-regression. Results: At baseline 81 out of 192 pts (43.3%) had at least 1 CTC/7.5 mL of peripheral blood, 40 pts (22.2%) had &gt;2 cells and 20 pts (11.6%) had &gt;3 cells. We found a weak association between OS and total CTCs number (p=0.069), a significant association with M30-positive CTCs (p=0.022) and no association between RFS and CTC status. Limiting the analysis to stage I-III (n=151), we found a strong association between OS and both CTCs number (p=0.021) and M30-positive cells (p=0.007). The multivariate analyses demonstrated a significant improvement of survival prediction adding baseline CTCs count to clinical and pathological characteristics (HR=1.51, 95% CI 1.16-1.96). Conclusions: We first demonstrated a significant association between OS and CTCs status in stage I-III CRC pts. This result is consistent with previous report in metastatic CRC. It highlights a potential advantage of CTCs count in stratifying CRC pts and identifying a subset of pts with a high metastatic risk that could benefit from an adjuvant therapy. Further studies are needed to explore this aspect, which might be particularly significant in stage II CRC where the indication for adjuvant therapy is still under debate.

Heterogeneity of prostate-specific membrane antigen (PSMA) expression in classic and apoptotic circulating tumor cells (CTC) in metastatic castration-resistant prostate cancer (mCRPC).

198 Background: Prostate-specific membrane antigen (PSMA) is a folate hydrolase expressed on the surface of PC cells that has been used as a target to detect disease and selectively deliver cytotoxic agents and radionuclides. The ability to detect PSMA levels on circulating tumor cells (CTCs) may identify patients likely to benefit from such targeted therapy. Technology developed by Epic Sciences utilizes high definition imaging of plated nucleated cells. Methods: Blood samples were obtained from patients (pts) with metastatic castration-resistant prostate cancer (mCRPC). Cells were stained for CK, CD45, PSMA and categorized as classic CTC (CK+, CD45-, intact/morphologically distinct nuclei) or apoptotic CTC (CK+, CD45-, morphology suggesting apoptosis). Clinical data including treatment, metastatic sites, Veridex CellSearch CTC enumeration, prostate-specific antigen, and alkaline phosphatase was collected. Results: Fourteen pts with mCRPC, including eight with serial samples were analyzed (33 samples in total). At the first draw (t1), classic CTC were detected in 13 pts (93%), (median two cells/ml, range 0 to 40 cells/ml) and apoptotic CTC in 14 pts (100%) (median four cells/ml, range 1 to 18 cells/ml), including six pts (42%) with no CTC by Veridex CellSearch. PSMA expression was detected in five pts (36%) with classic CTC of which a median of 32% of cells (range 5 to 100%) expressed the antigen. Similar intra-patient heterogeneity was seen for the 10 pts (71.4%) with PSMA+ apoptotic CTCs (median 33.5%, range 11 to 75% cells). During treatment, often with more complete androgen suppression, PSMA was detected in 3 of the 8 (38%) pts with no PSMA+ classic CTCs at t1. The presence of PSMA expression in apoptotic CTCs did not appear to change while on therapy. Conclusions: A larger percentage of PSMA expression was seen in mCRPC pts in apoptotic CTC (10 out of 14) than classic CTC (5 out of 14) at t1, with intra-patient cell heterogeneity of PSMA expression in both CTC populations. Serial measures suggest dynamic changes in PSMA expression over time. The threshold of detectable cells and proportion and degree of PSMA expression that associates with drug sensitivity is unknown. Larger samples of pts at discrete time points on therapy are underway to further elucidate the potential clinical relevance.

Apoptotic circulating tumor cells (CTCs) in the peripheral blood of metastatic colorectal cancer patients are associated with liver metastasis but not CTCs

Enumeration of circulating tumor cells (CTCs) by the CellSearch system provides prognostic information in metastatic colorectal cancer, regardless of metastatic site. We found that CTCs generally represent <1% of observed events with CellSearch analysis and adapted scoring criteria to classify other peripheral blood events. Examination of twenty two metastatic colorectal cancer patients' blood revealed that patients with high CEA or liver metastases, but not lung or distant lymph node metastases, possessed significant numbers of apoptotic CTCs prior to treatment initiation by Fischer's exact test. Six out of eleven patients with liver metastasis possessed apoptotic CTCs whereas one of nine patients with other metastases had measurable apoptotic CTCs. An elevated CTC number was not necessarily associated with apoptotic CTCs or CTC debris by Spearman's correlation, suggesting the metastatic site rather than CTCs per se as contributing to the origin of these events.

Apoptotic Circulating Tumor Cells in Early and Metastatic Breast Cancer Patients

The detection of circulating tumor cells (CTC) in breast cancer is strongly associated with disease relapse. Since it is unclear whether all CTCs are capable of generating metastasis, we investigated their apoptotic and proliferative status in 56 CTC-positive (29 early and 27 metastatic) patients with breast cancer. Double-staining immunofluorescence experiments were carried out in peripheral blood mononuclear cells (PBMC) cytospins, using the pancytokeratin A45-B/B3 antibody and either M30 (apoptotic marker) or Ki67 (proliferation marker) antibodies. Apoptosis was also evaluated using a polycaspase detection kit. Patients with metastatic disease had significantly lower numbers of apoptotic CTCs compared with patients with early breast cancer (polycaspase kit: 8.1% vs. 47.4% of the total CTC number; P = 0.0001; M30-antibody: 32.1% vs. 76.63%; P = 0.002). The median percentage of apoptotic CTCs per patient was also lower in patients with advanced compared with those with early disease (polycaspase kit: 0% vs. 53.6%; M30-antibody: 15% vs. 80%). Ki67-positive CTCs were identified in 51.7% and 44% of patients with early and metastatic disease, respectively. Adjuvant chemotherapy reduced both the number of CTCs per patient and the number of proliferating CTCs (63.9% vs. 30%). In conclusion, apoptotic CTCs could be detected in patients with breast cancer irrespective of their clinical status, though the incidence of detection is higher in early compared with metastatic patients. The detection of CTCs that survive despite adjuvant therapy implies that CTC elimination should be attempted using agents targeting their distinctive molecular characteristics.

Potential applications of circulating melanoma cells (CMCs) expressing γ-H2AX.

e22036 Background: The CMC analysis was recently improved by the CellSearch platform that offers an accurate (and reproducible) assessment of tumor burden, to be used as prognosticator and predictor of treatment response.Moreover, not only decreased total circulating cells numbers but also increased fraction of apoptotic cells may represent response-related markers. To monitor changes in the balance between live and apoptotic Circulating Tumor Cells (CTCs) ( Rossi, Basso et al. 2010 ) during therapy, a novel CTCs assay with a companion algorithm (the ΔAUC) was reported to be a pharmacodynamic marker, as documented by consistent radiological findings in MBC and mRCC patients. Methods: Fluorescent anti γ-H2AX mAb (specific for the phosphorylated form of H2AX) was integrated in the standard CMC assay to assess DNA damage and to measure cell viability and apoptosis in spreading melanoma cells. The integrated test was developed using MCF-7 and SK23mel cells line and tested in melanoma patients. From June-2011 till now, basal blood samples from 64 patients (pts) in stage IV were collected; median age 62 (range: 27-85). Both considering the cost of the CMC assay and the little already known about this new method in melanoma, to evaluate the feasibility of CMC retrieval and ΔAUC calculation an interim analysis was specified in the study protocol and conducted at September 2012 on the first 40 pts. Results: At baseline, in Padova’s cohort 72.6% of pts are CMC-positive. The CMC number ranged from 1 to 94. The γ-H2AX-positive CMCs ranged from 0% to 100% (median 67%). Shorter median Progression-free survival PFS was observed for patients who had &gt; 2 CMCs (n=26, 162 days) compared with &lt; 2 CMCs patients (n=14, 203 days, P value = 0.0270). No significant differences were found with the ΔAUC criterion (i.e. exceeding live vs apoptotic CMCs). Furthermore, a significant association was found between CMC-positive status at baseline and disease sites at progression (visceral and CNS, Chi-square = 8,521 with 3 df, P value = 0.036). Conclusions: Accrual is ongoing. Updated data including evaluations on prognostic value of total and apoptotic CMCs and correlation with clinical stage, tumor burden, metastatic sites and survival will be available and presented at the meeting.

Evaluation of proliferation and apoptosis markers in circulating tumor cells (CTCs) of women with early breast cancer who are candidates for tumor dormancy.

e22101 Background: Clinical dormancy is frequently observed in breast cancer (BC). In the present study, we aimed to characterize CTCs in dormancy candidates (DC) with BC in terms of proliferation and apoptosis. Methods: Cytospins of peripheral blood mononuclear cells (PBMCs) were obtained from DC (n=122) disease-free for ≥5 yrs and from metastatic pts (n=37) on relapse that occurred ≥5 yrs after surgery. Sequential samples (n=27) of 8 DC with late relapse and from 8 relapse-free (n=38), were also analyzed. 106 PBMCs were stained with pancytokeratin antibody along with ki-67 and M30 as proliferation and apoptosis markers, respectively. Results: CTCs were identified in 40 (32%) of 122 DC. In 25 (61.5%), all CTCs were ki-67(-)/M30(-), 7 (17.5%) had ki-67(+), 4 (10%) M30(+) CTCs and 4 had both phenotypes. Among 243 CTCs detected, 201 (82.5%) were ki-67(-)/M30(-), 14 (5.7%) ki-67(+) and 29 (11.9%) M30(+). Of 13 (35%) CTC(+) metastatic pts, 6 (46%) had ki-67(+) CTCs (p=0.037) whereas 54 (40%) of total CTCs were ki-67(+) (p&lt;0.001). No M30(+) CTCs were detected. When sequential samples of the 8 DC who relapsed were analyzed, 2 (25%), had only ki-67(-)/M30(-) CTCs, 6 (75%) had ki-67(+) and 4 (50%) M30(+) CTCs. The respective numbers in the non-relapsed group were 4 (50%), 3 (37.5%) and 3 (37.5%). Moreover, ki-67(+) CTCs were more frequently observed in samples of the relapsed pts (29.6% vs 13.1%). Among 382 CTCs detected in all sequential samples from the relapsed group, 337 (88.14%) were ki-67(-)/M30(-), 26 (6.7%) ki-67(+) and 20 (5.1%) M30(+), whereas the respective percentages among 78 CTCs in non-relapsed pts were 77.6%, 6.5% and 15.8% (p=0.001). Conclusions: The great majority of CTCs detected in DC with BC express neither proliferation, nor apoptosis markers. However, the apoptotic index in CTCs is increased during clinical dormancy, whereas the proliferation index is increased on relapse. In addition, apoptotic CTCs are more frequently encountered during follow-up in DC free of relapse. The above observations suggest that monitoring proliferation and apoptosis in CTCs during clinical dormancy could be used to predict subsequent late relapse.