Dynamics Of Circulating Tumor Cells Research Articles

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.

Circulating tumor cells dynamics in pancreatic adenocarcinoma correlate with disease status: Results of the prospective cluster study

OBJECTIVES Previous retrospective studies demonstrated that circulating tumor cells (CTCs) subtypes correlate with overall survival in patients with pancreatic ductal adenocarcinoma (PDAC). Herein, we report results of a prospective observational study on CTCs dynamics to assess their clinical significance. METHODS The CLUSTER study is a prospective longitudinal study on PDAC CTCs dynamics (NCT02974764). Multiple peripheral blood samples were collected from 200 consecutively enrolled patients with presumed PDAC diagnosis. CTCs were isolated and characterized by immunofluorescence. RESULTS Two major CTCs subtypes were identified in PDAC patients: epithelial CTCs (eCTCs) and epithelial/mesenchymal CTCs (mCTCs). Patients who received neoadjuvant chemotherapy had significantly lower total CTCs (tCTCs, P = 0.007), eCTCs (P = 0.007), and mCTCs (P = 0.034), compared with untreated patients eligible for upfront resection. Surgical resection of the primary tumor resulted in significant reduction, but not disappearance, of CTCs burden across all cell subtypes (P < 0.001). In multivariable logistic regression analysis, preoperative numbers of all CTCs subpopulations were the only predictors of early recurrence within 12 months from surgery in both chemo-naive and post-neoadjuvant patients (odds ratio 5.9 to 11.0). Alterations in CTCs were also observed longitudinally, before disease recurrence. A risk assessment score based on the difference of tCTCs increase accurately identified disease recurrence within the next 2 months, with an accuracy of 75% and 84% for chemo-naive and post-neoadjuvant patients, respectively. CONCLUSION We report novel findings regarding CTCs from a large prospective cohort of PDAC patients. CTCs dynamics reflect progression of disease and response to treatment, providing important information on clinical outcomes, not available by current tumor markers and imaging.

Circulating Tumor Cells Dynamics in Pancreatic Adenocarcinoma Correlate With Disease Status: Results of the Prospective CLUSTER Study.

Previous retrospective studies demonstrated that circulating tumor cells (CTCs) subtypes correlate with overall survival in patients with pancreatic ductal adenocarcinoma (PDAC). Herein, we report results of a prospective observational study on CTCs dynamics to assess their clinical significance. The CLUSTER study is a prospective longitudinal study on PDAC CTCs dynamics (NCT02974764). Multiple peripheral blood samples were collected from 200 consecutively enrolled patients with presumed PDAC diagnosis. CTCs were isolated and characterized by immunofluorescence. Two major CTCs subtypes were identified in PDAC patients: epithelial CTCs (eCTCs) and epithelial/mesenchymal CTCs (mCTCs). Patients who received neoadjuvant chemotherapy had significantly lower total CTCs (tCTCs, P = 0.007), eCTCs (P = 0.007), and mCTCs (P = 0.034), compared with untreated patients eligible for upfront resection. Surgical resection of the primary tumor resulted in significant reduction, but not disappearance, of CTCs burden across all cell subtypes (P < 0.001). In multivariable logistic regression analysis, preoperative numbers of all CTCs subpopulations were the only predictors of early recurrence within 12 months from surgery in both chemo-naive and post-neoadjuvant patients (odds ratio 5.9 to 11.0). Alterations in CTCs were also observed longitudinally, before disease recurrence. A risk assessment score based on the difference of tCTCs increase accurately identified disease recurrence within the next 2 months, with an accuracy of 75% and 84% for chemo-naive and post-neoadjuvant patients, respectively. We report novel findings regarding CTCs from a large prospective cohort of PDAC patients. CTCs dynamics reflect progression of disease and response to treatment, providing important information on clinical outcomes, not available by current tumor markers and imaging.

AB015. S015. Circulating tumor cells dynamics in pancreatic adenocarcinoma correlate with disease status: data from a prospective trial

AB015. S015. Circulating tumor cells dynamics in pancreatic adenocarcinoma correlate with disease status: data from a prospective trial

Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies.

Abstract P2-01-01: Trajectory patterns of circulating tumor cells (CTC) in chemotherapy-treated metastatic breast cancer (MBC) patients predict poor clinical outcomes: CALGB 40502 (Alliance)/NCCTG N063H study

Abstract Little is known about the dynamics of CTCs during treatment and its clinical significance. We examined the predictive utility of serial CTC analysis in ER+HER2- MBC patients (pts) treated with chemotherapy in the CALGB 40502/NCCTG N063H study, a randomized phase III trial of weekly paclitaxel compared to weekly nanoparticle albumin bound nab-paclitaxel or ixabepilone +/- bevacizumab as first-line therapy (ClinicalTrials.gov Identifier: NCT00785291, Support: U10CA180821, U10CA180882). Methods: Of the 783 pts treated, 469 had ≥3 serial blood samples (including baseline) successfully analyzed for CTCs by CellSearch® and were included in this analysis (n=2,202). Samples with ≥5 CTCs per 7.5 mLs of blood were considered CTC+. The prognostic and predictive performance of baseline CTCs (bCTC) and CTC status from baseline to cycle 2 (b2CTC) were compared to a novel latent mixture model classification based on trajectory of CTCs (tCTC). Akaike Information Criterion (AIC) was used to select the model (bCTC vs b2CTC vs tCTC) that best predicts overall survival (OS), progression-free survival (PFS), and time-to-treatment failure (TTF). Results: 53% of the pts were CTC+ at baseline. b2CTC status changed in 36% of the pts, most of whom were CTC+CTC- (35%), and very few CTC-CTC+ (1%); the rest of the pts did not experience a change in b2CTC status (46% CTC-CTC- and 19% CTC+CTC+). Mixture model analysis revealed 4 groups of pts that show distinct tCTC patterns over the course of treatment: consitently very low/undectectable CTCs (tCTCneg, 56%), low (tCTClo, 24%), intermediate (tCTCmid, 15%), or high (tCTChi, 5%). bCTC, b2CTC, and tCTC were significantly correlated with tumor subtype (all p &amp;lt;0.0022) and presence of bone metastasis (all p &amp;lt;0.0001). Multivariate analysis showed that pts who were CTC+ at baseline, and those whose b2CTC status remained positive (CTC+CTC+) had significantly reduced OS, PFS and TTF. OSPFSTTFModelsHR (95% CI)p-valueHR (95% CI)p-valueHR (95% CI)p-valuebCTC (vs CTC-) → CTC+2.5(1.8-3.3)&amp;lt;0.00011.6(1.3-2.0)&amp;lt;0.00011.3(1.1-1.6)0.0046b2CTC (vs CTC+CTC-) → CTC-CTC+1.6(0.5-5.4)0.41491.6(0.6-4.5)0.39051.6(0.6-4.3)0.3961→ CTC+CTC+2.7(1.9-3.8)&amp;lt;0.00011.8(1.4-2.5)&amp;lt;0.00011.8(1.3-2.4)&amp;lt;0.0001→ CTC-CTC-0.5(0.4-0.8)0.00020.8(0.6-0.9)0.01600.9(0.7-1.1)0.2771tCTC (vs tCTCneg) → tCTClo2.6(1.9-3.7)&amp;lt;0.00011.9(1.4-2.4)&amp;lt;0.00010.9(0.7-1.1)0.0033→ tCTCmid5.3(3.6-8.0)&amp;lt;0.00012.5(1.8-3.4)&amp;lt;0.00011.8(1.4-2.5)0.0001→ tCTChi10.8(6.1-19)&amp;lt;0.00013.0(1.8-5.0)&amp;lt;0.00012.3(1.4-3.7)0.0009CTC- (&amp;lt;5 CTCs per 7.5 mLs); CTC+ (≥5 CTCs per 7.5 mLs) Pts with tCTClo, tCTCmid and tCTChi had significantly shorter OS, PFS and TTF compared to those with tCTCneg. After adjustment for potential confounders, AIC analysis revealed that the tCTC model best predicts OS and PFS, while b2CTC best predicts TTF. AIC Score*ModelsOSPFSTTFbCTC243240514199b2CTC240540384186tCTC237940264188*The lowest AIC score indicates the best model. Conclusions: Analysis of CTC trajectory patterns identified pts with poor outcome who could potentially benefit from more effective treatment. Validation in independent cohorts is warranted to confirm the findings in this study. Citation Format: Magbanua MJ, Hendrix L, Hyslop T, Barry WT, Winer EP, Hudis C, Toppmeyer D, Burnstein H, Qadir M, Ma C, Scott JH, Park JW, Rugo HS. Trajectory patterns of circulating tumor cells (CTC) in chemotherapy-treated metastatic breast cancer (MBC) patients predict poor clinical outcomes: CALGB 40502 (Alliance)/NCCTG N063H study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-01.

Circulating tumor cells and survival in abiraterone- and enzalutamide-treated patients with castration-resistant prostate cancer.

The outcome to treatment administered to patients with metastatic castration-resistant prostate cancer (mCRPC) greatly differs between individuals, underlining the need for biomarkers guiding treatment decision making. To investigate the prognostic value of circulating tumor cell (CTC) enumeration and dynamics, in the context of second-line endocrine therapies (ie, abiraterone acetate or enzalutamide), irrespective of prior systemic therapies. In a prospective, multicentre study blood samples for CTC enumeration were collected from patients with mCRPC at baseline (n = 174). In patients who responded for minimally 10-12 weeks a follow-up sample was collected. For baseline analysis, patients were stratified in <5 or ≥5 CTCs/7.5 mL, whereas for the analysis of CTC dynamics at 10-12 weeks, in patients with stable, increasing or decreasing CTC counts. Progression-free survival (PFS), overall survival (OS), and PSA changes at 10-12 weeks were compared between groups. Patients demonstrating increasing CTCs on therapy had a shorter median PFS (4.03 vs 12.98 vs 13.67 months, HR 3.6, 95%CI 1.9-6.8; P < 0.0001) and OS (11.2 months vs not reached, HR 9.5, 95%CI 3.7-24; P < 0.0001), compared to patients with decreasing or stable CTCs. Multivariable Cox regression showed that prior chemotherapy (HR 4.1, 95%CI 1.9-8.9; P = 0.0003), a high baseline CTC count (HR 1.5, 95%CI 1.2-1.9; P = 0.002) and increasing CTCs at follow-up (HR 3.3, 95%CI 1.4-7.6; P = 0.005) were independent predictors of worse PFS. Previous chemotherapy (HR 7, 95%CI 1.9-25; P = 0.003), high baseline CTC counts (HR 2.2, 95%CI 1.4-3.7; P = 0.002) and increasing CTCs during therapy (HR 4.6, 95%CI 1.4-15; P = 0.01) were independently associated with shorter OS. ≥30% and ≥50% PSA responses less frequently occurred in patients with CTC inclines at 10-12 weeks on therapy (χ2 test: P < 0.01). CTC dynamics during therapy are associated with PSA response and provide independent clinical prognostication over PSA declines. Hence the study demonstrates the pharmacodynamic properties of CTCs.

Acute phase dynamics of circulating tumor cells after paclitaxel and doxorubicin chemotherapy in breast cancer mouse models.

Circulating tumor cells (CTCs) can provide a potentially minimal invasive source for monitoring chemotherapeutic effects. However, detailed in vivo dynamics of CTC after chemotherapy remain largely unknown. We monitored CTC number and morphology early after chemotherapy using a newly developed cytology-based CTC detection device and triple-negative breast cancer mouse CTC models with spontaneous lung metastatic potential. Paclitaxel inhibited cell growth of breast cancer cells by mainly G2/M cell cycle arrest and partly apoptosis, whereas doxorubicin inhibited cell growth mainly by apoptosis and partly G2 cell cycle arrest in vitro. The number of CTCs was significantly increased 3-10days after paclitaxel and doxorubicin chemotherapy and decreased thereafter in two mouse CTC models. The transiently increased CTCs early post-chemotherapy consisted of not only G2/M arrested cells (apoptotic cells), but also morphologically near-intact live cells. This heterogeneous cell population of CTCs was similar to that of primary tumor tissue after chemotherapy. These results indicate that CTCs can be mobilized from the primary tumor in rapid response to chemotherapy and suggest the possibility that CTC monitoring from both numerical and morphological viewpoints early after chemotherapy using a cytology-based CTC detection device would be a useful diagnostic tool for predicting drug sensitivity/resistance in preclinical and clinical setting.

Clinical relevance of circulating tumor cells in ovarian, fallopian tube and peritoneal cancer.

e17080 Background: Presence of circulating tumor cells (CTCs) in peripheral blood is associated with impaired clinical outcome in a variety of cancers. So far, limited data are available on the significance of CTCs in gynaecological malignancies. Aims of the present study were to evaluate the dynamics of CTCs in patients with ovarian, fallopian tube and peritoneal cancer during chemotherapy and to assess the clinical relevance of these changes. Methods: 38 patients with ovarian (n=31), fallopian tube (n=4) and peritoneal (n=3) cancer were included into the study. All patients received chemotherapy in the first-line setting (n=19) or tumor recurrence (n=19). CTC analysis was performed prior to systemic treatment, after three and six cycles of chemotherapy and analysed using CellSearch system (Veridex). A tumor cell was defined as EpCAM+, cytokeratin+, CD45-, and positive for the nuclear stain DAPI. CTC positivity was defined as detection of at least one CTC per 7.5 ml blood. Results: 11 out of 38 (29%) patients were CTC positive at baseline. Positivity rate was 20% in patients with first-line setting and 37% in those with tumor recurrence. 1of the 4 patients with fallopian tube cancer presented with CTCs in contrast to 1 out of 3 patients with peritoneal cancer and 10 out of 31 ovarian cancer patients. Presence of CTCs was not correlated with other prognostic factors, such as the FIGO stage, nodal status, or grading. CTC positivity declined to 8% after three cycles of cytotoxic therapy and no patient was CTC positive after 6 cycles of chemotherapy. 12 patients died during follow-up. Patients with CTCs at baseline had significantly shorter overall survival compared with CTC negative patients (p = 0.002; mean OS 5.8 [95%-CI 2.7-8.9] vs. 15.2 [10.5-19.8] months, median OS 3.0 [1.4-4.6] vs. not reached). In the subgroup of patients with primary cancer, CTC positivity was significantly associated with overall survival in the univariate analysis (p = 0.008). Conclusions: Hematogenous dissemination of single tumor cells is a common phenomenon in ovarian, fallopian tube and peritoneal cancer. Patients with CTCs in the peripheral blood at time of diagnosis are more likely to die than those who are CTC-negative at baseline.

Abstract OT1-02-04: The DETECT V-study – Comparison of dual HER2-targeted therapy with trastuzumab plus pertuzumab in combination with chemo- or endocrine therapy in patients with HER2-positive and hormone-receptor positive metastatic breast cancer

Abstract Background: Maintenance of quality of life (QoL) is one of the main aims of treatment of incurable diseases such as metastatic breast cancer (MBC). In patients with HER2-positive MBC, taxan-based chemotherapy in combination with dual HER2 targeted therapy with trastuzumab and pertuzumab has shown promising efficacy results in terms of prolonged survival. However, cytostatic treatment is often accompanied by adverse events of grade 3 or higher, seriously impacting the patients' QoL. In patients with HER2-positive and hormone-receptor positive MBC, the combination of trastuzumab with aromatase inhibitors was shown to be a safe and effective treatment option. The synergistic combination of dual HER2-targeted therapy with trastuzumab and pertuzumab plus endocrine therapy might offer an even better treatment option for these patients. DETECT V is the first prospective randomized phase III clinical trial comparing the safety and efficacy of the dual HER2-targeted therapy in combination with either endocrine therapy or chemotherapy. Trial design and eligibility criteria: Women with HER2-positive and hormone-receptor positive MBC with first to third line therapy are 1:1 randomized either to a dual HER2-targeted therapy with Pertuzumab and Trastuzumab plus endocrine therapy or to the dual HER2-targeted therapy plus chemotherapy. Specific aims: The primary objective of this study is to compare the safety and QoL in both arms, as assessed by the occurrence of AEs during the treatment period. We developed a modified adverse event score - including all adverse events grade 3 or higher, except neutropenia, which is included only if rated grade 4, and alopecia, rash, hand-foot-syndrome and peripheral neuropathy which are included if rated grade 2 or higher – in order to better reflect the clinical, physiological and psychological impact of AEs on patients' QoL. Key secondary endpoint, besides the efficacy endpoints progression free survival (PFS) and overall survival, is to compare quality-adjusted survival (QAS), as measured using the quality-adjusted time without symptoms and toxicity (Q-TWiST) method, between both treatment arms. QAS as measured using the Q-TWiST method provides a single metric value that is a composite measure of quantity of survival time and quality of survival as assessed by the patients themselves. Q-TWiST analyses account for possible trade-offs between quantity and quality of life (e.g. prolonged time to progression at the cost of higher toxicity, which adversely affects QoL), and provide an excellent tool to evaluate whether two treatment options differ with regard to the overall perceived value to the patients. Translational research projects focus on Circulating Tumor Cell(CTC)-enumeration (the presence of CTCs is not obligatory in DETECT V), prognostic role of CTC dynamics, and the assessment of marker expression on CTCs in order to calculate an endocrine responsiveness score which will be evaluated regarding its suitability to predict treatment success. Contact: For further information on the DETECT V study please contact www.detect-studien.de or studienzentrale.ufk@uniklinik-ulm.de. Citation Format: Polasik A, Schramm A, Friedl TWP, Rack B, Trapp E, Tzschaschel M, Fasching PA, Taran F-A, Hartkopf A, Schneeweiss A, Müller V, Aktas B, Pantel K, Meier-Stiegen F, Wimberger P, Janni W, Fehm T. The DETECT V-study – Comparison of dual HER2-targeted therapy with trastuzumab plus pertuzumab in combination with chemo- or endocrine therapy in patients with HER2-positive and hormone-receptor positive metastatic breast cancer [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 OT1-02-04.

In Vivo Long-Term Monitoring of Circulating Tumor Cells Fluctuation during Medical Interventions.

The goal of this research was to study the long-term impact of medical interventions on circulating tumor cell (CTC) dynamics. We have explored whether tumor compression, punch biopsy or tumor resection cause dissemination of CTCs into peripheral blood circulation using in vivo fluorescent flow cytometry and breast cancer-bearing mouse model inoculated with MDA-MB-231-Luc2-GFP cells in the mammary gland. Two weeks after tumor inoculation, three groups of mice were the subject of the following interventions: (1) tumor compression for 15 minutes using 400 g weight to approximate the pressure during mammography; (2) punch biopsy; or (3) surgery. The CTC dynamics were determined before, during and six weeks after these interventions. An additional group of tumor-bearing mice was used as control and did not receive an intervention. The CTC dynamics in all mice were monitored weekly for eight weeks after tumor inoculation. We determined that tumor compression did not significantly affect CTC dynamics, either during the procedure itself (P = 0.28), or during the 6-week follow-up. In the punch biopsy group, we observed a significant increase in CTC immediately after the biopsy (P = 0.02), and the rate stayed elevated up to six weeks after the procedure in comparison to the tumor control group. The CTCs in the group of mice that received a tumor resection disappeared immediately after the surgery (P = 0.03). However, CTC recurrence in small numbers was detected during six weeks after the surgery. In the future, to prevent these side effects of medical interventions, the defined dynamics of intervention-induced CTCs may be used as a basis for initiation of aggressive anti-CTC therapy at time-points of increasing CTC number.

Abstract 2039: Dynamic changes and regulation of circulating tumor cells that govern the metastasis of colorectal cancer

Abstract Despite the growing public awareness and recent progress in multimodality therapy to the colorectal cancer (CRC), CRC still ranks high on the list of the most common and deadly cancers in the worldwide. The cause of CRC mortality is due mainly to the occurrence of distant metastasis, upon which the circulating tumor cells (CTCs), is thought to play an active role. CTCs could serve as a prognostic marker for mortality in clinical stage IV patients; however, the prognostic value of CTCs in early-stage patients remains uncertain. Here, we used EpCAM-postive/CD45-negative immunoselection and flow-cytometry to identify and quantify CTCs in CRC patients. The number of mesenteric CTCs obtained from stage II patients was higher than those obtained for patients with stages I, III, or IV CRC. To explore the mechanisms underlying this clinical observation, we established an orthotopic (intra-cecal) tumor growth and metastasis mouse model. Cancer progression was monitored in real time with tumor cells expressing luciferase and GFP tag by using a bioluminescence imaging system and flow cytometery. We noticed that CTC dynamics was associated with a corresponding change in serum interleukin (IL)-17A and an opposing change in serum GM-CSF. Ablation of IL-17A by neutralizing antibodies and administration of GM-CSF effectively diminished the rise of CTCs and prevented metastasis in the mouse model. GM-CSF was through to help eliminating CTCs by boost host immunity, whereas IL-17A affected cancer cells intravasation by promoting cancer cell motility, matrix digestion and angiogenesis. We also noticed that IL-17 depletion could increase CD8+ to CD4+CD25+ ratio that might help CTCs clearance. In CRC patients, serum levels of IL-17A positively correlated with the number of mesenteric CTCs and the patients' disease-free survival. Our results show that CTCs and IL-17A can serve not only as prognostic markers, but also therapeutic targets for CRC metastasis. Citation Format: Ju-Yu Tseng, Chih-Yung Yang, Jeng-Kai Jiang, Chi-Hung Lin. Dynamic changes and regulation of circulating tumor cells that govern the metastasis of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2039. doi:10.1158/1538-7445.AM2014-2039

Detection and quantitation of circulating tumor cell dynamics by bioluminescence imaging in an orthotopic mammary carcinoma model.

Circulating tumor cells (CTCs) have been detected in the bloodstream of both early-stage and advanced cancer patients. However, very little is know about the dynamics of CTCs during cancer progression and the clinical relevance of longitudinal CTC enumeration. To address this, we developed a simple bioluminescence imaging assay to detect CTCs in mouse models of metastasis. In a 4T1 orthotopic metastatic mammary carcinoma mouse model, we demonstrated that this quantitative method offers sensitivity down to 2 CTCs in 0.1–1mL blood samples and high specificity for CTCs originating from the primary tumor, independently of their epithelial status. In this model, we simultaneously monitored blood CTC dynamics, primary tumor growth, and lung metastasis progression over the course of 24 days. Early in tumor development, we observed low numbers of CTCs in blood samples (10–15 cells/100 µL) and demonstrated that CTC dynamics correlate with viable primary tumor growth. To our knowledge, these data represent the first reported use of bioluminescence imaging to detect CTCs and quantify their dynamics in any cancer mouse model. This new assay is opening the door to the study of CTC dynamics in a variety of animal models. These studies may inform clinical decision on the appropriate timing of blood sampling and value of longitudinal CTC enumeration in cancer patients.

In Vivo Photoswitchable Flow Cytometry for Direct Tracking of Single Circulating Tumor Cells

Photoswitchable fluorescent proteins (PSFPs) that change their color in response to light have led to breakthroughs in studying static cells. However, using PSFPs to study cells in dynamic conditions is challenging. Here we introduce a method for invivo ultrafast photoswitching of PSFPs that provides labeling and tracking of single circulating cells. Using invivo multicolor flow cytometry, this method demonstrated the capability for studying recirculation, migration, and distribution of circulating tumor cells (CTCs) during metastasis progression. In tumor-bearing mice, it enabled monitoring of real-time dynamics of CTCs released from primary tumor, identifying dormant cells, and imaging of CTCs colonizing a primary tumor (self-seeding) or existing metastasis (reseeding). Integration of genetically encoded PSFPs, fast photoswitching, flow cytometry, and imaging makes invivo single cell analysis in the circulation feasible to provide insights into the behavior of CTCs and potentially immune-related and bacterial cells in circulation.

Dynamic Fluctuation of Circulating Tumor Cells during Cancer Progression

Circulating tumor cells (CTCs) are a promising diagnostic and prognostic biomarker for metastatic tumors. We demonstrate that CTCs’ diagnostic value might be increased through real-time monitoring of CTC dynamics. Using preclinical animal models of breast cancer and melanoma and in vivo flow cytometry with photoacoustic and fluorescence detection schematics, we show that CTC count does not always correlate with the primary tumor size. Individual analysis elucidated many cases where the highest level of CTCs was detected before the primary tumor starts progressing. This phenomenon could be attributed to aggressive tumors developing from cancer stem cells. Furthermore, real-time continuous monitoring of CTCs reveals that they occur at highly variable rates in a detection point over a period of time (e.g., a range of 0–54 CTCs per 5 min). These same fluctuations in CTC numbers were observed in vivo in epithelial and non-epithelial metastatic tumors, in different stages of tumor progression, and in different vessels. These temporal CTC fluctuations can explain false negative results of a one-time snapshot test in humans. Indeed, we observed wide variations in the number of CTCs in subsequent blood samples taken from the same metastatic melanoma patient, with some samples being CTC-free. If these phenomena are confirmed in our ongoing in vivo clinical trials, this could support a personalized strategy of CTC monitoring for cancer patients.

Abstract OT2-6-10: DETECT IV – A multicenter, single arm, phase II study evaluating the efficacy of everolimus in combination with endocrine therapy in patients with HER2-negative, hormone-receptor positive metastatic breast cancer and HER2-negative circulating tumor cells (CTCs)

Abstract BACKGROUND: The monitoring of therapy efficacy in metastatic breast cancer (MBC) is a very important component of treatment. Several studies have indicated that the determination of prevalence and number of circulating tumor cells (CTCs) at various times during treatment may be an effective tool for assessing treatment efficacy. Even if the prognostic value of CTCs in MBC is well understood, the role of CTC prevalence and - in particular - CTC phenotype in predicting treatment response requires further investigation. While the recently started DETECT III-trial is designed to evaluate the effect of HER2-targeted therapy in patients with HER2-negative MBC and HER2-postive CTCs, DETECT IV aims to assess the effect of an endocrine treatment in combination with the mTOR-inhibitor everolimus for patients with HER2-negative CTCs. SPECIFIC AIMS/TRIAL DESIGN: DETECT IV is a prospective, multicenter, open-label, single arm phase II study aimed at postmenopausal patients with hormone-receptor positive, HER2-negative metastatic breast cancer. Patients with HER2-negative CTCs will be treated with Everolimus in combination with an endocrine therapy according to the physician's choice (letrozole, anastrozole, exemestan or tamoxifen). The primary objective of the trial is to estimate the clinical efficacy of everolimus in combination with endocrine therapy as assessed by progression-free survival (PFS) in the targeted patient population. Additional research will be done on CTC dynamics and characteristics (e.g. PI3K-mutation-analysis). This will provide a better understanding of prognostic and predictive value of the CTCs. This clinical trial is one more step into a more personalized therapy for metastatic breast cancer and will provide further information on CTCs and their informative value during the therapy. ELIGIBILITY CRITERIA: In this trial postmenopausal female patients suffering from hormone-receptor positive, HER2-negative metastasizing breast cancer with HER2-negative circulating tumor cells (CTC) and indication for standard anticancer therapy will be included. STATISTICAL METHODS/TARGET ACCRUAL: DETECT IV will start in September 2013. The estimated number of patients is 400. Therefore, 2000 patients need to be screened for HER2-negative CTCs. This screening is combined with the DETECT III - trail, which started February 2012. The primary endpoint progression-free survival will be estimated using the Kaplan-Meier method. Prevalence and number of CTCs at various time points will be determined using the FDA-approved CellSearch System (Veridex, USA). In addition, different measures of CTC dynamics and their value for evaluating therapy efficacy or as a prognostic tool will be examined in detail by explorative data analyses. Contact information: fabienne.schochter@uniklinik-ulm.de. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT2-6-10.

Abstract OT1-1-11: DETECT III - A multicenter, randomized, phase III trial to compare standard therapy alone versus standard therapy plus lapatinib in patients with initially HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells

Abstract Background: In metastatic breast cancer (MBC) patients, HER2 status may be subject to change and it has been shown that 20-30% of initially HER2-negative patients have HER2-positive metastases. The discordance of the HER2 status between primary tumor and circulating tumor cells (CTCs) may be an important factor affecting the response to HER2-targeted treatments; however, it is unclear if therapy based on the HER2 status of CTC offers a clinical benefit for patients. Trial Design: DETECT III is a recruiting prospective, multicenter, open-label, two-arm, phase-III study for patients with HER2-negative MBC. Patients with HER2-positve CTCs are randomized to receive either a standard therapy of the physician's choice alone or a standard therapy plus additional HER2-targeted treatment with Lapatinib. Main Eligibility criteria: - Metastatic breast cancer; - HER2-negative primary tumor tissue and/or biopsies from metastatic sites; - Evidence of at least 1 HER2-positive CTC out of 7.5 ml blood; - Indication for systemic treatment - ≥1 lesion evaluable according to RECIST Specific aims: The objective of the DETECT III trial is to estimate the clinical efficacy of standard therapy plus lapatinib as assessed by progression free survival (PFS) in patients with initially HER2-negative MBC and HER2-positive CTCs. Additional research will be performed on CTC dynamic and characteristics. Present accrual and target accrual: The DETECT III – trial started February 2012 and has analyzed so far (2013-06-06) 506 patients. We found CTCs in 336 (66.4%) patients (median 7 CTCs, range 1-35078). 67 of these patients (19.9%) had at least one HER2 positive CTC. Estimated patient enrollment is 228. Methods: The primary endpoint progression-free survival will be estimated using the Kaplan-Meier method. Prevalence and number of CTCs at various time points as well as the HER2 status of CTCs will be determined using the FDA-approved CellSearch System (Veridex, USA). In addition, different measures of CTC dynamics and their value for evaluating therapy efficacy or as a prognostic tool will be examined in detail by explorative data analyses. Perspectives: The DETECT III-Trail is the first study where treatment is based on phenotypic characteristics of CTCs. If this trial succeeds in proving efficacy of lapatinib in patients with initially HER2-negative primary tumor but HER2-positive CTCs, it may lead to a new strategy for the treatment of metastatic breast cancer. Contact information: nikolaus.de-gregorio@uniklinik-ulm.de. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT1-1-11.

Real‐time monitoring of circulating tumor cell release during tumor manipulation using in vivo photoacoustic and fluorescent flow cytometry

Circulating tumor cells (CTCs) form metastases in distant organs. The purpose of this research was to determine if tumor manipulation could enhance cancer cell release from the primary tumor into the circulatory system. Nude mice were inoculated with melanoma or breast cancer cells. The implanted tumor underwent compression, biopsy, complete resection, or laser treatment. CTCs were monitored in the bloodstream using in vivo photoacoustic and fluorescence flow cytometry. We discovered that pressure, biopsy, and laser treatment can dramatically increase CTC counts (up to 60-fold), whereas proper tumor resection significantly decrease CTC counts. Standard medical procedures could trigger CTC release that may increase the risk of metastases. This finding suggests the guidance of cancer treatment and likely diagnosis by real-time monitoring of CTC dynamics followed by well-timed treatment to reduce CTCs in the blood. In vivo detection of intervention-amplified CTCs could be used for early diagnosis of a small tumor, which is undetectable with conventional methods.

In vivo flow cytometry: A powerful optical technology to detect circulating tumor cells and diagnose cancer metastasis in vivo/In-vivo-Durchflusszytometrie: Ein leistungsstarkes optisches Verfahren zur Detektion zirkulierender Tumorzellen und zur In-vivo-Diagnose von Metastasen

AbstractOptical methods have revolutionized medical diagnoses, therapies and research, and have penetrated many medical disciplines for example, tumor diagnosis and treatment. Tumor recurrence and metastasis are the major obstacles for long-term survival in cancer. The metastasis is reported to be correlated with the presence of circulating tumor cells (CTCs) in the vasculature as a consequence of either advanced tumor growth and invasion, or a therapeutic intervention. Thus, CTCs are considered to be important biomarkers for cancer progress and prognosis. The quantification of CTCs is an emerging tool used to diagnose, stratify and monitor patients with metastatic diseases. A number of methods have been developed to detect CTCs, some of which have been applied in clinical diagnosis. However, conventional methods are restricted by invasiveness, lower sensitivity caused by small blood sample volumes, and difficulty in recording the dynamics of CTCs. A novel technique named

Circulating tumor cell (CTC) counts and CTC telomerase activity (TA) as prognotic markers of overall survival (OS) in SWOG S0421: Docetaxel with or without atrasentan for metastatic castration-resistant prostate cancer (mCRPC).

1 Background: CTCs are promising biomarkers in mCRPC, and telomerase activity (TA) is a recognized cancer marker. In this phase III trial we analyzed CTCs using 2 methods: CellSearch for fixed cell enumeration, and a novel Parylene-C slot filter for live CTC capture and TA measurement. Methods: Blood samples (7.5 ml) were drawn at baseline (d1) and pre-cycle 2 (d21) of Rx and shipped overnight for central processing. For CellSearch enumeration, Cox regression was used to evaluate the association between OS, baseline CTC counts, and CTC dynamics (d1 to d21). For TA, filter-trapped cells were lysed and assayed for TA using qPCR-based telomeric repeat amplification. Cox regression evaluated the association between OS and TA overall and within subgroups characterized by good vs. poor (&lt;5 vs. &gt;=5) prognosis baseline CTC counts. For all measurements, receiver operator characteristic (ROC) analysis and characteristics and regression trees (CART) were used to explore further prognostic cutpoints. Results: Samples were obtained from 263 men. Median d1 CTC count was 5, and there was a significant difference in OS for d1 CTC &lt; vs. &gt;=5, hazard ratio (HR) 2.92 (p&lt;0.001) after adjustment for other factors. D1 CTC and OS had ROC AUC of 0.781. In men with low d1 CTC (&lt; 5), an increase in CTC was associated with shorter OS, HR 4.04 (p=0.004); in men with high d1 CTC (&gt;=5), a &gt;=2-fold decrease in CTC was associated with longer OS, HR 0.45 (p=0.012); adjusting for risk factors. For TA, men with baseline CTC &gt;=5 (41% of cohort) who had high CTC TA had HR 1.14 (p&lt;0.005) for OS after adjustment for other factors including CTC counts. CART identified additional risk subgroups based on CTC counts and TA. Conclusions: In this phase III trial, d1 CTC and d1 to d21 CTC dynamics were prognostic of OS after risk factor adjustment, comprising the largest docetaxel-based prospective cohort to date which validates a 5 CTC prognostic threshold and identifies new potentially useful enumeration subgroups. In men with CTCs &gt;=5, TA from cells live-captured on a new slot filter constitutes the first CTC-derived biomarker prognostic of OS in a prospective clinical trial.