Prognostic Value Of Circulating Tumor Cells Research Articles

Abstract P4-11-21: Prevalence of circulating tumor cells (CTCs) after five years of zoledronate treatment in the adjuvant SUCCESS-A study

Abstract Aim: The prognostic value of CTCs at primary diagnosis has recently been confirmed by the SUCCESS A Study (Rack et al. JNCI 2014). Key questions on the role of adjuvant bisphosphonate treatment, including patient populations deriving benefit and optimal timing/scheduling of therapy are still controversial. Aim of this study was therefore to evaluate CTCs in the context of two different zoledronate regimens. Methods: The SUCCESS A trial is a large, randomized, open-label, 2x2 factorial design Phase III study in patients with high risk breast cancer (stage N1 or T2–T4 or grade 3 or age ≤ 35 or hormone-receptor negative). Patients were first randomized to adjuvant chemotherapy treatment with 3 cycles of Epirubicin-Fluorouracil-Cyclophosphamide (FEC) followed by either 3 cycles of Docetaxel or 3 cycles of Gemcitabine-Docetaxel. In addition, patients were randomized to 2 years vs. 5 years of zoledronate treatment. CTC status 5 years after primary diagnosis was assessed using the FDA-approved CellSearch System (Veridex, USA), and CTC positivity was defined as ≥ 1 CTC. We studied the influence of zoledronate treatment duration on CTC prevalence at 5 years in addition to well-known patient and tumour characteristics using a multiple logistic regression analysis on the ITT population. Results: Data on CTC status at 5 years after primary diagnosis were available for 728 (19.4%) out of 3754 randomized patients. 310 patients had been randomized to 2 years of zoledronate treatment and 418 patients had been randomized to 5 years of zoledronate treatment. In these patients a difference in CTC positivity after 5 years could not be shown between patients randomized to 2 or 5 years of zoledronate (p = 0.13, Wald test). The adjusted odds ratio for 2 years vs 5 years was 0.65 (95%CI: 0.37 to 1.13). Conclusions: The final survival analysis of the SUCCESS A trial will give further insight, whether CTCs can be used as early predictive marker for the efficacy of adjuvant zoledronate treatment. Citation Format: Brigitte Rack, Peter A Fasching, Lothar Häberle, Thomas Friedl, Mahdi Rezai, Jörn Hilfrich, Hans Tesch, Georg Heinrich, Helmut Forstbauer, Julia Neugebauer, Elisabeth Trapp, Susanne Albrecht, Bernadette Jäger, Tanja Fehm, Volkmar Müller, Andreas Schneeweiss, Klaus Friese, Werner Lichtenegger, Matthias W Beckmann, Wolfgang Janni. Prevalence of circulating tumor cells (CTCs) after five years of zoledronate treatment in the adjuvant SUCCESS-A study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-21.

Abstract P4-01-04: Circulating tumor cells (CTC) are associated with defects in innate and adaptive immunity in inflammatory breast cancer (IBC) patients

Abstract Background: CTCs play a crucial role in tumor dissemination and are prognostic factor in primary and metastatic breast cancer patients. Immune cells in peripheral blood (PB) contribute to an unfavorable microenvironment for the CTCs survival. As such, effective host innate and adaptive immune surveillance systems could adversely influence tumor dissemination whereas dysfunctional immune systems could provide a favorable microenvironment for the dissemination of CTCs and cancer progression. This study aimed to correlate CTCs with the functions of innate [natural killer (NK) cells] and adaptive (T-cells) immune effector cells in PB of IBC patients. Methods: This prospective study included 65 IBC (21 non-metastatic, 14 de novo metastatic and 30 recurrent metastatic) patients treated between October 2008 and April 2012 at the MD Anderson Cancer Center. CTCs were enumerated before patients started a new line of chemotherapy using the CellSearch® system, 33 (50.8%) of patients were treatment naïve at the time of blood collection. The phenotype of T cells, their ability to secrete cytokines following activation through the T-cell receptor (TCR) and the NK cell subsets were analyzed by multiparameter flow cytometry and the results were correlated with CTCs and clinical outcome. For survival analysis immune cell counts were dichotomized to low or high category using the median count. Results: At least 1 CTC (≥ 1) or ≥ 5 CTCs per 7.5 mL of PB was detected in 40 (61.5%) or 21 (32.3%) of patients, respectively. Patients with at least 1 CTC or ≥ 5 CTCs had a significantly inferior overall survival (OS) [HR=2.48, p=0.003 and HR=1.85, p=0.045] than patients with no CTCs or with <5 CTCs, respectively. There was no correlation between CTCs count and total lymphocytes; however, patients with at least 1 CTC or ≥5 CTCs had significantly lower percentages of CD3+ and CD4+ T-cells compared with patients with no CTCs or < 5 CTCs, respectively. Patients with ≥ 1 CTC, had a lower percentage of TCR-activated CD8+ T-cells producing TNF-α (p=0.03) and IFN-γ (p=0.08), and a higher percentage of T regulatory lymphocytes (p=0.05) compared to patients with undetectable CTCs. Moreover, CTCs ≥5 was inversely associated with the percentage of the following NK cells subsets: non-ADCC NK (Spearman rho' = -0.30, p=0.02), ADCC NK (rho' = -0.15, p=0.20) and exhausted NK (rho = -0.24, p=0.04). We also observed increased prognostic value of CTCs in the context of adaptive immune cells, with worse OS for patients with ≥ 5 CTCs and low count of TCR-activated CD8+ T cells producing TNF-α (HR=6.72, p=0.0007) compared with patients with < 5 CTCs and high count of TCR-activated CD8+ that produced TNF-α. Conclusions: IBC patients with CTCs in PB had abnormalities in both innate and adaptive immunity as evidenced by low percentages of NK cell subsets, and low percentage of TCR-activated CD8+ T cells producing TNF-α, respectively. These data illustrate an inverse relationship between CTCs and both innate and adaptive immune cells in the PB microenvironment that could potentially impact tumor cell dissemination and initiation of the metastatic cascade. Moreover, immune cell profiling could add further prognostic value to CTCs in IBC patients. Citation Format: Michal Mego, Hui Gao, Evan N Cohen, Simone Anfossi, Antonio Giordano, Sanda Tin, Tamer M Fouad, Wendy A Woodward, Ricardo H Alvarez, Vicente Valero, Naoto T Ueno, Gabriel N Hortobagyi, Massimo Cristofanilli, James M Reuben. Circulating tumor cells (CTC) are associated with defects in innate and adaptive immunity in inflammatory breast cancer (IBC) patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-04.

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

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 P3-12-09: The prevalence and quantity of circulating tumor cells (CTCs) after adjuvant chemotherapy with and without anthracyclines in patients with HER2-negative early breast cancer (EBC)

Abstract Background: The use of anthracycline based chemotherapy in early breast cancer (EBC) patients has been well established. However, adverse effects like cardiotoxicity and efficacy in certain subgroups continue to be subjects of discussion. Based on data suggesting a limited benefit of anthracyclines in HER2-negative patients, the German SUCCESS C study randomly assigned patients with EBC to be treated with either anthracycline-containing or anthracycline-free chemotherapy. Since the prognostic value of CTCs in EBC has already been demonstrated in several trials, we compared the prevalence of CTCs after the completion of chemotherapy between both treatment arms. Methods: The SUCCESS C trial was a randomized, open-label, Phase III study comparing disease free survival (DFS) in patients with HER2-negative EBC. Treatments were either 3 cycles epirubicin, 5-fluorouracil and cyclophosphamide followed by 3 cycles of docetaxel (FEC–DOC), or 6 cycles of an anthracycline-free regimen with docetaxel and cyclophosphamide (DOC-C). The CTC status at chemotherapy cycle 6 was prospectively evaluated using the FDA-approved CellSearch System (Veridex, USA). Results: Data on CTC status after chemotherapy are available for 1757 patients. Overall, CTCs were found in 220 (12.5%) patients (median 1, range 1 – 18 CTCs). One CTC was detected in 123 (55.9%), two CTCs in 53 (24.1%), three to five CTCs in 37 (16.8%), and more than five CTCs in 7 (3.2%) of these patients. Univariate analyses revealed that CTC prevalence was not significantly associated with tumor size (pT1, pT2, pT3, pT4), nodal stage (pN0, pN1, pN2, pN3), grading (G1, G2, G3), histological type (invasive ductal, invasive lobular, other), estrogen-receptor status, or progesterone-receptor status (Chi-square tests, all p > 0.1). There was no significant difference with respect to the prevalence of CTCs after chemotherapy between the two treatment arms (Chi-square test, p = 0.23), as CTCs were detected in 11.6% (103 out of 889) of patients treated with the anthracycline-containing chemotherapy regimen and in 13.5% (117 out of 868) of patients treated with the anthracycline-free chemotherapy regimen. In addition, there was no significant difference between the two treatment arms with regard to the number of CTCs detected after chemotherapy in CTC-positive patients (FEC-DOC: median = 1, range 1 – 18; DOC-C: median = 1, range 1 – 8; Mann-Whitney U test, p = 0.30). Conclusions: The comparable prevalence and number of CTCs after the completion of chemotherapy may indicate that anthracycline-free chemotherapy is not inferior to anthracycline-containing chemotherapy in this study. This however, has to be confirmed by survival analyses, which will be available in 2014. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-12-09.

716 Differential Survival in Patients With Hepatitis B Virus-Related Hepatocellular Carcinoma (HBV/HCC) Compared to Hepatitis C Virus-Related Hepatocellular Carcinoma (HCV/HCC)

Background/Aims: Detection of circulating tumor cells (CTC) is associated with poor progression free and overall survival in patients with breast, prostate, and colon cancer. There is limited information about the prognostic value of CTC in patients with cholangiocarcinoma (CCA). The aim of this study are (1) to quantify circulating tumor cells in the peripheral blood of patients with CCA; and (2) to test the hypothesis that the presence of circulating tumor cells is associated with larger tumor burden of CCA and poor overall survival. Methods: A total of 45 patients with CCA seen at Mayo Clinic Rochester were prospectively enrolled between June 2010 and September 2012. 10 ml peripheral blood was drawn into CellSave Preservative Tubes. Blood samples were processed within 96 hours after the blood draw. The CellSearch system by Veridex was used for the capture, enrichment, identification and enumeration of CTC of epithelial origin in 7.5 ml of peripheral blood from the patients. The CellSearch system has an estimated sensitivity of approximately 20-30% for detecting CTC at a prognostic level . The association between baseline characteristics and detection of CTC was analyzed. Overall survival was estimated by the Kaplan Meier method and compared using the Log Rank test. Results: The mean age of patients was 61 and 29 (64%) patients were male. CTC were detected in 13 (28%) patients. Patients with CTC tended to have larger tumor size, more tumor nodules, a higher CA19-9 level, increased CEA, lymph node involvement, and metastatic disease compared to patients without CTC. (Table) There was no significant association between CTC and demography (age, gender, and race) or underlying liver dysfunction . There was a trend towards poorer survival in patients with CTC compared to patients without CTC (p=0.09). Conclusion: CTC are detectable in patients with cholangiocarcinoma. The presence of CTC was associated with tumor burden and metastatic cancer. The prognostic implications of CTC in patients with CCA need further validation in a larger patient group.

631 Circulating Tumor Cells Are Associated With More Advanced Disease in Cholangiocarcinoma

Background/Aims: Detection of circulating tumor cells (CTC) is associated with poor progression free and overall survival in patients with breast, prostate, and colon cancer. There is limited information about the prognostic value of CTC in patients with cholangiocarcinoma (CCA). The aim of this study are (1) to quantify circulating tumor cells in the peripheral blood of patients with CCA; and (2) to test the hypothesis that the presence of circulating tumor cells is associated with larger tumor burden of CCA and poor overall survival. Methods: A total of 45 patients with CCA seen at Mayo Clinic Rochester were prospectively enrolled between June 2010 and September 2012. 10 ml peripheral blood was drawn into CellSave Preservative Tubes. Blood samples were processed within 96 hours after the blood draw. The CellSearch system by Veridex was used for the capture, enrichment, identification and enumeration of CTC of epithelial origin in 7.5 ml of peripheral blood from the patients. The CellSearch system has an estimated sensitivity of approximately 20-30% for detecting CTC at a prognostic level . The association between baseline characteristics and detection of CTC was analyzed. Overall survival was estimated by the Kaplan Meier method and compared using the Log Rank test. Results: The mean age of patients was 61 and 29 (64%) patients were male. CTC were detected in 13 (28%) patients. Patients with CTC tended to have larger tumor size, more tumor nodules, a higher CA19-9 level, increased CEA, lymph node involvement, and metastatic disease compared to patients without CTC. (Table) There was no significant association between CTC and demography (age, gender, and race) or underlying liver dysfunction . There was a trend towards poorer survival in patients with CTC compared to patients without CTC (p=0.09). Conclusion: CTC are detectable in patients with cholangiocarcinoma. The presence of CTC was associated with tumor burden and metastatic cancer. The prognostic implications of CTC in patients with CCA need further validation in a larger patient group.

Meta-Analysis of the Prognostic Value of Circulating Tumor Cells in Breast Cancer

The prognostic value of circulating tumor cells (CTC) detected in breast cancer patients is currently under debate. Different time points of blood collections and various CTC assays have been used in the past decades. Here, we conducted the first comprehensive meta-analysis of published literature on the prognostic relevance of CTC, including patients with early and advanced disease. A comprehensive search for articles published between January 1990 and January 2012 was conducted; reviews of each study were conducted and data were extracted. The main outcomes analyzed were overall survival (OS) and disease-free survival (DFS) in early-stage breast cancer patients, as well as progression-free survival (PFS) and OS in metastatic breast cancer patients. Pooled hazard ratio (HR) and 95% confidence intervals (CIs) were calculated using the random and the fixed-effects models. Subgroup and sensitivity analyses were also conducted. Forty-nine eligible studies enrolling 6,825 patients were identified. The presence of CTC was significantly associated with shorter survival in the total population. The prognostic value of CTC was significant in both early (DFS: HR, 2.86; 95% CI, 2.19-3.75; OS: HR, 2.78; 95% CI, 2.22-3.48) and metastatic breast cancer (PFS: HR, 1.78; 95% CI, 1.52-2.09; OS: HR, 2.33; 95% CI, 2.09-2.60). Further subgroup analyses showed that our results were stable irrespective of the CTC detection method and time point of blood withdrawal. Our present meta-analysis indicates that the detection of CTC is a stable prognosticator in patients with early-stage and metastatic breast cancer. Further studies are required to explore the clinical utility of CTC in breast cancer.

Circulating tumor cells in immunohistochemical subtypes of metastatic breast cancer: lack of prediction in HER2-positive disease treated with targeted therapy

Circulating tumor cells (CTCs) are associated with inferior prognosis in metastatic breast cancer (MBC). We hypothesized that the relationship between CTCs and disease subtype would provide a better understanding of the clinical and biologic behavior of MBC. We retrospectively analyzed 517 MBC patients treated at a single institution. Subtypes of primary tumors were analyzed by immunohistochemical (IHC) or fluorescent in situ hybridization analyses and CTCs were enumerated by CellSearch(®) at starting a new therapy. Overall survival (OS) and progression-free survival durations for each IHC subtype were determined. At a median follow-up of 24.6 months, 276 of 517 (53%) patients had died. The median OS for patients with <5 and ≥ 5 CTCs were 32.4 and 18.3 months, respectively (P < 0.001). Except in HER2+ patients, the prognostic value of CTCs was independent of disease subtype and disease site. In this large retrospective study, CTCs were strongly predictive of survival in all MBC subtypes except HER2+ patients who had been treated with targeted therapy. Our results clearly demonstrate the value of enumerating CTCs in MBC and strongly suggest an interesting biological implication in the HER2+ subset of patients that need to be further explored.

P4-07-10: Circulating Tumor Cells in Newly Diagnosed Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer and is associated with special clinical and biological features. Circulating tumor cells (CTC) are an independent prognostic factor in metastatic breast cancer for progression-free and overall survival. Previously we showed that metastatic IBC patients treated with first and subsequent lines of chemotherapy had a lower number of CTC when compared with CTC levels of non-IBC patients; hence the prognostic value of CTC in mIBC patients is limited. In the present study, we investigated the prognostic value of baseline CTC in newly diagnosed IBC patients. Patients and methods: This retrospective study included 84 newly diagnosed IBC (37 stage III B or C and 47 stage IV) patients treated with neoadjuvant therapy or first line chemotherapy between January 2004 and July 2009 at the MD Anderson Cancer Center. The median age was 55 years (range, 23–78). Twenty-four (28.8%) patients were HER2 positive, 36 (42.9%) hormone receptor positive and 33 (39.3%) were triple receptor negative. CTC were detected and enumerated using the CellSearch® system before patients started chemotherapy. Progression free survival (PFS) and overall survival (OS) were calculated from the date of CTC measurement and estimated by the Kaplan-Meier product limit method. Results: At baseline, 64 (76.2%) patients had at least ≥1 CTC and 29 (34.5%) ≥5 CTC. Proportions of patients with stage III IBC with ≥1 CTC and ≥5 CTC were lower than those of newly diagnosed stage IV IBC patients (67.6% vs. 86%; p = 0.12 and 24.3% vs. 42.6%; p = 0.11, respectively). At a median follow up of 22.4 months (range: 4.8 - 58.3 months), 48 (57.1%) patients experienced disease progression and 26 (31.0%) had died. Patients with &amp;lt; 5 CTC had similar PFS (p = 0.36) and OS (p = 0.80) to those of patients with ≥5 CTC, respectively. We investigated the prognostic value of CTC ≥ 1 in stage III IBC patients, as well. There was no difference in PFS (p = 0.71) and OS (p = 0.15) in patients with CTC = 0 vs. those of patients with CTC ≥ 1, respectively. Moreover, we correlated CTC levels with prognosis, using different CTC threshold, but we observed no correlation between baseline CTC and patients’ outcome (Table 1). Conclusions: CTC can be detected in a large proportion of patients with newly diagnosed IBC; however, baseline CTC was not prognostic for PFS and OS. Collective dissemination of cancer cells via lymphovascular tumor emboli and/or epithelial-mesenchymal transition accompanied by inadequate detection of CTC due to lack of sensitivity of current assays may account for limited prognostic value of CTC in newly diagnosed IBC. Nevertheless, sample size and study power could influence study results as well. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-10.

Determination of cut-offs for circulating tumor cell measurement in metastatic cancer

Evaluation of: Pierga JY, Hajage D, Bachelot T et al. High independent prognostic and predictive value of circulating tumor cells compared with serum tumor markers in a large prospective trial in first-line chemotherapy for metastatic breast cancer patients. Ann. Oncol. DOI: 10.1093/annonc/mdr263 (2011) (Epub ahead of print).The metastatic transformation of epithelial tumors progresses through various steps leading to the generation of circulating tumor cells (CTCs). Measurement of CTCs in the peripheral blood is being increasingly recognized as a promising tool in breast oncology. Several studies have evaluated the prognostic significance of CTCs in newly diagnosed metastatic breast cancer (MBC) patients. The IC 2006-04 was a high-powered, prospective, multicenter, observational study conceived to assess CTC changes in women with MBC treated with first-line chemotherapy. Levels ≥5 CTCs/7.5 ml blood at baseline and before the second cycle of treatment were independent prognostic factors associated with shorter progression-free and overall survival. This study provides further level II evidence for the clinical and prognostic value of CTCs in MBC, confirming data from earlier small studies. It also provides proof that CTCs should be investigated in ongoing interventional trials to see if better patient outcomes can be attained by altering treatment based on CTC levels.

Circulating tumor cells count and characterization in a male breast cancer patient

A 64-y-old man presented to Medical Oncology Department a metastatic invasive ductal breast carcinoma, positive for estrogen (ER) and progesterone receptors (PR) and Her2/neu negative. The patient was treated with different lines of therapy, with rapid radiological progression of disease. After four courses of a third-line chemotherapy, a radiological stable disease was maintained. The patient was followed by serial blood drawings for the characterization and count of circulating tumor cells (CTC). This is the first report concerning the predictive and prognostic value of CTC in a male breast cancer patient.

Abstract P3-02-03: Prognostic Value of Circulating Tumor Cells (CTC) in HER-2 Positive Metastatic Breast Cancer (MBC) Patients (pts) Treated with Anti-HER2-Based Therapy

Abstract Background. Detection of ≥5 CTC per 7.5 mL of blood before starting systemic treatment is a strong and independent predictor of worse progression-free survival (PFS) and overall survival (OS) in MBC. Trastuzumab (Herceptin®) was shown to be highly effective in reducing CTC number in all HER-2 amplified MBC cases after the first dose (De Giorgi et al., SABCS 2009). Furthermore, trastuzumab can target chemotherapy-resistant CTC in pts with breast cancer (Bozionellou et al. 2004). To date no significant analysis exploring the effect of another anti-HER2 therapy, lapatinib (Tykerb®) on CTC has been published.We hypothesized that anti-HER2-based therapy may modify the detection rate and prognostic value of CTC in pts with HER-2 amplified MBC. Patients and Methods. We retrospectively evaluated 91 HER-2 positive MBC pts, treated at the M. D. Anderson Cancer Center with trastuzumab-based (n=59), or lapatinib-based therapy (n=32). In all pts, CTC were enumerated before starting systemic treatments, using CellSearch™. Follow-up CTC counts were available for 41 (69%) pts treated with trastuzumab and 25 (78%) pts treated with lapatinib. PFS and OS were estimated by Kaplan-Meier product limit, and compared between groups according to baseline CTC count (&amp;lt;5 vs ≥5) by log-rank test. Results. Median follow-up was 27 months (range, 8-65) and 16 months (range, 3-33) for pts receiving trastuzumab and lapatinib, respectively. Treatments administered and CTC counts are reported in table 1. Two (5%) pts receiving trastuzumab and 4 (16%) pts treated with lapatinib had CTC ≥5 at the follow-up assessment. Estimated median PFS and OS according to baseline CTC value, for both treatments are shown in table 2. An elevated CTC count was associated with poor prognosis in pts treated with lapatinib (median OS: 25.8 vs 13.6 months in pts with CTC &amp;lt;5 and ≥5, respectively, P=.072). Surprisingly, in the trastuzumab group, PFS showed a non-statistically significant trend in favor of pts with CTC ≥5 compared to those with &amp;lt;5. Conclusions. According to previously published data, our study confirms that trastuzumab-based therapies can drastically reduce the number of CTC and neutralize their negative prognostic value. Furthermore, we found that lapatinib had a limited effect on the detection rate and prognostic value of CTC, although the low number of pts and the high percentage of pre-treated women do not allow us to draw definitive conclusions. Table 1 Table 2 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-03.

Abstract PD04-04: Cytokeratin-19 mRNA Positive Circulating Tumor Cells during Follow-Up of Patients with Early Breast Cancer: Prognostic Relevance for Late Relapse

Abstract PURPOSE: We reported previously that the detection of CK-19 mRNA-positive circulating tumor cells (CTCs) after adjuvant chemotherapy in patients with early breast cancer is associated with poor clinical outcome. Reliable prognostic markers for late disease relapse are not available. In this study we investigated the prognostic value of CTCs’ detection during the first 5 years of follow-up in predicting late disease relapse. PATIENTS AND METHODS: Blood was obtained from 281 patients with early breast cancer who had not experienced disease relapse during the first two years of follow up. CTCs were detected using a real-time reverse transcriptase polymerase chain reaction for CK 19 mRNA after the completion of adjuvant chemotherapy and every six months thereafter for a 5-year follow up period. Patients were classified into four groups based on the CTCs’ status during the first two years and the subsequent three years of follow up (persistently negative, persistently positive, negative turned to positive and the opposite). RESULTS: The vast majority of patients (92.9%) received some kind of hormonal therapy in addition to chemotherapy. Sixty-nine (24.6%) patients remained CTCs-free throughout the 5-year period. A change in CK-19 mRNA status was observed in 126 (44.9%) patients; 60 (21.4%) patients with initially CK-19 mRNA-positive CTCs during the first 24 months turned CTC-negative afterwards while 66 (23.5%) patients who were initially CTC-negative became CTC-positive. Eighty-six (30.6%) patients remained persistently CK-19 mRNA-positive for the entire 5 years. After 94 months of median follow up (range: 51-147), the relapse rate for each of the above groups of patients is illustrated in the following table. Table 1: CTCs’ detection status and risk of late disease repapse. Compared to the persistently negative, only the group of CK-19 mRNA-persistently positive patients had a statistically significant higher risk of relapse (38.4% versus 7.2%, p&amp;lt; 0.001), shorter progression-free survival (not reached vs 135 months, P&amp;lt;0.001) and significant difference in overall survival (not reached vs 146 months, p=0.045). CONCLUSION: Persistent detection of CK-19 mRNA-positive CTCs in the blood during the first 5 years of follow up after adjuvant chemotherapy is associated with an increased risk of late disease relapse indicating the presence of chemotherapy-and hormonetherapy-resistant residual disease. This prognostic evaluation may be useful when deciding on subsequent adjuvant systemic therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-04.

Abstract S6-6: High Independent Prognostic and Predictive Value of Circulating Tumor Cells in a Large Prospective Multicenter Trial Including Serum Tumor Markers in First Line Chemotherapy Metastatic Breast Patients

Abstract Background: Circulating tumor cells (CTCs) in peripheral blood and decrease during treatment has been reported as an independent prognostic factor in metastatic breast cancer (MBC) (Cristofanilli M., NEJM 2004). Data comparing CTC with circulating tumor markers are limited. We conducted a multicenter prospective study to validate these markers in a large series of patients receiving specifically first line chemotherapy (CT) for MBC including assessment of CA15.3, CEA and LDH. Methods: CTC were measured in 7.5ml of whole blood at baseline, before cycle 2, at clinical and/or radiological evaluation (C3-C4) and at tumor progression. Analysis was performed using the CellSearch™ System, combining EpCAM immunomagnetic selection (IMS) followed by anti-cytokeratin (A45B/B3). Double labeling for HER2 was performed in a subgroup of patients. Tumor markers were assessed locally with ELISA kit and high if above normal. Results: From 06/07 to 9/09, 267 patients with measurable or evaluable disease starting first line CT for MBC were included in five French Cancer Centers. Median follow-up was 16 months, 57 deaths and 160 progressions were observed. Median age was 57 years, 39 patients (15%) had HER2+ tumor and received trastuzumab, 55 (21%) had triple negative tumor, 125 received bevacizumab associated with CT, 260 were evaluable for CTC levels at baseline, 170 (65%) had ≥1CTC and 115 (44%) ≥5CTC. CTC HER2 status was assessed in 113 patients at baseline and was positive in 64% of the cases in at least one cell. CA15.3 was high in159/247 (64%), CEA in 109/212 (51%) and LDH in 99/220 (45%). CTC were highly correlated with tumor markers, tumor burden, performans status (PS), number of metastatic sites, liver metastasis) but were independent of tumor biology (HER2 status, Triple Negative BC (TNBC), grade). At first evaluation (C3-C4), 110/225 patients (49%) had objective response. At univariate analysis, high CTC level with a threshold of 1 or 5 at base line, before C2 (3-4w) and at radiological evaluation (C3-C4, 9-12w) was predictive of poor progression-free survival (PFS) and overall survival (OS) (p&amp;lt;10-5). Baseline CA15.3, CEA and LDH were prognostic for PFS and LDH only for OS. At multivariate analysis, TNBC, PS ≥2, CTC ≥5, and high CEA were prognostic for PFS and TNBC, high LDH, CTC ≥5 and PS ≥2 for OS. Multivariate analyses including CTC and tumor markers variations during treatment are ongoing. Conclusions: This is the largest prospective series validating the prognostic value of CTC in first line chemotherapy MBC independently from serum tumor marker for PFS and OS. Persistence of a high level of CTC before second cycle of chemotherapy is a strong and early predictive marker of poor PFS, before radiological evaluation. A prospective multicenter randomized trial (CIRCE01) to assess the use of CTC to modify treatment after second line chemotherapy is ongoing. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S6-6.

Predictive Value of Circulating Tumor Cells (CTCs) in Metastatic Breast Cancer Patients Treated by Bevacizumab-Based Therapy.

Abstract Background:Circulating tumor cells (CTC) are involved in cancer dissemination and are an independent prognostic factor in metastatic breast cancer (MBC). Antiangiogenic, bevacizumab-based chemotherapy improves response rate and progression free survival in patients with metastatic breast cancer (MBC), without impact on overall survival. Preclinical data suggest the possibility of increased metastatic potential of tumor cells pretreated by anti-angiogenic therapy (Ebos et al. Cancer Cell 2009,15: 232–9). The aim of this study was to determine the prognostic value of CTC in MBC patients treated by bevacizumab-based therapy.Patients and Methods: This retrospective study included 48 MBC treated with bevacizumab combined chemotherapy regimens and 46 patients treated with chemotherapy alone between January 2004 and December 2008 at M.D.Anderson Cancer Center. CTCs were detected and enumerated before patients started therapy using the CellSearch™ system (Veridex, LLC, NJ, USA). Progression free survival (PFS) and overall survival (OS) were calculated from the date of CTC measurement, estimated by the Kaplan-Meier product limit method, and compared between groups with the log-rank test.Results: At a median follow up of 10.1 months (range: 1-26 months), 22 patients (45.8%) had died. The estimated medians of PFS in bevacizumab-treated patients were 8.1 vs. 5.2 months (p = 0.42) in patients with baseline &amp;lt; 5 CTCs vs. ≥ 5 CTCs. Moreover, the OS for the two subgroups were 18.3 vs. 12.4 months (p = 0.41), respectively. Twenty-three patients had CTC measurements at the time of progression. Median CTC counts at baseline and at time of disease progression were 6 (range: 0-230) and 7 (range: 0-359) respectively in the bevacizumab-treated group. The median CTC counts in the control group at same time points were 7 (range: 0-724) and 2 (range:0-999), respectively. Thirteen (56.5%) and 12 (52.2%) patients had CTC ≥ 5 at baseline and at time of disease progression in bevacizumab-treated group compared to CTC counts of 24 (52.2%) and 17 (37%) in control group treated with chemotherapy without bevacizumab. Post progression overall survival in patients with CTC &amp;lt; 5 and CTC ≥ 5 measured at time of disease progression were 11.0 and 10.4 months (p = 0.36), in the bevacizumab treated group vs. 27 and 12.4 months (p = 0.04), in the control group respectively.Conclusion: Our data support the prognostic value of CTC measured before therapy in MBC. The detection of higher CTC counts at time of disease progression and the limited prognostic value of CTC after failure of bevacizumab-based chemotherapy although intriguing, warrants further prospective investigations. Moreover, a comparison between the differential effects of monoclonal antibodies and tyrosine kinases inhibitors on CTCs detection and monitoring will better clarify the role of specific targeted therapies on micrometastatic disease. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3013.

Circulating tumor cells in solid cancer: Tumor marker of clinical relevance?

Circulating tumor cells (CTC) can be detected in the peripheral blood of patients with a variety of solid cancers. Because of their very low frequency, these tumor cells are not easily detected using conventional cytology methods. In the past decade, numerous groups have attempted to detect CTC of solid malignancies using the highly sensitive reverse transcriptase polymerase chain reaction, which has been shown to be superior to conventional techniques. However, the biological significance of CTC and the therapeutic relevance of their detection are still debated. This article reviews the most recent findings on this subject, and discusses the potential of identification and molecular characterization of the subset of CTC responsible for metastasis development. Confirming the prognostic value of CTC would provide clinicians with a unique tool for better stratification of patients' risk and provide basic researchers with a new target for the development of novel therapeutic approaches.

Circulating tumor cells (CTCs) in metastatic breast cancer: Biological value beyond tumor burden

615 Background: The detection of circulating tumor cells (CTCs) can predict progression-free (PFS) and overall survival (OS) in patients with metastatic breast cancer (MBC). We evaluated the prognostic significance of baseline CTCs in relation to standard measures of tumor burden. Furthermore, we assessed if the prognostic value of CTCs was related to any particular molecular phenotype. Patients and Methods: One hundred twenty-three consecutive MBC patients (pts) evaluated prospectively between 12/2000 and 5/2005 were included in this analysis. CTCs from 7.5 mL of whole blood were isolated and enumerated using CellSearch system. Prognostic value was determined by analyzing the following factors, baseline level of CTCs (negative: &lt;5 CTCs/7.5 mL; positive: ≥ 5 CTCs/7.5 mL), age (50 yrs&lt;vs. &gt;/=50 yrs), hormonal receptor (HR), Her-2/neu status, metastatic site (visceral vs. non-visceral), Swenerton score, CA27.29 levels, and previous chemotherapy for MBC (none vs. pre-treated), Results: Median age was 52 years (range 24–88) and the median follow-up was 7.7 months (range 0–53.4 months). The median OS for the alive pts was 17.5 months. Fifty-two pts (42%) had positive CTCs and abnormal CA27.29 was detected in 80 pts (66%). The median OS for patients with negative vs. positive CTCs were 28.3 months (range 1.28 - 31.24) and 12.8 months (range 1.71 to 36.83) respectively (p=0.0001). In the multivariable model, HR status, CTCs, and CA27.29 level were the only factors significantly related to OS. CTCs demonstrated the strongest predictor for OS and were associated with 2.53 times the risk of death (p = 0.003). The prognostic value of CTCs was independent of line of treatment, site of recurrence and phenotype of the disease. Conclusions: CTCs demonstrated prognostic value independent of standard measures of tumor burden and phenotypic characteristics of the disease. CTCs are an important marker of tumor biology in metastatic breast cancer. No significant financial relationships to disclose.