Marker In Metastatic Breast Cancer Research Articles

Diagnostic, prognostic, and treatment monitoring value of plasma X in patients with metastatic breast cancer.

37 Background: Metastasis is the main course of death in breast cancer (BC) patients. Reliable prognostic markers are very much appreciated to evaluate possible outcome of patients. Circulating tumor cells (CTC) is a circulating prognostic tumor marker of metastatic breast cancer patients. Despite the big technical challenges in CTC detection, the reliability of CTC enumeration for the prognosis of BC is still on debate. Some studies have proved that the plasma level of X, a major component in extracellular matrix, was increased in patients with metastatic breast cancer. However, the association between the plasma X level and the prognosis of metastatic breast cancer is still unknown. Methods: Plasma X was measured by ELISA in 60 healthy controls, 48 primary breast cancer (PBC) patients, and 212 metastatic breast cancer (MBC) patients. Progression free survival (PFS) and overall survival (OS) were analyzed. An independent cohort with 210 primary and 69 patients with metastatic breast cancer were further investigated to validate our findings. Results: A 2-fold elevation was presented in MBC patients when compared with PBC patients and controls, regardless of their CTC status. A multivariate Cox regression demonstrated that lower X level at base line was associated with longer PFS (HR: 2.50, 95% CI: 1.72–3.63, p = 1.607 × 10-6), as well as longer OS (HR: 3.74, 95% CI: 1.65 – 8.51, p = 0.002). After 1stcycle of chemotherapy, plasma X level was still prognostic and was dramatically decreased in patients who had responded to the treatment (54%, IQR: 36%–64%). In the independent validation round, a 2-fold increase of plasma X was observed again in MBC patients, compared with PBC patients. The prognostic value was also validated for PFS (HR: 2.12, 95% CI: 1.43– 3.84, p = 0.001) and OS (HR: 2.91, 95% CI: 1.73–7.65, p = 0.002) in MBC patients. Conclusions: Plasma X could be used as a diagnostic and prognostic marker for metastatic breast cancer. Its reduction after 1st cycle of chemotherapy could be an indicator of treatment efficacy. This finding may further prevent unnecessary systemic therapy by facilitating personalized medicine in the treatment of metastatic breast cancer.

Lack of prognostic value of mean corpuscular volume with capecitabine therapy in metastatic breast cancer.

Capecitabine is an oral fluoropyrimidine derivative which is frequently used alone or in combination regimens for the treatment of metastatic breast cancer. Although overall and progression free survivals have increased in recent years with the use of new generation drugs, predictive factors that would further improve the outcomes are needed. Previous studies have demonstrated the relation between post-treatment increase in mean corpuscular volume (MCV) and predicting therapy response as well as survival. The present study investigated the clinical impact of MCV elevation in metastatic breast cancer patients treated with capecitabine. The data of a total of 82 patients from three centers followed between June 2005 and June 2013 were retrospectively analyzed. The demographic data and hormone receptor status of the patients, as well as initial examination before and after treatment and data concerning progression were recorded. MCV ≥ 100 fl was considered as macrocytosis. Capecitabine was given at a dose of 2500 mg/m2 daily for 14 days every three weeks. Pre-treatment and post-treatment MCV and other parameters of complete blood count were recorded. Post-treatment initial evaluation was performed after 2 cycles of therapy. The median age of the patients was 46.5 years (range 26-72 years) and 54% were premenopausal. Performance status was ECOG 0 and 1 in 81 (99%) patients. The median number of cycles for capecitabine therapy was 5 (min-max: 2-18). The median ΔMCV level (post-treatment values at sixth week - baseline) was 6.4. Whilst ΔMCV was ≥ 6.4 in 42 patients, it was <6.4 in 40 patients. Clinical benefit (complete response+partial response+stable disease) was observed in 37 (88%) of 42 patients with a median ΔMCV ≥ 6.4 and in 30 (75%) of 40 patients with ΔMCV <6.4 with no statistically significant difference (p=0.158). No significant difference was determined between the group with ΔMCV ≥ 6.4 and the group with ΔMCV <6.4 in terms of progression-free survival (11 vs 12 months) (p=0.55) and overall survival (20 months vs. 24 months) (p=0.11). The identification of new predictive markers in metastatic breast cancer is very important. In some recent studies, increase in MCV has been suggested as a marker in tumor response. In the present study, however, no significant difference was determined between tumor response and increase in MCV. Further studies including higher numbers of patients are needed to determine whether increase in MCV is a predictive marker or not.

Inflammatory markers in metastatic breast cancer.

e11545 Background: The effects of inflammation on the prognosis, life expectancy and several parameters such as response to treatment of breast cancer have been previously studied. The aim of this study is to find out the importance of serum markers as a prognostic indicator in metastatic breast cancer Methods: Women with metastatic breast cancer at the time of diagnosis were included. The study was conducted in Hacettepe University, Institute of Oncology, Department of Medical Oncology. Patient data were collected between December 2009 and January 2013. For all studied parameters Kaplan-Meier survival estimates and p values computed by log-rank test were calculated. A p value of &lt; 0.05 was considered statistically significant. Results: Median follow-up time was 26 months. There were 29 mortalities due to disease progression during the follow up. The levels of serum albumin, ESR and LDH were not associated with a significant effect on overall survival. Among patients with a higher serum CRP estimated median survival was 84±36 months, compared to 278±113 months among patients with a normal serum CRP (p = 0.49). Among patients with higher serum ferritin levels estimated median survival was 29±10 months, compared to 212±113 months for normal serum ferritin (p = 0.01). Among patients with higher serum beta-2 microglobulin estimated median survival was 28±8 months, compared to 84±57 months for normal levels (p&lt;0.01). Conclusions: Serum CRP, ferritin and beta-2 microglobulin can be useful prognostic factors for overall survival among metastatic breast cancer patients.

Abstract 2410: Circulating tumor cells to guide the choice between chemotherapy and hormone therapy as first line treatment for metastatic breast cancer patients: the STIC CTC METABREAST trial.

Abstract Background Baseline CTC count already demonstrated its very good performance as an independent prognostic marker in metastatic breast cancer (MBC), before the start of any treatment (Cristofanilli, NEJM 2004; Pierga Ann Oncol 2011). Multivariate analyses performed in both a pooled analysis (Liu, ASCO 2011) and in the IC 2006-04 study showed that the other independent prognostic factors were the performance status and hormone-receptor (HR) status. Oppositely, the other criteria that are frequently used to choose between hormone therapy and chemotherapy for the treatment of first line hormone receptor-positive MBC patients (e.g. metastatic sites, metastasis-free interval…) were not independent prognostic factors. On the basis that CTC count may be a better criterion for this important choice than the currently used empiric criteria, we designed a large pivotal phase III trial. Methods N=996 HR+ M+ breast cancer patients will be randomized between the clinician choice and CTC count-driven choice. Main inclusion criteria include hormone receptor-positive MBC patients with no previous treatment for the metastatic disease, and who can receive either hormone therapy or chemotherapy as first line treatment. In the CTC arm, patients with ≥5 CTC/7.5ml will receive chemotherapy whereas patients with &amp;lt;5 CTC/7.5ml will receive endocrine therapy as first line treatment. As every patient will receive a treatment, this pivotal trial has been designed to show a non-inferiority of the CTC arm for PFS (primary clinical endpoint) and a superiority of the CTC arm for the medico-economics study (co-primary endpoint). The STIC CTC METABREAST trial has been funded by the French Ministry of Health (STIC program) and Veridex. The promoter is the Institut Curie (Paris). Results This trial began in February 2012 and more than 15 French cancer centers participate to this study. The accrual should be completed in 2 to 3 years. Conclusion This large pivotal phase III trial is intended to demonstrate the clinical relevance of baseline CTC count. Citation Format: Jean-Yves Pierga, Sandrine Baffert, David Hajage, Etienne Brain, Sebastien Armanet, Cécile Simondi, Laurent Mignot, Bernard Asselain, Patricia Tresca, François-Clément Bidard. Circulating tumor cells to guide the choice between chemotherapy and hormone therapy as first line treatment for metastatic breast cancer patients: the STIC CTC METABREAST trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2410. doi:10.1158/1538-7445.AM2013-2410

Abstract P2-02-03: The Collagen Receptor Endo180: A Metastatic Plasma Marker In Breast Cancer Modulated By Bisphosphonate Treatment

Abstract Purpose: To establish whether the collagen remodelling receptor, Endo180, should be given consideration as a useful plasma marker in metastatic breast cancer. Patients and Methods: Analysis of MCF-7 and MDA-MB-231 cell conditioned medium validated the anti-human (39.10) monoclonal antibody as suitable for the detection of soluble Endo180 in plasma. Eighty-seven breast cancer patients with early primary, locally advanced and metastatic disease were included in the study. Correlations between Endo180, CA 15–3 antigen (MUC1; mucin 1) and bisphosphonate treatment were investigated. Results: Endo180 was elevated in metastatic compared to early breast cancer (P &amp;lt;.0001) and was able to differentiate locoregional disease and visceral and/or osseous metastasis (P = .0005). In combination CA 15–3 antigen (cut-off: 28 U/mL) and Endo180 (relative plasma level cut-off range: 0.95–1.65) had sensitivity of 94–97% and specificity of 52–68%. Endo180 levels were significantly higher in patients who were treatment naive (2.17 ± 0.82, N = 13) compared to those previously (1.82 ± 0.33, N = 5) or currently (1.37 ± 0.74, N = 24) receiving bisphosphonates (P = .011). In the bisphosphonate naive setting (N = 57) the combination of Endo180 (relative plasma level cut-off range: 0.95–1.65) and CA 15–3 antigen (cut-off: 28 U/mL) had a sensitivity of 87–92% and specificity of 72–79%. Conclusion: Endo180 is a plasma marker with high sensitivity in metastatic breast cancer that can be modulated by bisphosphonate treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-02-03.

Abstract OT3-4-06: Circulating tumor cells to guide the choice between chemotherapy and hormone therapy as first line treatment for hormone receptors positive metastatic breast cancer patients: the STIC CTC METABREAST trial

Abstract Background: Baseline CTC count already demonstrated its accuracy as an independent prognostic marker in metastatic breast cancer (MBC), before the start of any treatment (Cristofanilli, NEJM 2004; Pierga Ann Oncol 2011) for progression free survival and overall survival. Multivariate analyses performed in both a pooled analysis (Liu, ASCO 2011) and in the IC 2006–04 study showed that the other independent prognostic factors were the performance status and hormone-receptor (HR) status. Oppositely, the other criteria that are frequently used to choose between hormone therapy and chemotherapy for the treatment of first line hormone receptor-positive MBC patients (e.g. number of metastatic sites, visceral disease, metastasis-free interval…) were not independent prognostic factors. As CTC count is highly correlated to PFS, we designed a large pivotal phase III trial to evaluate the use of CTC to determine the disease aggressiveness and the choice of first line treatment in potentially hormonosensitive MBC. Trial design: First line metastatic breast cancer patients will be randomized between the clinician choice and CTC count-driven choice. In the CTC arm, patients with ≥5 CTC/7.5ml will receive chemotherapy whereas patients with &amp;lt;5 CTC/7.5ml will receive endocrine therapy as first line treatment. Eligibility criteria: Main inclusion criteria include hormone receptor-positive metastatic breast cancer patients with no previous treatment for the metastatic disease, and who can receive either hormone therapy or chemotherapy as first line treatment. Statistical methods: As every patient will receive a treatment, this pivotal trial has been designed to show a non-inferiority of the CTC arm for PFS (primary clinical endpoint) and a superiority of the CTC arm for the medico-economics study (co-primary endpoint). Present accrual and target accrual: This trial began in March 2012 and more than 15 French cancer centers participate to this study. The accrual of 996 metastatic breast cancer patients should be completed in 2 to 3 years. 20 patients have been included in 2 months in the first opened center. Contact information for people with a specific interest in the trial The STIC CTC METABREAST trial has been funded by the French Ministry of Health (STIC program) and Veridex. The promoter is the Institut Curie (Paris). Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-4-06.

Circulating miRNAs as Surrogate Markers for Circulating Tumor Cells and Prognostic Markers in Metastatic Breast Cancer

The use of circulating tumor cells (CTC) as a prognostic marker in metastatic breast cancer (MBC) has been well established. However, their efficacy and accuracy are still under scrutiny mainly because of methods of their enrichment and identification. We hypothesized that circulating miRNAs can predict the CTC status of patients with MBC, and tested for the same. Furthermore, we aimed at establishing a panel of circulating miRNAs capable of differentiating MBC cases from healthy controls. Circulating miRNAs from plasma of CTC-positive and CTC-negative patients with MBC, and healthy controls, were profiled by TaqMan Human MicroRNA arrays. Candidates from the initial screen were validated in an extended cohort of 269 individuals (61 CTC-positive, 72 CTC-negative, 60 CTC-low MBC cases, and 76 controls). CTC-positive had significantly higher levels of miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-375, and miR-801 than CTC-negative MBC and controls (P < 0.00001), whereas miR-768-3p was present in lower amounts in MBC cases (P < 0.05). miR-200b was singled out as the best marker for distinguishing CTC-positive from CTC-negative patients (AUC 0.88). We identified combinations of miRNAs for differentiating MBC cases from controls (AUC 0.95 for CTC-positive; AUC 0.78 for CTC-negative). Combinations of miRNAs and miR-200b alone were found to be promising prognostic marker for progression-free and overall survival. This is the first study to document the capacity of circulating miRNAs to indicate CTC status and their potential as prognostic markers in patients with MBC.

WE-C-217BCD-04: Multimodality Imaging of Breast Cancer Experimental Lung Metastasis.

Metastatic breast cancer (MBC) is incurable. The clinical gold standard for assessing tumor microvessel density (MVD), an independent prognostic marker in MBC, is CD 105 staining. The goal of this study is to develop a positron emission tomography (PET)/near-infrared fluorescent (NIRF) probe for imaging of CD105 expression in MBC (i.e. non-invasive measurement of MVD), as well as other applications such as early detection of metastasis, intraoperative guidance, etc. TRC105, a chimeric anti-CD105 mAb, was dual-labeled with a NIRF dye and 89 Zr to yield 8 9 Zr-Df-TRC105-800CW. Luciferase-transfected 4T1 murine breast cancer cells were injected intravenously into female BALB/c mice to establish a lung MBC model. Bio luminescence imaging (BLI) was carried out to non- invasively monitor the lung tumor burden. Comprehensive in vivo/ex vivo studies were performed to investigate 8 9 Zr-Df-TRC105-800CW in this MBC model. Cetuximab was used as an isotype-matched control. Radiolabeled TRC105 has high tumor uptake in many tumor types in addition to MBC (e.g. pancreatic/prostate cancer and brain tumor), revealing broad clinical potential for TRC105-based agents. FACS analysis of HUVECs showed no difference in CD 105 binding between TRC105 and Df- TRC105-800CW. PET imaging revealed that 4T1 lung tumor uptake of 89 Zr-Df-TRC105-800CW was 8.7±1.4,10.9±0.5, and 9.7±1.1 %ID/g at 4, 24, and 48 h post-injection (n = 4), with excellent tumor contrast. Bio distribution studies, blocking, control studies with 8 9 Zr-Df-cetuximab- 800CW, ex vivo BLI/PET/NIRF imaging, and histology all confirmed CD 105 specificity of the tracer. NIRF imaging-guided removal of 4T1 tumors with Df-TRC105-800CW in a subcutaneous model was also straightforward. We report the first PET/NIRF imaging of CD105 expression in a MBC model. Broad clinical potential of TRC105- based agents was shown in many tumor types, which also enabled early detection of small metastases and provided intraoperative guidance for tumor removal.

Abstract 2371: Circulating cancer associated cells co-express epithelial and hematopoietic markers in metastatic breast cancer (MBC) patients (pts)

Abstract Background: Circulatingtumor cells (CTCs) are most commonly isolatedby using positive selection enrichment targeting the epithelial cell adhesion activating molecule (EpCAM). Using positive selection, a CTC is defined as EpCAM and cytokeratins (CK) positive (+) and CD45 negative (-). However, other rare cancer associated cells (RCACs) including EpCAM - and/or CD45 + cells have been reported (van de Stolpe, 2011). In order to investigate these RCACs, a prospective study in MBC using multi-parameter flow cytometry and immunocytochemistry (ICC) was undertaken. Methods:Forty blood samples from MBC pts were collected prior to initiation of a new line of chemotherapy, after one cycle, or at time of progression. Twenty one (52%) samples were from pts with estrogen receptor (ER) -, progesterone receptor (PR) -, and HER2neu non-overexpressing, or triple negative breast cancer (TNBC); nineteen (47%) were from pts with ER/PR +, HER2neu nonoverexpressing disease. Blood was processed using immunomagnetic negative depletion (Yang, 2009). Multiparameter, flow cytometry analysis was conducted prior to, and after depletion of CD45 + cells. Multistep, sequential labeling and fixation for surface markers was performed, followed by permeablizition for CK. ICC staining for EpCAM, CD45, CK 8/ 18/19 and DAPI was performed. Results: Either very low or no CK + events were present in blood samples from healthy volunteers (n=5). Various post-enrichment events were identified. These populations were also present by ICC with confocal microscopy. Conclusions: In addition to CTCs, RCACS that are CD45 + and/or EpCAM - are detectable in MBC pts and more frequently present in TNBC pts. These populations, only detectable by negative depletion, may be clinically significant as ongoing studies evaluate the predictive capabilities of blood based biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2371. doi:1538-7445.AM2012-2371

Abstract 698: Overexpression of the aryl hydrocarbon receptor correlates with high tumor grade in human breast invasive carcinomas

Abstract The aryl hydrocarbon receptor (AhR) is a ligand-activated basic helix-loop-helix transcription factor which binds environmental poly aromatic hydrocarbons (PAH) and mediates their carcinogenic effects. Recent reports, including data from our laboratory implicate AhR in breast cancer development and progression independent of the receptor occupancy by PAH. In this study we investigated the potential of AhR as a stage specific marker of breast cancer. We examined the expression of AhR by immunohistochemistry in tissue microarrays (TMA) containing 192 specimens of clinically defined three stages of invasive breast cancer: node-negative (NN), node-positive (NP) and metastatic (Met) carcinoma. The TMA were obtained from the National Cancer Institute Cooperative Breast Cancer Tissue Resource [NCI-CBCTR] and were stained for AhR using high affinity antibody. The AhR staining was then scored by three evaluators, including two pathologists who were blinded to the study. Statistical analysis showed a significant correlation between the AhR expression and the carcinoma case type (NN, NP or Met) (p-value of ANOVA is &amp;lt; 0.0003), and although there is a significant difference of AhR expression among carcinoma case type of white women (p&amp;lt; 0.0017) there is no significant difference for black women (p&amp;lt;0.0968). There is also a strong positive correlation between the receptor expression and the tumor grade (p &amp;lt;0.0001). While there is no correlation with the status of estrogen or progesterone receptors (p= 0.1643 and 0.1884, respectively), there is strong correlation of high AhR expression and invasive breast carcinoma in women over 50 years old (p&amp;lt;0.0043). In conclusion, our findings identify the AhR as a new predictive clinical marker for metastatic breast cancer and a unique target for the design of novel selective inhibitors for therapeutic intervention of metastatic breast cancer. More importantly, the AhR overexpression identifies a subset of patients who could benefit from therapy targeting this receptor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 698. doi:1538-7445.AM2012-698

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

Circulating tumor cells (CTCs) are a prognostic marker in metastatic breast cancer, but comparisons with serum tumor markers (CA 15-3, carcinoembryonic antigen and lactate dehydrogenase) variations are needed. CTCs were counted with CellSearch® at baseline, before cycle 2 (C2) and cycle 3 or 4 (C3/4) in 267 metastatic breast cancer patients on first-line chemotherapy with/without targeted therapy. Baseline CTC detection rate was 65% with ≥1 CTC/7.5 ml threshold and 44% with ≥5 CTC/7.5 ml and was independent of subtypes (luminal, triple negative, human epithelial growth factor receptor 2 (HER2)+). CTCs were associated with tumor markers, bone/liver involvement, tumor burden and performance status. CTC detection ≥1 CTC/7.5 ml was a strong prognostic factor for progression-free survival (PFS), P < 0.0001. Threshold of CTC ≥5 was statistically significant for PFS and overall survival (OS), P = 0.03 on multivariate analysis. Among patients with ≥5 CTC/7.5 ml at baseline, 50% had <5 CTC/7.5 ml at C2. Changes were correlated with both PFS and OS (P < 0.0001). All patients receiving anti-HER2 therapy had <5 CTC/7.5 ml after three cycles of treatment. This is the largest prospective series validating the prognostic value of CTC independently from serum tumor marker. Elevated CTCs before C2 are an early predictive marker of poor PFS and OS, which could be used to monitor treatment benefit. CTC decrease under treatment seems stronger with targeted therapy.

P4-07-06: Correlation of Two Analytical Methods for Circulating Tumor Cells in Peripheral Blood of Patients with Primary Breast Cancer.

Abstract Background While the evidence for circulating tumor cells (CTCs) as a prognostic marker in metastatic breast cancer has been well established, there is still a lack of data in primary disease. In the SUCCESS A trial two different techniques for the detection of CTCs in early breast cancer were prospectively evaluated. Material and Methods: SUCCESS A compared FEC-Docetaxel vs. FEC-Docetaxel-Gemcitabine and 5 vs. 2 years of treatment with zoledronic acid in primary breast cancer patients and node positive or high-risk node negative disease. Two different techniques to detect CTCs were prospectively evaluated in two consecutive, but comparable subgroups of the whole study population. In 3515 samples the CellSearch® System (Veridex, Warren, USA) was used for CTC detection. Immunomagnetic enrichment with an EPCAM-antibody was followed by labeling with monoclonal antibodies specific for cytokeratin (8, 18, 19) and leukocytes (CD45). 2165 samples were evaluated with a manual immunocytochemistry (MICC) protocol. Cytospins were prepared after mononuclear cell enrichment based on Oncoquick® centrifugation (greiner bio-one, Frickenhausen, Germany). Staining was performed with the monoclonal pancytokeratin antibody A45-B/B3 (Micromet, Munich, Germany) and the APAAP technique. Conventional light field microscopy (Axiophot; Zeiss, Oberkochen, Germany) was used for the detection of stained cells. For both methods, the cut-off value for positivity was ≥ 1 CTC. All events were evaluated by two independent observers. Results: CTCs were examined in a total number of 3243 patients before and after chemotherapy (CHT). The two subgroups evaluated with one or the other method were well-balanced regarding clinical parameters as tumor size, grading, lymph node-status, hormone receptors and Her2. Furthermore there was no significant correlation between the CTC positivity and one of these clinical parameters using CellSearch or the MICC, respectively (p &amp;gt; 0,05 using the chi square test each time). Before adjuvant CHT 21. 3% (424 out of 1994) and 21.1 % (264 out of 1249) of the patients were found positive for CTCs using CellSearch® or the MICC respectively, with a mean CTC level of 5.9 (range: 1 to 827) and 3.1 (range: 1 to 256). Immediately after CHT 21.9% (333 out of 1521) and 16.5% (151 out of 916) of the patients were positive for CTCs using CellSearch® or the MICC. The mean CTC level decreased to 3.0 (range: 1 to 124) and 2.1 (range: 1 to 23) in both analytical methods. Using CellSearch® there was a significant correlation between the presence of CTCs before CHT and disease progression (p = 0.0044), as well as survival (p = 0.0001), whereas the MICC did not predict any of these (p = 0.3143 and p = 0.0801 respectively; the chi-square test was used each time). Conclusion: We found comparable prevalence of CTCs before and after adjuvant chemotherapy both with the CellSearch® System or the MICC. However, prognostic relevance could only be shown for CTCs detected with the CellSearch® System. This may be attributed to the high standardization and reproducibility of the automated system, as well as the additional CD45 counterstaining. According to our findings, the FDA approved CellSearch® System should be used as gold standard for CTC detection in future clinical trials. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-06.

P4-16-04: An Open-Label, Phase IIa, Non-Randomized Study of Radium-223 in Breast Cancer Patients with Bone Dominant Disease No Longer Considered Suitable for Endocrine Therapy.

Abstract Background Radium-223 (Alpharadin™) is a 1st-in-class alpha-pharmaceutical. It targets bone metastases (mets) with high-energy alpha-radiation of extremely short range that spares bone marrow. These characteristics generate highly localized radiation zones that may inhibit tumor progression and induce pain relief. Radium-223 has a profound effect on markers of bone metabolism and has shown a survival advantage over standard of care in pts with castration-resistant prostate cancer. The study's main objective was to investigate whether multiple IV injections (inj) of radium-223 have any clinically relevant effect on bone markers in metastatic breast cancer (MBC) pts with bone dominant disease (BDD). Methods: Study included MBC pts with BDD who had progressed (based on imaging or other clinically relevant information) on endocrine therapy and were no longer considered suitable for endocrine therapy. In this open-label, multicenter, single-arm, phase IIa study (EudraCT #2009-012189-30), 23 pts were scheduled to receive 4 IV inj of radium-223 50 kBq/kg every 4 wk. Bone markers were assessed at baseline, prior to every treatment, and thereafter at each follow-up visit. Primary efficacy endpoint was change in urine levels of NTX (uNTX) and bALP at 16 wk. Functional imaging with FDG-PET was performed in 20 pts at baseline and wk 8 and 16. Symptomatic response was assessed using validated questionnaires. Results: Histologic types were ductal carcinoma (n=12), lobular carcinoma (n=7), and others (n=2). Median interval from diagnosis of breast cancer to 1st relapse was 3 (1, 11) years. All except 1 pt received endocrine therapies, and adjuvant chemotherapy was given to approximately 60%. 21 of 23 pts were treated concurrently with bisphosphonates. 15 of 23 pts received all 4 inj of radium-223; 4/23 received 3 inj; and 4/23 received 2 inj. Pts who received &amp;lt;4 inj had disease progression (bone and visceral mets [n=3], visceral mets [n=3]) or withdrawn consent (n=1) and were not eligible to continue radium-223. One pt died before 4th inj due to atrial fibrillation. Median age was 58 (41, 83) years. Median uNTX levels were reduced by 20% (from 36 to 29 ***nmo1BCE/mmo1 creatinine; P=0.03) and 5% (from 36 to 23 ***nmo1BCE/mmo1 creatinine; P=NS) at wk 8 and 16 respectively; 17/23 and 9/13 pts (for whom data were available) had a decrease in uNTX at wk 8 and 16 respectively. Median bALP levels were reduced by 33% (from 22.1 to 12.1 ng/mL; P=0.0001) at wk 8 and 33% (from 22.1 to 10.4 ng/mL; P=0.04) at wk 16. Bone-ALP levels were reduced in 20/22 pts at wk 8 and in 10/12 pts (for whom data were available) at wk 16. Radium-223 was found to be safe and well tolerated. 3 pts had serious AEs, none related to study drug; 1 of them died due to disease progression. Functional imaging results, additional bone marker data, and pt-reported outcomes are being analyzed. Conclusions: Radium-223 consistently reduced uNTX and bALP during the 16-wk treatment period, results observed in addition to bisphosphonate use. Results show radium-223 targets the areas of increased bone metabolism caused by bone mets. Radium-223 found to be safe and well tolerated, confirming highly tolerable SE profile seen in other studies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-16-04.

P4-07-04: Nomogram Including Circulating Tumor Cells (CTC) Count before and during Chemotherapy for Individual Survival Prediction of Metastatic Breast Cancer Patients.

Abstract Background CTC count before a new line of treatment and CTC count early changes under chemotherapy have been reported as an independent prognostic marker in metastatic breast cancer in a recent pooled analysis in 841 pts (Liu M. ASCO 2011). The aim of this study was to build a prognostic tool including CTC and other parameters to assesse its predictive value for progression-free survival (PFS) and overall survival (OS). Methods: Data from the IC 2006–04 study were used. This prospective multicentre study included 267 metastatic breast cancer patients treated by first line chemotherapy with or without targeted therapy, in whom appropriate pre-treatment prognostic variables (age, performance status, number of metastatic sites, disease-free interval, ER, PR and HER2 status, tumor grade, LDH, serum markers, CTC count by CellSearch technique before treatment and before cycle 2) were available for statistical analysis. We constructed a multivariate Cox regression model for PFS and OS prediction. A stepwise selection process was applied to achieve the most informative and parsimonious models. Performance was measured with the C-index statistic. Internal validation was performed using leave-two-out technique. Results: Four nomograms have been obtained, in two clinical settings: at inclusion (before the start of any treatment) taking into account the initial CTC count, and during treatment (before cycle 2) taking into account CTC changes under treatment. Their accuracy was good for PFS and OS prediction, with C-index ranging from 0.72 to 0.88. Internal validations allow considering a good accuracy of the models in an external population. Conclusion: These clinically relevant nomograms are a simple tool for a personalized prognostic assessment including CTC assessment. Validation on independent series of patients are ongoing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-04.

Prognostic value of HER2-positive circulating tumor cells in patients with metastatic breast cancer

The presence of ≥5 circulating tumor cells (CTCs) in 7.5 ml blood is a poor prognostic marker in metastatic breast cancer (MBC). However, the role of human epidermal growth factor receptor 2 (HER2) status in CTCs is not known. We prospectively assessed the prognostic value of this parameter for patients with MBC who started a new line of systemic therapy. The CTC count (≥5 or <5) and the HER2 status in CTCs at the initiation of the therapy and 3-4 weeks later (first follow-up) were determined. The median follow-up time of the 52 enrolled patients was 655.0 days (18-1,275 days). HER2-positive CTCs were present in 14 of the 52 patients (26.9%) during the study period. Eight of 33 patients (24.2%) with HER2-negative primary tumors had HER2-positive CTCs during the study period. At first follow-up, patients with HER2-positive CTCs had significantly shorter progression-free (n = 6; P = 0.001) and overall (P = 0.013) survival than did patients without HER2-positive CTCs (n = 43) in log-rank analysis. In multivariate analysis, HER2-positive CTCs at first follow-up (P = 0.029) and the number of therapies patients received before this study (P = 0.006) were independent prognostic factors in terms of progression-free survival. The number of therapies (P = 0.001) and a count of ≥5 CTCs (P = 0.043) at baseline were independent prognostic factors in terms of overall survival. We showed that HER2 status in CTCs may be a prognostic factor for MBC. Well-powered prospective studies are necessary to determine the potential role of HER2-targeted therapies for patients with HER2-positive CTCs and HER2-negative primary tumors.

Abstract 3208: Impact of targeted therapy on circulating tumor cells in metastatic breast cancer treated by first-line chemotherapy: IC 2006-04 study

Abstract Circulating tumor cells (CTC) have been reported to be a prognostic marker in metastatic breast cancer. This study was designed to test whether changes in CTC counts during treatment are associated with outcome of first-line chemotherapy with/without targeted therapy. Methods: CTC were counted (CellSearch®) at baseline, before cycle 2 (C2) and C3/4 in 267 metastatic breast cancer patients. Usual prognostic variables, serum tumor markers, tumor response according to RECIST criteria and events were prospectively registered and compared with CTC status and outcome over time. Results: Median follow-up was 16 months, 45 patients (17%) had HER2 positive tumor and received trastuzumab, 55 (21%) had triple negative tumor, 125 received bevacizumab associated with CT. Baseline CTC positivity (65%; ≥1CTC/7.5ml threshold, 45% ≥5CTC/7.5ml threshold) was correlated with tumour markers, bone/liver involvement and number of metastatic sites, as well as performance status but was not statistically different according to breast cancer subtypes (hormone-positive, HER2-positive or triple-negative). At multivariate analysis, CTC ≥5CTC/7.5ml was an independent prognostic factor for progression-free survival (PFS), RR=1.9 (CI95% 1.26-2.88), p=0.002, with triple negative status, poor performance status and elevated CEA. For overall survival, CTC ≥5CTC/7.5ml was an independent prognostic factor with triple negative status and poor performance status (RR=2.41, p=0.02). Changes with a threshold ≥5CTC/7.5ml, between baseline and Cycle 2 were highly correlated with both PFS (p=5e-07) and OS (p=3e-07). Persistent CTC positivity at C2 was associated with lower tumor response (p=4e-03). Among CTC-positive patients at baseline treated by chemotherapy only, chemotherapy/bevacizumab and chemotherapy/anti-HER2, CTC counts decreased respectively below the ≥5CTC/7.5ml positivity threshold before cycle 2 in 53%, 64% and 83% of patients, and before cycle 3 or 4, in 62%, 77% and 100% of patients (p=0.04). Prognostic value of CTC count at baseline and C2 remained in all subgroups. Conclusions: CTC count appeared to decrease more markedly in the patients who received also bevacizumab or trastuzumab. However, CTC remain a reliable independent prognostic marker in metastatic breast cancer treated by first-line chemotherapy with/without targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3208. doi:10.1158/1538-7445.AM2011-3208

Symposium zeroes in on new discoveries in breast cancer

Symposium zeroes in on new discoveries in breast cancer

Circulating Tumor Cells in Breast Cancer Patients: An Evolving Role in Patient Prognosis and Disease Progression

In this paper, we examine the role of circulating tumor cells (CTCs) in breast cancer. CTCs are tumor cells present in the peripheral blood. They are found in many different carcinomas but are not present in patients with benign disease. Recent advances in theories regarding metastasis support the role of early release of tumor cells in the neoplastic process. Furthermore, it has been found that phenotypic variation exists between the primary tumor and CTCs. Of particular interest is the incongruency found between primary tumor and CTC HER2 status in both metastatic and early breast cancer. Overall, CTCs have been shown to be a poor prognostic marker in metastatic breast cancer. CTCs in early breast cancer are not as well studied, however, several studies suggest that the presence of CTCs in early breast cancer may also suggest a poorer prognosis. Studies are currently underway looking at the use of CTC level monitoring in order to guide changes in therapy.

Abstract P3-01-01: Cyfra 21-1 Is Correlated with Circulating Tumor Cells Detection and Patient Outcome in Metastatic Breast Cancer: Results of a Substudy of the Prospective IC 2004-06 Trial

Abstract Background: The Cyfra 21-1 epitope is a polypeptide originating from cytokeratin-19 fragments possibly released by cell death and was an interesting serum marker in small and/or retrospective studies in breast cancer. On the back of circulating tumor cell (CTC) detection, the prospective IC 2006-04 study planned serum markers (CEA, Ca15.3, Cyfra 21-1) measurements. Methods: Metastatic breast cancer patients were included in a multicentric non-interventional study before the start of the 1st line chemotherapy. Blood biomarkers, including CTC detection (CellSearch®, ≥5CTC/7.5ml), were analyzed at 4 different times: before the start of the chemotherapy, before cycle 2 (ie mostly at day 21), at first tumor evaluation (ie before cycle 3/4) and at tumor progression. Results: 191 of the 267 patients included had Cyfra 21-1 assessed at inclusion. CTC level was ≥5CTC/7.5ml in 44% of the cases. Cyfra 21-1&amp;gt;ULNV (in 65% of pts) was not correlated with tumor ER/PR/HER2 status but strongly correlated with PS (p=8e-04), number of metastatic sites (p=0.0001), Ca15.3 (p=0.009), CEA (p=0.005), LDH (p=7e-08) and CTC detection (p=2e-06). CTC (≥5CTC/7.5ml) and Cyfra21-1 (&amp;gt;ULNV) correlation (N patients) The effectiveness of curcumin in the treatment of HER-2 overexpressed breast cancer was further confirmed through in vivo BT-474 xenograft model. The comparable ability of curcumin to inhibit cell proliferation, mobility, and angiogenesis as herceptin, combined with the potential ability to overcome the herceptin resistance, curcumin may be a novel agent in the treatment of HER-2 overexressed breast cancer. Logistic regression showed that LDH (p=0.0005) and CTC (p=0.0004) were the two independent predictors of Cyfra 21-1 elevation. Baseline Cyfra 21-1 &amp;gt;ULNV was associated with early tumor progression (RECIST, p=0.03). In multivariate analysis including standard biomarkers (without CTC), baseline Cyfra 21-1 was independently associated with PFS (p=0.008, RR), together with PS (P&amp;lt;0.001), triple-negativity (P&amp;lt;0.001) and CEA (p=0.02). Multivariate analysis with CTC, subsequent Cyfra 21-1 analyses (cycles 2, 3-4 and progression) and correlation with tumor response and PFS will be will be shown at the meeting. Conclusion: Cyfra 21-1 is a commonly elevated serum marker in metastatic breast cancer and has an independent prognostic value. Although highly correlated, Cyfra 21-1 appears to be more frequently positive in metastatic breast cancer patients than CTC count. Multivariate analyses comparing CTC and Cyfra 21-1 at different time points will be shown at the meeting. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-01-01.

Abstract P3-02-06: Correlation of BCL-2 and Apoptosis on Circulating Tumor Cells and Breast Cancer Tissue

Abstract Background: Circulating Tumor Cell (CTC) levels are prognostic markers in metastatic breast cancer (MBC). Further phenotypic characterization of CTC may provide an opportunity for non-invasive evaluation of predictive and prognostic markers. Apoptosis is a common form of chemotherapy-induced cell death. Monoclonal antibody (MAb) M30 recognizes a neo-epitope on fragmented cytokeratin and is a marker of apoptosis. BCL-2 is an anti-apoptotic marker and may predict for resistance to anti-neoplastic therapy. We have previously reported the results of a pilot clinical trial to estimate M30 and BCL-2 expression in CTC from patients with MBC. The current study was performed to correlate the expression of both BCL-2 and M30 in CTC with breast cancer tissue samples. Methods: Of the 85 patients in the original pilot study, 52 (61%) had evaluable tissue available (39 primary and 12 metastatic lesions; one unknown) and were included in this analysis. CTC were collected at baseline and were isolated, enumerated, and phenotypically characterized for M30 and BCL-2 using the CellSearch® System. CTC phenotype is reported as percentage of cells with positive staining. Tissue Microarrays (TMA) were constructed and immunohistochemically stained for M30 (anti-Cytodeath™ M30, Roche) and Bcl-2 (M-0887, DAKO) and scored using the Allred method. Results: Tissue staining for M30 and Bcl-2 were inversely correlated. Of the 52 patients with TMA available for investigation, 22 (42%) had &amp;gt; 5 CTC/7.5 ml whole blood. A positive, but non-significant, correlation was observed between increasing numbers of CTC levels and tissue BCL-2 Allred Score (Spearman r=0.36; p=0.1310). M30 staining was detected in ≥10% of CTC in 20 of the 22 (91%) patients with elevated CTC, while BCL-2 staining was detected in ≥10% of CTC in 19 of the 22 (86%) patients. Little if any correlation was observed between CTC M30 and tissue M30 expression, however CTC BCL-2 was positively correlated with tissue Bcl-2 expression (Spearman r=0.47; p=0.0440). Conclusions: BCL-2 and M30 can be characterized in both CTC and breast cancer tissue. The likelihood of having elevated CTC may be associated with higher BCL-2 expression, and CTC BCL-2 was associated with tissue BCL-2 expression. Although the clinical implications of these findings are unknown, they offer the opportunity for future trials to investigate whether CTC BCL-2 and M30 might be useful to identify patients with cancers that are resistant to standard therapies. Furthermore, CTC BCL-2 and M30 might be monitored in clinical trials of novel, apoptosis-inducing therapeutic agents. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-06.