Does the Presence of Circulating Tumor Cells in High-Risk Early Breast Cancer Patients Predict the Site of First Metastasis-Results from the Adjuvant SUCCESS A Trial.

The prognostic relevance of circulating tumor cells (CTCs) in breast cancer is well established. However, little is known about the association of CTCs and site of first metastasis. In the SUCCESS A trial, 373 out of 3754 randomized high-risk breast cancer patients developed metastatic disease. CTC status was assessed by the FDA-approved CellSearch®-System (Menarini Silicon Biosystems, Bologna, Italy) in 206 of these patients before chemotherapy and additionally in 159 patients after chemotherapy. CTCs were detected in 70 (34.0%) of 206 patients before (median 2 CTCs, 1-827) and in 44 (27.7%) of 159 patients after chemotherapy (median 1 CTC, 1-124); 16 (10.1%) of 159 patients were CTC-positive at both timepoints. The site of first distant disease was bone-only, visceral-only, and other-site-only in 44 (21.4%), 60 (29.1%), and 74 (35.9%) patients, respectively, while 28 (13.6%) patients had multiple sites of first metastatic disease. Patients with CTCs at both timepoints more often showed bone-only first distant disease (37.5% vs. 21.0%) and first distant disease at multiple sites (31.3% vs. 12.6%) than patients without CTCs before and/or after chemotherapy (p = 0.027). In conclusion, the presence of CTCs before and after chemotherapy is associated with multiple-site or bone-only first-distant disease and may trigger intensified follow-up and perhaps further treatment.

The impact of anthracyclines in intermediate and high-risk HER2-negative early breast cancer-a pooled analysis of the randomised clinical trials PlanB and SUCCESS C.

BACKGROUND: Anthracycline/cyclophosphamide-taxane-containing chemotherapy (AC-T) is the standard of care in the adjuvant treatment of HER2-negative early breast cancer (EBC), but recent studies suggest omission of anthracyclines for reduced toxicity without compromising efficacy. METHODS: Based on individual patient data (n = 5924) pooled from the randomised Phase III trials PlanB and SUCCESS C, we compared disease-free survival (DFS) and overall survival (OS) between intermediate to high-risk HER2-negative EBC-patients treated with either six cycles of docetaxel/cyclophosphamide (TC6) or an AC-T regime using univariable and adjusted multivariable Cox regression models. RESULTS: AC-T conferred no significant DFS or OS advantage in univariable (DFS: hazard ratio (HR) for TC vs. AT 1.05, 95% confidence interval (CI): 0.89-1.24, P = 0.57; OS: HR 1.00, 95% CI: 0.80-1.26, P = 1.00) and adjusted multivariable analysis (DFS: HR 1.01, 95% CI: 0.86-1.19, P = 0.91; OS: HR 0.97, 95% CI: 0.77-1.22, P = 0.79). Patients receiving TC6 had significantly fewer grade 3-4 adverse events. Exploratory subgroup analysis showed that AC-T was associated with significantly better DFS and OS in pN2/3 patients, specifically in those with lobular histology. CONCLUSION: For most patients with HER2-negative EBC, AC-T is not associated with a survival benefit compared to TC6. However, patients with lobular pN2/pN3 tumours seem to benefit from anthracycline-containing chemotherapy.

Retrospective, Multicenter Analysis Comparing Conventional with Oncoplastic Breast Conserving Surgery: Oncological and Surgical Outcomes in Women with High-Risk Breast Cancer from the OPBC-01/iTOP2 Study.

INTRODUCTION: Recent data suggest that margins ≥2 mm after breast-conserving surgery may improve local control in invasive breast cancer (BC). By allowing large resection volumes, oncoplastic breast-conserving surgery (OBCII; Clough level II/Tübingen 5-6) may achieve better local control than conventional breast conserving surgery (BCS; Tübingen 1-2) or oncoplastic breast conservation with low resection volumes (OBCI; Clough level I/Tübingen 3-4). METHODS: Data from consecutive high-risk BC patients treated in 15 centers from the Oncoplastic Breast Consortium (OPBC) network, between January 2010 and December 2013, were retrospectively reviewed. RESULTS: A total of 3,177 women were included, 30% of whom were treated with OBC (OBCI n = 663; OBCII n = 297). The BCS/OBCI group had significantly smaller tumors and smaller resection margins compared with OBCII (pT1: 50% vs. 37%, p = 0.002; proportion with margin <1 mm: 17% vs. 6%, p < 0.001). There were significantly more re-excisions due to R1 ("ink on tumor") in the BCS/OBCI compared with the OBCII group (11% vs. 7%, p = 0.049). Univariate and multivariable regression analysis adjusted for tumor biology, tumor size, radiotherapy, and systemic treatment demonstrated no differences in local, regional, or distant recurrence-free or overall survival between the two groups. CONCLUSIONS: Large resection volumes in oncoplastic surgery increases the distance from cancer cells to the margin of the specimen and reduces reexcision rates significantly. With OBCII larger tumors are resected with similar local, regional and distant recurrence-free as well as overall survival rates as BCS/OBCI.

Interleukin 15 and Eotaxin correlate with the outcome of breast cancer patients vice versa independent of CTC status.

BACKGROUND: Circulating tumor cells (CTC) in the peripheral blood in women with breast cancer has been found to be an indicator of prognosis before the start of systemic treatment. The aim of this study is the assessment of specific cytokine profiles as markers for CTC involvement that could act as independent prognostic markers in terms of survival outcome for breast cancer patients. METHODS: Patients selected for this study were defined as women with breast cancer of the SUCCESS study. A total of 200 patients' sera were included in this study, 100 patients being positive for circulating tumor cells (CTC) and 100 patients being CTC negative. The matching criteria were histo-pathological grading, lymph node metastasis, hormone receptor status, TNM classification, and patient survival. Commercial ELISA with a multi cytokine/chemokine array was used to screen the sera for Interleukin 15 (IL-15) and eotaxin. RESULTS: Statistically significant concentrations were exposed for IL-15 levels regardless of the CTC-Status, lymph node involvement, or hormone receptor status. Significantly enhanced serum IL-15 concentrations were observed in those patients with worse overall survival (OS) and disease-free survival (DFS). Elevated serum concentrations of IL-15 significantly correlate with patients diagnosed with Grade 3 tumor and worse OS. In contrast, patients with a Grade 3 tumor with a favourable OS and DFS demonstrated significantly decreased IL-15 values. The CTC negative patient subgroup with a favourable OS and DFS, showed statistically significant elevated eotaxin values. CONCLUSION: These findings suggest a potential functional interaction of increased IL-15 concentrations in the peripheral blood of patients with a worse OS and DFS, regardless of prognostic factors at primary diagnosis. The increased levels of the chemokine eotaxin in CTC negative patients and a favourable OS and DFS, on the other hand, suggest that the overexpression inhibits CTCs entering the peripheral blood, thus emphasizing a significant inhibition of circulation specific metastasis. To sum up, IL-15 could be used as an independent prognostic marker in terms of survival outcome for breast cancer patients and used as an early indicator to highlight high-risk patients and consequently the adjustment of cancer therapy strategies.

Differential prognostic relevance of patho-anatomical factors among different tumor-biological subsets of breast cancer: Results from the adjuvant SUCCESS A study.

OBJECTIVES: In breast cancer, large tumor size, positive nodal stage and a triple-negative tumor subtype are associated with reduced survival, but the interactions between these prognostic factors are not well understood. MATERIAL AND METHODS: Here we re-evaluated the impact of tumor size, nodal stage and tumor subtype on disease-free survival (DFS), overall survival (OS), distant disease-free survival (DDFS) and breast cancer specific survival (BCSS) in a retrospective analysis using data from the adjuvant SUCCESS A trial. Subgroup analyses were conducted to assess whether the effect of tumor size and nodal stage on survival depended on tumor subtype. RESULTS: Increasing tumor size, higher nodal stage and triple negative breast cancer (TNBC) were associated with unfavorable prognosis (all p < 0.001). There was no significant interaction between tumor subtype and tumor size (p > 0.5 for all four survival endpoints), but we found significant interactions between tumor subtype and nodal stage (p < 0.05 for all four survival endpoints), with no differences in survival among tumor subtypes for patients with pN0 tumors (all p > 0.05) and pronounced differences in survival among tumor subtypes for patients with positive nodal stage (all p < 0.001). CONCLUSIONS: This analysis confirms tumor size, nodal stage and tumor subtype as independent prognostic factors in high-risk early breast cancer. Nodal-positive patients with TNBC had a considerably worse outcome compared to nodal-positive patients with another tumor subtype. This underlines the importance for early detection particularly for patients with TNBC. TRIAL REGISTRATION: EudraCT 2005-000490-21; ClinicalTrials.gov Identifier: NCT02181101.

Presence of Circulating Tumor Cells in High-Risk Early Breast Cancer During Follow-Up and Prognosis.

BACKGROUND: The prognostic relevance of circulating tumor cells (CTCs) at the time of primary diagnosis has been well established. However, little information is available regarding their prognostic relevance to follow-up care. METHODS: The multicenter, open-label, phase III SUCCESS A trial compared two adjuvant chemotherapy regimens followed by 2 vs 5 years of zoledronate for early-stage, high-risk breast cancer patients. The presence of CTCs was assessed before and 2 years after chemotherapy using the FDA-approved CellSearch System. Overall survival (OS) and disease-free survival (DFS) were analyzed using univariate log-rank tests and multivariable Cox regressions. OS and DFS were measured starting from an assessment of CTCs 2 years after the completion of chemotherapy. All statistical tests were two-sided. RESULTS: The sample included 1087 patients who participated in the translational research program of the SUCCESS A trial and for whom sufficient translational data were available regarding CTC status at baseline and at the 2-year follow-up visit. Two years after chemotherapy, 198 (18.2%) patients were CTC-positive. The median follow-up after this timepoint was 37 months. Cox regressions that included CTC status at baseline revealed that CTC status 2 years after chemotherapy had statistically significant and independent prognostic relevance for OS (hazard ratio [HR] = 3.91, 95% confidence interval [CI] = 2.04 to 7.52, P < .001) and DFS (HR = 2.31, 95% CI = 1.50 to 3.55, P < .001). CONCLUSION: The presence of CTCs 2 years after chemotherapy was associated with decreased OS and DFS. Based on these results, active individualized surveillance strategies for breast cancer survivors based on biomarkers should be reconsidered.

EpCAMhigh and EpCAMlow circulating tumor cells in metastatic prostate and breast cancer patients.

The presence of high expressing epithelial cell adhesion molecule (EpCAMhigh) circulating tumor cells (CTC) enumerated by CellSearch® in blood of cancer patients is strongly associated with poor prognosis. This raises the question about the presence and relation with clinical outcome of low EpCAM expressing CTC (EpCAMlow CTC). In the EU-FP7 CTC-Trap program, we investigated the presence of EpCAMhigh and EpCAMlow CTC using CellSearch, followed by microfiltration of the EpCAMhigh CTC depleted blood. Blood samples of 108 castration-resistant prostate cancer patients and 22 metastatic breast cancer patients were processed at six participating sites, using protocols and tools developed in the CTC-Trap program. Of the prostate cancer patients, 53% had ≥5 EpCAMhigh CTC and 28% had ≥5 EpCAMlow CTC. For breast cancer patients, 32% had ≥5 EpCAMhigh CTC and 36% had ≥5 EpCAMlow CTC. 70% of prostate cancer patients and 64% of breast cancer patients had in total ≥5 EpCAMhigh and/or EpCAMlow CTC, increasing the number of patients in whom CTC are detected. Castration-resistant prostate cancer patients with ≥5 EpCAMhigh CTC had shorter overall survival versus those with <5 EpCAMhigh CTC (p = 0.000). However, presence of EpCAMlow CTC had no relation with overall survival. This emphasizes the importance to demonstrate the relation with clinical outcome when presence of CTC identified with different technologies are reported, as different CTC subpopulations can have different relations with clinical outcome.

Endocrine Treatment with 2 Years of Tamoxifen versus 2 Years of Exemestane in Postmenopausal Patients with High-Risk Early Breast Cancer and Persisting Circulating Tumor Cells - First Results of the SUCCESS C Endocrine Treatment Sub-Study.

BACKGROUND: Optimal choice and sequence of endocrine treatment following adjuvant chemotherapy in postmenopausal early breast cancer patients are still under discussion and treatment stratification factors are missing. PATIENTS AND METHODS: Postmenopausal women with HER2-negative, hormone receptor-positive tumors and persisting circulating tumor cells (CTCs; assessed using the FDA-approved CellSearch® System, Janssen Diagnostics, LLC) after chemotherapy were randomized to 2 years of tamoxifen followed by 3 years of exemestane (tamoxifen-exemestane group, n = 54) or 5 years of exemestane (exemestane-only group, n = 54). CTCs were again assessed after the first 2 years of endocrine treatment. In addition, safety data were compared between the 2 groups. RESULTS: The 2 groups were well-balanced with regard to baseline characteristics. The CTC clearance rate after 2 years was 89% in the exemestane-only group and 97% in the tamoxifen-exemestane group (exact Fisher test, p = 0.36). The safety profile showed good tolerability with few grade 3 or 4 adverse events in both groups. CONCLUSION: The similar CTC clearance rate after 2 years of endocrine therapy with exemestane or tamoxifen, and the safety profiles obtained may indicate comparable efficacy and tolerability of both endocrine treatment regimens. However, these results have to be confirmed by final survival and safety analysis.

Whole blood microRNAs as potential biomarkers in post-operative early breast cancer patients.

BACKGROUND: microRNAs (miRNAs) are considered promising cancer biomarkers, showing high reliability, sensitivity and stability. Our study aimed to identify associations between whole blood miRNA profiles, presence of circulating tumor cells (CTCs) and clinical outcome in post-operative early breast cancer patients (EBC) to assess the utility of miRNAs as prognostic markers in this setting. METHOD: A total of 48 post-operative patients, recruited in frame of the SUCCESS A trial, were included in this retrospective study and tested with a panel of 8 miRNAs (miR-10b, -19a, - 21, - 22, -20a, - 127, - 155, -200b). Additional 17 female healthy donors with no previous history of cancer were included in the study as negative controls. Blood samples were collected at different time points (pre-adjuvant therapy, post-adjuvant therapy, 2 years follow up), total RNA was extracted and the relative concentration of each miRNA was measured by quantitative PCR and compared in patients stratified on blood collection time or CTC detection. Furthermore, we compared miRNA profiles of patients, for each time point separately, and healthy donors. CTCs were visualized and quantified with immunocytochemistry analysis. Data were analyzed using non-parametric statistical tests. RESULTS: In our experimental system, miR-19a, miR-22 and miR-127 showed the most promising results, differentiating patients at different time points and from healthy controls, while miR-20a, miR-21 and miR-200b did not show any difference among the different groups. miR-10b and miR-155 were never detectable in our experimental system. With respect to patients' clinical characteristics, we found a significant correlation between miR-200b and lymph node status and between miR-20a and tumor type. Furthermore, miR-127 correlated with the presence of CTCs. Finally, we found a borderline significance between Progression Free Survival and miR-19a levels. CONCLUSIONS: This pilot study suggests that profiling whole blood miRNAs could help to better stratify post-operative EBC patients without any sign of metastasis to prevent later relapse or metastatic events.

Anti-Müllerian hormone (AMH) levels in premenopausal breast cancer patients treated with taxane-based adjuvant chemotherapy - A translational research project of the SUCCESS A study.

BACKGROUND: Premenopausal women undergoing chemotherapy are at high risk for premature ovarian failure and its long-term consequences. Data on potential markers to evaluate ovarian reserve pre- and posttreatment are limited. Anti-Müllerian hormone (AMH) known for ovarian reserve in reproductive medicine could be a surrogate marker and was assessed in premenopausal breast cancer patients of the SUCCESS A study (EUDRA-CT no. 2005-000490-21). METHODS: We identified 170 premenopausal patients, age ≤ 40 years at trial entry, who received FEC-Doc as taxane-anthracylince based chemotherapy. Blood samples were taken at three time points: Before, four weeks after and two years after adjuvant chemotherapy. Serum AMH-levels were evaluated in a central laboratory by a quantitative immunoassay AMH Gen II ELISA (Beckman Coulter, Brea, USA). RESULTS: Median age was 36 years (21-40 years). Median serum AMH-level before chemotherapy was 1.37 ng/ml (range < 0.1-11.3 ng/ml). Four weeks after chemotherapy AMH-levels dropped in 98.6% of the patients to <0.1 ng/ml (range < 0.1-0.21 ng/ml). After two years, 73.3% (n = 101) showed no evidence of ovarian function recovery (AMH <0.1 ng/ml, range < 0.1-3.9 ng/ml). Permanent chemotherapy induced amenorrhea occurred only in 50.6% of the patients. CONCLUSIONS: In this analysis, premenopausal patients showed a high rate of ovarian impairment reflected by low AMH-levels after chemotherapy.

LKB1 pro-oncogenic activity triggers cell survival in circulating tumor cells.

During intravasation, circulating tumor cells (CTCs) detach from the epithelium of origin and begin the epithelial-to-mesenchymal transition (EMT) process, where they lose epithelial features and pass through the endothelium to enter circulation. Although detachment from the extracellular matrix is a strong source of metabolic stress, which induces anoikis, CTCs can survive. Recently, the tumor suppressor liver kinase B1 (LKB1) has gained attention for its role as a proto-oncogene in restoring the correct ATP/AMP ratio during metabolic stress. The aim of this study was to assess LKB1 expression in epithelial-negative CTCs isolated from patients with metastatic breast cancer and to characterize its possible association with EMT and stemness features. Transcriptome analysis of EpCAM-negative CTCs indicated that over 25% of patients showed enhanced LKB1 levels, while almost 20% of patients showed enhanced levels of an EMT transcription factor known as ZEB1. Transcriptome and immunofluorescence analyses showed that patients with enhanced LKB1 were correspondingly ZEB1 negative, suggesting complementary activity for the two proteins. Only ZEB1 was significantly associated with cancer stem cell (CSC) markers. Neither LKB1 nor ZEB1 upregulation showed a correlation with clinical outcome, while enhanced levels of stemness-associated CD44 correlated with a lower progression-free and overall survival. Ex vivo models showed that MDA-MB-231, a mesenchymal tumor cell line, grew in suspension only if LKB1 was upregulated, but the MCF-7 epithelial cell line lost its ability to generate spheroids and colonies when LKB1 was inhibited, supporting the idea that LKB1 might be necessary for CTCs to overcome the absence of the extracellular matrix during the early phases of intravasation. If these preliminary results are confirmed, LKB1 will become a novel therapeutic target for eradicating metastasis-initiating CTCs from patients with primary breast cancer.

Prevalence of Circulating Tumor Cells After Adjuvant Chemotherapy With or Without Anthracyclines in Patients With HER2-negative, Hormone Receptor-positive Early Breast Cancer.

BACKGROUND: Use of anthracycline-based chemotherapy in patients with early breast cancer (EBC) has been well-established but is often associated with cardiotoxicity. Based on data suggesting a limited benefit of anthracyclines in human epidermal growth factor receptor 2 (HER2)-negative patients, the Simultaneous Study of Docetaxel Based Anthracycline Free Adjuvant Treatment Evaluation, as well as Life Style Intervention Strategies (SUCCESS) C study randomized patients to either anthracycline-containing or anthracycline-free chemotherapy. Given the proven prognostic value of circulating tumor cells (CTCs) in EBC, we compared the prevalence of CTCs after chemotherapy between both treatment arms for a preliminary efficacy assessment. METHODS: The SUCCESS C trial (NCT00847444) is an open-label, phase III study randomizing 3547 patients with HER2-negative EBC to either 3 cycles of epirubicin, 5-fluorouracil, and cyclophosphamide followed by 3 cycles of docetaxel (FEC-DOC) or 6 cycles of docetaxel and cyclophosphamide (DOC-C). CTC status was prospectively evaluated in hormone receptor-positive patients at the time of last chemotherapy cycle using the US Food and Drug Administration-approved CellSearch System (Janssen Diagnostics). RESULTS: Data on CTC status were available for 1766 patients. Overall, CTCs were found in 221 (12.5%) patients. Univariate analyses revealed that presence of CTCs at time of last chemotherapy cycle was not significantly associated with tumor or patient characteristics (all P > .1). There was no significant difference with respect to presence of CTCs between patients randomized to FEC-DOC or DOC-C (11.5% vs. 13.6%; P = .18). CONCLUSIONS: The comparable prevalence of CTCs at the time of last chemotherapy cycle may indicate that anthracycline-free chemotherapy is equally effective to anthracycline-containing chemotherapy in HER2-negative, hormone receptor-positive EBC. However, efficacy data from the final survival analysis of SUCCESS C have to be awaited to confirm these preliminary findings.

Discordance in Human Epidermal Growth Factor Receptor 2 (HER2) Phenotype Between Primary Tumor and Circulating Tumor Cells in Women With HER2-Negative Metastatic Breast Cancer.

PURPOSE: Discordance in human epidermal growth factor receptor 2 (HER2) status between primary tumor and metastases might have important implications for treatment response and therapy decisions. Here, we evaluate both the frequency of circulating tumor cells (CTCs) and the factors predicting HER2 discordance between primary tumor and CTCs as a potential surrogate for tumor biology and tumor heterogeneity in patients with metastatic breast cancer. PATIENTS AND METHODS: The number of CTCs in 7.5 mL of peripheral blood and HER2 status were evaluated in 1,123 women with HER2-negative metastatic breast cancer. HER2 discordance was defined as the presence of at least one CTC with a strong immunocytochemical HER2 staining intensity. Factors predicting discordance in HER2 phenotype were assessed using multivariable logistic regression. RESULTS: Overall, 711 (63.3%) of 1,123 screened patients were positive for CTCs (≥ one CTC). Discordance in HER2 phenotype between primary tumor and CTCs was observed in 134 patients (18.8%) and was significantly associated with histologic type (lobular v ductal; odds ratio [OR], 2.67; 95% CI, 1.63 to 4.39; P < .001), hormone receptor status (positive v negative; OR, 2.84; 95% CI, 1.15 to 7.02; P = .024), and CTC number (≥ five v one to four; OR, 7.64; 95% CI, 3.97 to 14.72; P < .001). CONCLUSION: HER2 discordance between primary tumor and CTCs was observed in 18.8% of patients and was associated with histologic type, hormone receptor status of the primary tumor, and CTC number. The clinical utility of CTCs as liquid biopsy to assess tumor heterogeneity of metastatic disease and guide treatment decisions must be evaluated in prospective randomized trials.

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

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

CA27.29 as a tumour marker for risk evaluation and therapy monitoring in primary breast cancer patients.

Several trials showed that tumour markers are associated with an impaired prognosis for breast cancer. Whether earlier treatment can improve the course of the disease remains controversial. The SUCCESS Trial compares FEC (500/100/500)-docetaxel (100) vs. FEC (500/100/500)-docetaxel/gemcitabine (75/2000) as well as 2 vs. 5 years of zoledronate in high-risk primary breast cancer patients. In 2669 patients, CA27.29 was measured before and after chemotherapy with the ST AIA-PACK CA27.29 reagent for the AIA-600II automated enzyme immunoassay (Tosoh Bioscience, Belgium). Values above 31 U/ml were considered positive. Of the patients, 7.6 % (n = 202, mean 19, range 3-410) and 19.1 % (n = 511, mean 21, range 3-331) had elevated marker levels before and after chemotherapy, respectively. Of the patients, 4.9 and 78 % showed elevated and low CA27.29, respectively, at both time points. After treatment, 35 % of the pre-therapy positive patients were negative, and 15 % of the initially negative patients became positive. The correlation between both time points was significant (p < 0.0001). No correlations among nodal status, grading, hormonal status, HER2 status and CA27.29 levels were found. However, tumour size (p = 0.02), older age (p < 0.001) and post-menopausal status (p = 0.006) were significantly associated with higher CA27.29 levels. Before treatment, the prevalence of elevated CA27.29 was equally distributed between both treatment arms, whereas after chemotherapy, 13.7 % of the patients in the FEC-doc arm showed an increased level vs. 25.4 % of the patients in the FEC-doc/gemcitabine arm (p < 0.0001). However, we could not show a significant association between the G-CSF application (yes vs. no) and CA27.29 status before/after chemotherapy (p = 0.75). These results indicate a close relationship between CA27.29 levels and tumour mass. Increased values after the completion of chemotherapy might be attributed to treatment effects and should be considered with caution.

Detection of circulating tumor cells using manually performed immunocytochemistry (MICC) does not correlate with outcome in patients with early breast cancer - Results of the German SUCCESS-A- trial.

BACKGROUND: Recently, the prognostic significance of circulating tumor cells (CTCs) in primary breast cancer as assessed using the Food-and-Drug-Administration-approved CellSearch® system has been demonstrated. Here, we evaluated the prognostic relevance of CTCs, as determined using manually performed immunocytochemistry (MICC) in peripheral blood at primary diagnosis, in patients from the prospectively randomized multicenter SUCCESS-A trial (EudraCT2005000490-21). METHODS: We analyzed 23 ml of blood from 1221 patients with node-positive or high risk node-negative breast cancer before adjuvant taxane-based chemotherapy. Cells were separated using a density gradient followed by epithelial cell labeling with the anti-cytokeratin-antibody A45-B/B3, immunohistochemical staining with new fuchsin, and cytospin preparation. All cytospins were screened for CTCs, and the cutoff for positivity was at least one CTC. The prognostic value of CTCs with regard to disease-free survival (DFS), distant disease-free survival (DDFS), breast-cancer-specific survival (BCSS), and overall survival (OS) was assessed using both univariate analyses applying the Kaplan-Meier method and log-rank tests, and using multivariate Cox regressions adjusted for other predictive factors. RESULTS: In 20.6 % of all patients (n = 251) a median of 1 (range, 1-256) CTC was detected, while 79.4 % of the patients (n = 970) were negative for CTCs before adjuvant chemotherapy. A pT1 tumor was present in 40.0 % of patients, 4.8 % had G1 grading and 34.6 % were node-negative. There was no association between CTC positivity and tumor stage, nodal status, grading, histological type, hormone receptor status, Her2 status, menopausal status or treatment. Univariate survival analyses based on a median follow-up of 64 months revealed no significant differences between CTC-positive and CTC-negative patients with regard to DFS, DDFS, BCSS, or OS. This was confirmed by fully adjusted multivariate Cox regressions, showing that the presence of CTCs (yes/no) as assessed by MICC did not predict DFS, DDFS, BCSS or OS. CONCLUSIONS: We could not demonstrate prognostic relevance regarding CTCs that were quantified using the MICC method at the time of primary diagnosis in our cohort of early breast cancer patients. Further studies are necessary to evaluate if the presence of CTCs assessed using MICC has prognostic relevance, or can be used for risk stratification and treatment monitoring in adjuvant breast cancer. TRIAL REGISTRATION: The ClinicalTrial.gov registration ID of this prospectively randomized trial is NCT02181101 ; the (retrospective) registration date was June 2014 (study start date September 2005).

Determination of Interleukin-4, -5, -6, -8 and -13 in Serum of Patients with Breast Cancer Before Treatment and its Correlation to Circulating Tumor Cells.

BACKGROUND/AIM: Circulating tumor cells (CTCs) in women with breast cancer are an indication of prognosis before starting systemic treatment. The aim of this study was the evaluation of cytokine profiles as marker for CTC involvement. MATERIALS AND METHODS: The analysis of CTCs, the time of blood sampling and the methodology were prospectively designed. There were two groups of patients: 100 women with a positive result for presence of CTCs and 100 women negative for CTCs. These groups were matched into pairs by tumor factors and survival/death. A multi-array ELISA was used to screen T-helper cell (Th) 2 cytokines. The results were analyzed by Spearman correlation coefficient and Mann-Whitney U-test. RESULTS: In patients who were CTC-negative, expression of interleukin-8 (IL-8) and IL-13 was increased (p=0.017 and p=0.045, respectively) if they were negative for progesterone receptor. In patients who died from their tumor, correlation between hormone receptor negativity and an increase in IL-4 was found. IL-5 was increased in patients with lymph node-positive and human epidermal growth factor receptor 2 (HER2)-positive disease (p=0.042). Moreover IL-4 was increased in patients with progesterone receptor-positive and estrogen receptor-negative status (p=0.024). Furthermore, the level of IL-6 was increased in patients with tumor grade G3 without progesterone receptor expression. CONCLUSION: Th2 cytokines are significantly modified in patients who are CTC-negative and progesterone receptor-positive. We suppose that an increase of IL-4 depends on hormone receptor status. In literature, a correlation between IL-4 and resistance to apoptosis is described. We suspect that IL-4 is responsible for the poor outcome of these cases.

Liquid biopsy-based clinical research in early breast cancer: The EORTC 90091-10093 Treat CTC trial.

There is increasing evidence that breast cancer evolves over time under the selection pressure of systemic treatment. Today, treatment decisions in early breast cancer are based on primary tumour characteristics without considering the disease evolution. Chemoresistant micrometastatic disease is poorly characterised and thus it is not used in current clinical practice as a tool to personalise treatment approaches. The detection of chemoresistant circulating tumour cells (CTCs) has been shown to be associated with worse prognosis in early breast cancer. The ongoing Treat CTC trial is the first international, liquid biopsy-based trial evaluating the concept of targeting chemoresistant minimal residual disease: detection of CTCs following adjuvant chemotherapy (adjuvant cohort) or neoadjuvant chemotherapy in patients who did not achieve pathological complete response (neoadjuvant cohort). This article presents the rational and design of this trial and the results of the pilot phase after 350 patients have been screened and provides insights that might provide information for future trials using the liquid biopsy approach as a tool towards precision medicine (NCT01548677).

Comparison of HER2 Expression in Primary Tumor and Disseminated Tumor Cells in the Bone Marrow of Breast Cancer Patients.

OBJECTIVE: The aim of this study was to measure the human epidermal growth factor receptor 2 (HER2) status of disseminated tumor cells (DTCs) from bone marrow (BM) aspirates and to assess correspondence or discrepancy with the primary tumor. METHODS: DTCs were isolated from the BM of 156 breast cancer patients. Cytokeratin-positive DTCs were further analyzed by the chromogenic in situ hybridization method to detect HER2 gene amplification. RESULTS: A significant correlation (p = 0.021) was found between the HER2 status of DTCs and the primary tumors. Sixty-one (68.5%) patients had a corresponding status. However, a shift of phenotype between primary tumor and DTCs was found in the remaining patients. CONCLUSION: This study showed a significant grade of discordance of the HER2 status between primary tumors and DTCs in the BM of a relevant subgroup of patients. Detection of HER2 amplification on DTCs could therefore help to better stratify patients for a more tailored therapy, since they would benefit from a HER2-targeted therapy.