Tanja Fehm

Systemic Spread Is an Early Step in Breast Cancer

It is widely accepted that metastasis is a late event in cancer progression. Here, however, we show that tumor cells can disseminate systemically from earliest epithelial alterations in HER-2 and PyMT transgenic mice and from ductal carcinoma in situ in women. Wild-type mice transplanted with single premalignant HER-2 transgenic glands displayed disseminated tumor cells and micrometastasis in bone marrow and lungs. The number of disseminated cancer cells and their karyotypic abnormalities were similar for small and large tumors in patients and mouse models. When activated by bone marrow transplantation into wild-type recipients, 80 early-disseminated cancer cells sufficed to induce lethal carcinosis. Therefore, release from dormancy of early-disseminated cancer cells may frequently account for metachronous metastasis.

Circulating Tumor Cells in Patients with Breast Cancer Dormancy

Purpose: The purpose of this study was to test the hypothesis that circulating tumor cells (CTCs) are present in patients many years after mastectomy without evidence of disease and that these CTCs are shed from persisting tumor in patients with breast cancer dormancy. Experimental Design: We searched for CTCs in 36 dormancy candidate patients and 26 age-matched controls using stringent criteria for cytomorphology, immunophenotype, and aneusomy. Results: Thirteen of 36 dormancy candidates, 7 to 22 years after mastectomy and without evidence of clinical disease, had CTCs, usually on more than one occasion. Only 1 of 26 controls had a possible CTC (no aneusomy). The statistical difference of these two distributions was significant (exact P = 0.0043). The CTCs in patients whose primary breast cancer was just removed had a half-life measured in 1 to 2.4 hours. Conclusions: The CTCs that are dying must be replenished every few hours by replicating tumor cells somewhere in the tissues. Hence, there appears to be a balance between tumor replication and cell death for as long as 22 years in dormancy candidates. We conclude that this is one mechanism underlying tumor dormancy.

Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients.

IntroductionThe persistence of circulating tumor cells (CTC) in breast cancer patients might be associated with stem cell like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, these cells also may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT), which allows them to travel to the site of metastasis formation without getting affected by conventional treatment. Here we evaluated 226 blood samples of 39 metastatic breast cancer patients during a follow-up of palliative chemo-, antibody – or hormonal therapy for the expression of the stem cell marker ALDH1 and markers for EMT and correlated these findings with the presence of CTC and response to therapy.Methods2 × 5 ml blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1 and HER2 transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [Twist1, Akt2, PI3Kα] and separately for the tumor stem-cell markers ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample.Results97% of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts. CTC were detected in 69/226 (31%) cancer samples. In the CTC (+) group, 62% were positive for at least one of the EMT markers and 69% for ALDH1, respectively. In the CTC (-) group the percentages were 7% and 14%, respectively. In non-responders, EMT and ALDH1 expression was found in 62% and 44% of patients, in responders the rates were 10% and 5%, respectively.ConclusionsOur data indicate that a major proportion of CTC of metastatic breast cancer patients shows EMT and tumor stem cell characteristics. Further studies are needed to prove whether these markers might serve as an indicator for therapy resistant tumor cell populations and, therefore, an inferior prognosis.

Sentinel-lymph-node biopsy in patients with breast cancer before and after neoadjuvant chemotherapy (SENTINA): a prospective, multicentre cohort study.

BACKGROUND The optimum timing of sentinel-lymph-node biopsy for breast cancer patients treated with neoadjuvant chemotherapy is uncertain. The SENTINA (SENTinel NeoAdjuvant) study was designed to evaluate a specific algorithm for timing of a standardised sentinel-lymph-node biopsy procedure in patients who undergo neoadjuvant chemotherapy. METHODS SENTINA is a four-arm, prospective, multicentre cohort study undertaken at 103 institutions in Germany and Austria. Women with breast cancer who were scheduled for neoadjuvant chemotherapy were enrolled into the study. Patients with clinically node-negative disease (cN0) underwent sentinel-lymph-node biopsy before neoadjuvant chemotherapy (arm A). If the sentinel node was positive (pN1), a second sentinel-lymph-node biopsy procedure was done after neoadjuvant chemotherapy (arm B). Women with clinically node-positive disease (cN+) received neoadjuvant chemotherapy. Those who converted to clinically node-negative disease after chemotherapy (ycN0; arm C) were treated with sentinel-lymph-node biopsy and axillary dissection. Only patients whose clinical nodal status remained positive (ycN1) underwent axillary dissection without sentinel-lymph-node biopsy (arm D). The primary endpoint was accuracy (false-negative rate) of sentinel-lymph-node biopsy after neoadjuvant chemotherapy for patients who converted from cN1 to ycN0 disease during neoadjuvant chemotherapy (arm C). Secondary endpoints included comparison of the detection rate of sentinel-lymph-node biopsy before and after neoadjuvant chemotherapy, and also the false-negative rate and detection rate of sentinel-lymph-node biopsy after removal of the sentinel lymph node. Analyses were done according to treatment received (per protocol). FINDINGS Of 1737 patients who received treatment, 1022 women underwent sentinel-lymph-node biopsy before neoadjuvant chemotherapy (arms A and B), with a detection rate of 99.1% (95% CI 98.3-99.6; 1013 of 1022). In patients who converted after neoadjuvant chemotherapy from cN+ to ycN0 (arm C), the detection rate was 80.1% (95% CI 76.6-83.2; 474 of 592) and false-negative rate was 14.2% (95% CI 9.9-19.4; 32 of 226). The false-negative rate was 24.3% (17 of 70) for women who had one node removed and 18.5% (10 of 54) for those who had two sentinel nodes removed (arm C). In patients who had a second sentinel-lymph-node biopsy procedure after neoadjuvant chemotherapy (arm B), the detection rate was 60.8% (95% CI 55.6-65.9; 219 of 360) and the false-negative rate was 51.6% (95% CI 38.7-64.2; 33 of 64). INTERPRETATION Sentinel-lymph-node biopsy is a reliable diagnostic method before neoadjuvant chemotherapy. After systemic treatment or early sentinel-lymph-node biopsy, the procedure has a lower detection rate and a higher false-negative rate compared with sentinel-lymph-node biopsy done before neoadjuvant chemotherapy. These limitations should be considered if biopsy is planned after neoadjuvant chemotherapy. FUNDING Brustkrebs Deutschland, German Society for Senology, German Breast Group.

Neoadjuvant Chemotherapy and Bevacizumab for HER2-Negative Breast Cancer

BACKGROUND Bevacizumab, a monoclonal antibody against vascular endothelial growth factor A, has shown clinical efficacy in patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer. We evaluated the efficacy, measured according to the rate of pathological complete response (absence of invasive and intraductal disease in the breast and the axillary lymph nodes), and the safety of adding bevacizumab to neoadjuvant chemotherapy in patients with early-stage breast cancer. METHODS We randomly assigned 1948 patients with a median tumor size of 40 mm on palpation to receive neoadjuvant epirubicin and cyclophosphamide followed by docetaxel, with or without concomitant bevacizumab. Patients with untreated HER2-negative breast cancer were eligible if they had large tumors, hormone-receptor-negative disease, or hormone-receptor-positive disease with palpable nodes or positive findings on sentinel-node biopsy, and no increased cardiovascular or bleeding risk. RESULTS Overall, the rates of pathological complete response were 14.9% with epirubicin and cyclophosphamide followed by docetaxel and 18.4% with epirubicin and cyclophosphamide followed by docetaxel plus bevacizumab (odds ratio with addition of bevacizumab, 1.29; 95% confidence interval, 1.02 to 1.65; P=0.04); the corresponding rates of pathological complete response were 27.9% and 39.3% among 663 patients with triple-negative tumors (P=0.003) and 7.8% and 7.7% among 1262 patients with hormone-receptor-positive tumors (P=1.00). Breast-conserving surgery was possible in 66.6% of the patients in both groups. The addition of bevacizumab, as compared with neoadjuvant therapy alone, was associated with a higher incidence of grade 3 or 4 toxic effects (febrile neutropenia, mucositis, the hand-foot syndrome, infection, and hypertension) but with a similar incidence of surgical complications. CONCLUSIONS The addition of bevacizumab to neoadjuvant chemotherapy significantly increased the rate of pathological complete response among patients with HER2-negative early-stage breast cancer. Efficacy was restricted primarily to patients with triple-negative tumors, in whom the pathological complete response is considered to be a reliable predictor of long-term outcome. (Funded by Sanofi-Aventis and Roche, Germany; ClinicalTrials.gov number, NCT00567554.).

Detection and HER2 Expression of Circulating Tumor Cells: Prospective Monitoring in Breast Cancer Patients Treated in the Neoadjuvant GeparQuattro Trial

Purpose: This study was aimed at detecting and characterizing circulating tumor cells (CTC) before and after neoadjuvant therapy (NT) in the peripheral blood of patients with breast cancer. Experimental Design: The clinical trial GeparQuattro incorporated NT approaches (epirubicin/cyclophosphamide prior to randomization to docetaxel alone, docetaxel in combination with capecitabine, or docetaxel followed by capecitabine) and additional trastuzumab treatment for patients with HER2-positive tumors. We used the Food and Drug Administration–approved CellSearch system for CTC detection and evaluation of HER2 expression and developed HER2 immunoscoring for CTC. Results: We detected ≥1 CTC/7.5 mL in 46 of 213 patients (21.6%) before NT and in 22 of 207 patients (10.6%) after NT (P = 0.002). Twenty (15.0%) initially CTC-positive cases were CTC-negative after NT, whereas 11 (8.3%) cases were CTC-positive after NT, although no CTC could be found before NT. CTC detection did not correlate with primary tumor characteristics. Furthermore, there was no association between tumor response to NT and CTC detection. HER2-overexpressing CTC were observed in 14 of 58 CTC-positive patients (24.1%), including 8 patients with HER2-negative primary tumors and 3 patients after trastuzumab treatment. CTC scored HER2-negative or weakly HER2-positive before or after NT were present in 11 of 21 patients with HER2-positive primary tumors. HER2 overexpression on CTC was restricted to ductal carcinomas and associated with high tumor stage (P = 0.002). Conclusion: CTC number was low in patients with primary breast cancer. The decrease in CTC incidence during treatment was not correlated with standard clinical characteristics and primary tumor response. Information on the HER2 status of CTC might be helpful for stratification and monitoring of HER2-directed therapies. Clin Cancer Res; 16(9); 2634–45. ©2010 AACR.

Circulating Tumor Cells Predict Survival in Early Average-to-High Risk Breast Cancer Patients

Background Circulating tumor cells (CTCs) have been shown to predict reduced survival outcomes in metastatic breast cancer. Methods CTCs were analyzed in 2026 patients with early breast cancer before adjuvant chemotherapy and in 1492 patients after chemotherapy using the CellSearch System. After immuno-magnetic enrichment for cells expressing the epithelial-cell adhesion molecule, CTCs were defined as nucleated cells expressing cytokeratin and lacking CD45. The patients were followed for a median of 35 months (range = 0–54). Kaplan–Meier analyses and the log-rank test were used for survival analyses. All statistical tests were two-sided. Results Before chemotherapy, CTCs were detected in 21.5% of patients (n = 435 of 2026), with 19.6% (n = 136 of 692) of node-negative and 22.4% (n = 299 of 1334) of node-positive patients showing CTCs (P < .001). No association was found with tumor size, grading, or hormone receptor status. After chemotherapy, 22.1% of patients (n = 330 of 1493) were CTC positive. The presence of CTCs was associated with poor disease-free survival (DFS; P < .0001), distant DFS (P < .001), breast cancer-specific survival (P = .008), and overall survival (OS; P = .0002). CTCs were confirmed as independent prognostic markers in multivariable analysis for DFS (hazard ratio [HR] = 2.11; 95% confidence interval [CI] = 1.49 to 2.99; P < .0001) and OS (HR = 2.18; 95% CI = 1.32 to 3.59; P = .002). The prognosis was worst in patients with at least five CTCs per 30mL blood (DFS: HR = 4.51, 95% CI = 2.59 to 7.86; OS: HR = 3.60, 95% CI = 1.56 to 8.45). The presence of persisting CTCs after chemotherapy showed a negative influence on DFS (HR = 1.12; 95% CI = 1.02 to 1.25; P = .02) and on OS (HR = 1.16; 95% CI = 0.99 to 1.37; P = .06) Conclusions These results suggest the independent prognostic relevance of CTCs both before and after adjuvant chemotherapy in a large prospective trial of patients with primary breast cancer.

Effect of Luteinizing Hormone–Releasing Hormone Agonist on Ovarian Function After Modern Adjuvant Breast Cancer Chemotherapy: The GBG 37 ZORO Study

PURPOSE Observational studies suggested that luteinizing hormone-releasing hormone agonists (LHRHa) might prevent premature ovarian failure resulting from adjuvant chemotherapy in premenopausal patients. We aimed to test the efficacy of ovarian function preservation with the LHRHa goserelin in patients with breast cancer. PATIENTS AND METHODS In a prospective, randomized, open-label, controlled multicenter study, 60 patients younger than age 46 years with hormone-insensitive breast cancer were allocated to receive anthracycline/cyclophosphamide (with or without taxane) -based neoadjuvant chemotherapy with or without goserelin. The first goserelin injection was administered at least 2 weeks before the first chemotherapy cycle, continuing at 3.6 mg subcutaneously every 4 weeks until the end of the last cycle. The primary objective was the reappearance of normal ovarian function, defined as two consecutive menstrual periods within 21 to 35 days at 6 months after end of chemotherapy. RESULTS Fifty-three patients (88.3%) experienced temporary amenorrhea (93.3% with v 83.3% without goserelin). No significant difference was observed regarding the reappearance of menstruation at 6 months after chemotherapy (70.0% with v 56.7% without goserelin; difference of 13.3%; 95% CI, -10.85 to 37.45; P = .284). All but one evaluable patient reported regular menses at 2 years after chemotherapy. Time to restoration of menstruation was 6.8 months (95% CI, 5.2 to 8.4) with goserelin and 6.1 months (95% CI, 5.3 to 6.8) without goserelin (P = .304). Chemotherapy resulted in a decreased ovarian reserve measured by inhibin B and anti-Müllerian hormone during follow-up, supporting the other findings. CONCLUSION Premenopausal patients with breast cancer receiving goserelin simultaneously with modern neoadjuvant chemotherapy did not experience statistically significantly less amenorrhea 6 months after end of chemotherapy compared with those receiving chemotherapy alone.

Detection and characterization of circulating tumor cells in blood of primary breast cancer patients by RT-PCR and comparison to status of bone marrow disseminated cells

IntroductionThe role of circulating tumor cells (CTCs) in blood of primary breast cancer patients is still under investigation. We evaluated the incidence of CTCs in blood, we evaluated the correlation between CTCs and disseminated tumor cells (DTCs) in the bone marrow (BM), and we characterized CTCs for the expression of HER2, the estrogen receptor (ER) and the progesterone receptor (PR).MethodsBlood of 431 patients with primary breast cancer were analyzed for EpCAM, MUC1 and HER2 transcripts with the AdnaTest BreastCancer™ (AdnaGen AG, Germany). Expression of the ER and PR was assessed in an additional RT-PCR. BM aspirates from 414 patients were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.ResultsDTCs were found in 107/414 patients (24%), CTCs were detected in 58/431 (13%) patients. DTCs were associated with PR status of the primary tumor (P = 0.04) and CTCs significantly correlated with nodal status (P = 0.04), ER (P = 0.05), and PR (P = 0.01). DTCs in the BM weakly correlated with CTCs (P = 0.05) in blood. Interestingly, the spread of CTCs was mostly found in triple-negative tumors (P = 0.01) and CTCs in general were mostly found to be triple-negative regardless of the ER, PR and HER2 status of the primary tumor.Conclusions(1) Due to the weak concordance between CTCs and DTCs the clinical relevance may be different. (2) The biology of the primary tumor seems to direct the spread of CTCs. (3) Since the expression profile between CTCs and the primary tumor differs, the consequence for the selection of adjuvant treatment has to be evaluated.

Epithelial Cells in Bone Marrow of Breast Cancer Patients at Time of Primary Surgery: Clinical Outcome During Long-Term Follow-Up

PURPOSE To evaluate the detection of epithelial cells in bone marrow of breast cancer patients as an indicator of metastatic disease. PATIENTS AND METHODS Between 1989 and 1994, bone marrow biopsies were performed on 393 breast cancer patients during primary surgery. Specimens were stained immunocytochemically for epithelial cells expressing cytokeratins or the epithelial membrane antigen. The long-term outcomes of these patients were analyzed in this study. RESULTS In 166 of 393 patients, epithelial cells were found in bone marrow (BM) aspirates. These patients were designated BM+. The rate of tumor recurrence or cancer-related death was significantly higher in BM+ patients than in BM- patients. Multivariate analysis using the Cox regression model revealed BM status as a prognostic parameter independent of tumor size and axillary lymph node status. However, tumor size and axillary lymph node status were clearly superior prognostic parameters. CONCLUSION Disseminated epithelial cells in BM are associated with poor clinical outcome in breast cancer patients. However, the presence of these cells is not a sufficient parameter to predict growing metastases in the majority of patients, suggesting that epithelial cells in the BM of breast cancer patients at the time of surgery have limited metastatic potential. The role of these cells needs to be further evaluated.