Volkmar Mueller

Determination of HER2 status using both serum HER2 levels and circulating tumor cells in patients with recurrent breast cancer whose primary tumor was HER2 negative or of unknown HER2 status

IntroductionAt the time when metastatic disease is identified, assessment of human epidermal growth factor receptor (HER)2 status might help to optimize treatment decisions if HER2 status was not determined at first diagnosis and if HER2 positivity has been acquired during disease progression. Within this context, determination of serum HER2 or evaluation of HER2 status in circulating tumor cells (CTCs) may be of clinical relevance because metastatic tissue may be difficult to obtain for analysis as a result of its localization. The aim of this study was therefore to determine the HER2 status in serum and corresponding CTCs in patients with metastatic breast cancer whose primary tumors were HER2 negative or of unknown HER2 status.MethodsBlood samples were obtained from 77 metastatic breast cancer patients with negative (n = 44) or unknown (n = 33) HER2 status. Serum HER2 was determined using a commercial HER2/neu ELISA kit. CTCs were detected by slide-based assay using immunomagnetic enrichment and characterized by phenotyping and genotyping. Alternatively, a commercial kit, based on RT-PCR, was used to detect and characterize CTCs.ResultsTwenty out of 77 patients with metastatic disease had elevated serum levels of HER2. Blood samples could be analyzed for the presence of CTCs in 67 patients. Eight out of 21 patients with detectable CTCs exhibited HER2 amplification. Twenty-three out of 77 patients were HER2 positive using at least one method. Concordance between HER2 status of CTCs and serum HER2 was observed in 15 of 21 patients (71%). In six patients conflicting results were obtained. Three patients with elevated serum HER2 status had HER2-negative CTCs, whereas three patients with HER2-positive CTCs had normal serum HER2 levels.ConclusionA subgroup of patients with initially negative or unknown HER2 status can have elevated serum HER2 levels and/or HER2-positive CTCs at the time of development of metastatic disease. Although only a small number of patients were studied, our observations are of clinical relevance because, currently, these patients do not have access to HER2-targeted therapy.

Carbonic anhydrase IX in tumor tissue and sera of patients with primary cervical cancer

BackgroundCarbonic anhydrase IX (CAIX) is a membranous expressed metalloenzyme involved in pH homeostasis and cell adhesion. The protein is overexpressed in a variety of tumors and potentially associated with negative outcome. This study was designed to investigate the prognostic role of CAIX in serum and tumor tissue of patients with primary cervical cancer.MethodsTumor samples of 221 consecutive patients with primary cervical cancer who underwent surgery between 1993 and 2008 were analyzed for CAIX expression by immunohistochemistry. Additionally, preoperative serum CAIX concentrations were determined by ELISA in a subset of patients. Correlation with intratumoral CAIX expression as well as clinicopathological factors and outcome was analyzed.ResultsCAIX expression was observed in 81.9% of the tumor specimens; 62.0% showed a moderate or strong staining intensity. Moderate/strong expression was associated with squamous histology (p = 0.024), advanced tumor stage (p = 0.001), greater invasion depth (p = 0.025), undifferentiated tumor grade (p < 0.001) and high preoperative SCC-Ag values (p = 0.042). Furthermore patients with moderate/strong intratumoral CAIX expression had a higher number of metastatic lymph nodes compared to those with none/weak intratumoral expression levels (p = 0.047) and there was a non-significant association between high intratumoral CAIX expression and shorter survival (p = 0.118). Preoperative serum concentrations of CAIX ranged between 23 and 499 pg/mL and did not correlate with intratumoral expression or other clinicopathological variables.ConclusionCAIX is associated with advanced tumor stages and lymph node metastases in cervical cancer, potentially representing a new target in this disease. In contrast to other epithelial cancers we could not observe a correlation between serum CAIX and its intratumoral expression.

TIMP-1 and VEGF-165 serum concentration during first-line therapy of ovarian cancer patients

BackgroundAngiogenesis appears to play an important role in ovarian cancer. Vascular endothelial growth factor (VEGF) has recently been implicated as a therapeutic target in ovarian cancer. The tissue inhibitor of metalloproteinase 1 (TIMP-1) is involved in tissue invasion and angiogenesis. The application of serum TIMP-1 and VEGF to monitor primary therapy and predict clinical outcome of patients with ovarian cancer is unclear.MethodsPatients with epithelial ovarian cancer who presented for primary surgery were included in this study. A total of 148 serum samples from 37 patients were analyzed. Samples were prospectively collected at 4 predefined time points: 1. before radical debulking surgery, 2. after surgery and before platinum/taxane based chemotherapy, 3. during chemotherapy, 4. after chemotherapy. Serum VEGF-165 and TIMP-1 as well as CA-125 were quantified by ELISA or ECLIA and correlation with response and long-term clinical outcome was analyzed.ResultsSerum levels of all markers changed substantially during first-line therapy. High CA-125 (p = 0.002), TIMP-1 (p = 0.007) and VEGF-165 (p = 0.02) after chemotherapy were associated with reduced overall survival. In addition, elevated CA-125 (p < 0.001) and VEGF-165 (p = 0.006) at this time point predicted poor progression-free survival. TIMP-1 and VEGF-165 were closely correlated at all time-points during therapy.ConclusionsTIMP-1 and VEGF serum levels changed significantly during first-line therapy of ovarian cancer patients and predicted prognosis. These findings support the role of angiogenesis in ovarian cancer progression and the use of antiangiogenic therapy.

Serum Carbonic Anhydrase IX and Its Prognostic Relevance in Vulvar Cancer

Introduction: Therapeutic options in advanced or recurrent vulvar cancer are limited. The identification of new prognostic factors and markers for therapy stratification is therefore highly desirable. Carbonic anhydrase IX (CAIX) is up-regulated in various solid tumors and a promising new target. We therefore determined CAIX serum concentration and its prognostic relevance in correlation to intratumoral CAIX expression in patients with primary vulvar cancer. Methods: Thirty-one serum samples of patients with primary vulvar cancer were prospectively collected before surgery and analyzed for CAIX by enzyme-linked immunosorbent assay. In addition, intratumoral CAIX expression was determined by immunohistochemistry and correlation with serum CAIX and clinicopathological factors, and outcome was analyzed. Results: Preoperative serum concentration of CAIX ranged between 56 and 879 pg/mL (median, 147 pg/mL; mean, 237.29) and was significantly higher in patients with high intratumoral expression (median, 269 pg/mL vs 126 pg/mL, P = 0.03). High serum CAIX was not associated with any of the analyzed clinicopathological parameters. However, disease-free survival was shorter in patients with high preoperative serum CAIX (above median; P = 0.012). By immunohistochemistry, 26% of the tumors showed a moderate or strong expression of CAIX, whereas 74% showed weak or no expression. High intratumoral expression of CAIX was also associated with unfavorable disease-free survival (P = 0.043). Conclusions: Carbonic anhydrase IX serum concentration is higher in patients with high intratumoral expression, and elevated preoperative serum values are associated with unfavorable prognosis. Serum CAIX might therefore be an easily assessable marker to stratify patients for adjuvant therapy and potentially monitor response. Carbonic anhydrase IX is differentially expressed in vulvar cancer and potentially associated with negative outcome.

Serum carbonic anhydrase IX during first-line therapy of ovarian cancer

OBJECTIVE Carbonic anhydrase IX (CAIX) is primarily involved in maintaining the extracellular pH. It is overexpressed in a variety of tumors including ovarian cancer. To evaluate the potential prognostic and predictive role of serum CAIX for therapy response in ovarian cancer, we analyzed longitudinal serum samples. METHODS One hundred forty-eight serum samples from 37 patients with primary epithelial ovarian cancer were analyzed. Samples were prospectively collected at 4 time points: (1) before radical surgery, (2) after surgery and before platinum/taxane chemotherapy, (3) during chemotherapy, and (4) after chemotherapy. Serum CAIX was quantified by ELISA and expression in tumor tissue was verified by immunohistochemistry. Correlation with response and clinical outcome as well as the tumor marker CA-125 was analyzed. RESULTS Serum concentration of CAIX ranged between 30 and 1687 pg/mL and showed no significant changes during first-line therapy (median level before and after surgery 204 and 198 pg/mL, during and after chemotherapy 175 and 181 pg/mL). There was no association between serum CAIX and progression-free or overall survival. CA-125 decreased significantly after surgery (median serum level before and after surgery 413 and 84 kU/L, p<0.001) and further during and after first-line chemotherapy (median serum levels 21 and 15 kU/L, p<0.001). No intermarker correlation was observed. CONCLUSIONS CAIX is upregulated in ovarian cancer and serum CAIX could be a marker to stratify patients for therapy response. However, CAIX serum levels did not change significantly during first-line therapy and were not prognostically relevant. Based on the findings of the current study, CAIX cannot be recommended for therapy monitoring in this context.

Systemic treatment of vulvar cancer

Squamous cell carcinoma of the vulva is a rare disease, accounting for approximately 5% of cancers of the female genital tract. Standard therapy for early-stage vulvar cancer mainly comprises of surgery of the vulva and groins. In locally advanced or metastatic vulvar cancer, neoadjuvant or definitive chemoradiation is often considered as an alternative treatment option. Given its rarity, the level of evidence for different treatment modalities is poor and few clinical trials have been performed on this disease. Therefore indication criteria for systemic treatment in advanced stage vulvar cancer vary widely among countries and institutions. This review focuses on the different systemic treatment options for patients with locally advanced, recurrent or metastatic vulvar cancer, and highlights the need for an international multicenter approach to identify the most effective therapeutic options.

A comparative study of tissue inhibitor of metalloproteinases-1 levels in plasma and tumour tissue from patients with primary breast cancer and in plasma from patients with metastatic breast cancer.

Objective: Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been investigated as a potential tumour marker in breast cancer. Here we investigated the correlation between TIMP-1 in tumour tissue and plasma to evaluate whether TIMP-1 in plasma is actually a surrogate marker for TIMP-1 in primary tumours. Furthermore, we assessed whether increased TIMP-1 levels in plasma could be indicative of tumour progression in patients with advanced breast cancer. Methods: Tumour tissue and preoperatively collected plasma samples from 96 primary breast cancer patients were included together with plasma samples from 46 patients with advanced disease. TIMP-1 levels were measured by ELISA. Results: TIMP-1 levels in plasma (median 81.5 ng/ml, range 41.9–174.9) and tumour tissue (median 25.4 ng/mg of total protein, range 0–110.2) from primary breast cancer patients were not correlated (r = 0.05, p = 0.6). Plasma levels of TIMP-1 in primary breast cancer patients were significantly lower than levels in patients with advanced disease (median 108.7 ng/ml, range 59.7–560.7; p < 0.0001). Conclusions: Our findings suggest that TIMP-1 release into the blood might be controlled by an active mechanism. They also point to plasma TIMP-1 as a potential marker for predicting tumour progression and for monitoring tumour burden in metastatic breast cancer patients.

Abstract PD04-06: Changes in Circulating Tumor and Endothelial Cells in Peripheral Blood of Patients Treated in the Neoadjuvant Chemotherapy Plus Targeted Treatment Breast Cancer Study “GeparQuinto”

Background: The GeparQuinto study is a phase III neoadjuvant therapy (NT) study exploring the integration of Trastuzumab and Lapatinib for HER2-positive patients and Bevacizumab and Everolimus (RAD001) for HER2-negative patients. Predictive factors for these new treatment options are required. Therefore, we investigated in a subanalysis the potential of circulating tumor cells (CTC) and circulating endothelial cells (CEC) in peripheral blood during the course of NT. Material and Methods: Whole blood was taken prior to start of NT, after 4 cycles of NT and after NT prior to surgery. CTC and CEC were prospectively measured with the CellSearch® system using 15 mL and 4 mL blood, respectively that was also applied for HER2 status determination of CTC. Study recruitment will finish approximately in July 2010. Results: From 419 patients currently included in this subanalysis (23% with HER2-positive primary tumor), 364 patients had a sample prior to NT, 268 patients after 4 cycles NT and 213 patients after completion of NT. Before therapy, ≥1 CTC/15mL were detected in 82/364 patients (22.5%, range 1-112; mean 8.01; median 2), after 4 cycles of NT in 37/268 patients (13.8%, range 1-170; mean 6.4; median 1) and before surgery in 22/210 patients (10.5%; range 1-7; mean 1.73; median 1). Five or more CTC/15mL were found at these three time points in 8%, 2.2% and 0.5%, respectively. Significant decreases in CTC incidence and CTC numbers per patient were observed during therapy from baseline to the 2nd analysis after 4 cycles of NT (p=0.0081), and these decreases further continued to the 3rd analysis after completing NT (P Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-06.

Prognostic relevance of ubiquitin C-terminal hydrolase L1 (UCH-L1) mRNA and protein expression in breast cancer patients

PurposeThe ubiquitin C-terminal hydrolase L1 (UCH-L1) belongs to the family of deubiquitinating enzymes. It is overexpressed in various tumour entities and associated with metastases formation in some solid tumours. However, only limited information about its role in breast cancer is available. The aim of this study was to examine the UCH-L1 expression in primary breast cancer and to determine its relevance as a potential prognostic marker.MethodsWe investigated both UCH-L1 mRNA expression in microarray data from 182 primary mammary carcinomas and UCH-L1 protein expression using a tissue microarray containing samples from 1,622 breast cancer patients.ResultsWith both methods, high UCH-L1 expression correlated significantly with negative oestrogen receptor and progesterone receptor status and advanced tumour stage. Moreover by Kaplan–Meier analysis, high UCH-L1 mRNA and protein expression correlated with a significantly shorter overall survival.ConclusionThe data of our study suggest that high levels of UCH-L1 expression indicate a more aggressive tumour behaviour and might represent a potential target in breast cancer treatment.

Abstract S2-03: Persistence of circulating tumor cells in high risk early breast cancer patients during follow-up care suggests poor prognosis – Results from the adjuvant SUCCESS A trial

Background: Recent data suggest that circulating tumor cells (CTCs) are of prognostic relevance in early as well as metastatic breast cancer (BC). While persisting CTCs immediately after chemotherapy are known to indicate poor prognosis, there is a lack of data regarding the prognostic role of CTCs assessed during long-term follow-up care. Hence the prognostic value of CTCs two years after chemotherapy was analyzed. Methods: The SUCCESS A trial is a randomized, open-label, 2x2 factorial design Phase III study in high-risk breast cancer patients (≥N0 or T2–T4 or grade 3 or age ≤ 35 or hormone-receptor negative). Patients were first randomized to adjuvant chemotherapy treatment with 3 cycles of epirubicin-fluorouracil-cyclophosphamide followed by either 3 cycles of docetaxel or 3 cycles of gemcitabine-docetaxel. In addition, patients were randomized to 2 vs. 5 years of zoledronate treatment. Presence of CTCs was assessed using the FDA-approved CellSearch System (Janssen Diagnostics, LLC). CTC positivity was defined as ≥ 1 CTC in 7.5 ml whole blood. To investigate if CTC status 2 years after chemotherapy is of prognostic relevance independent from CTC status before chemotherapy and to evaluate the prognostic relevance of changed CTC status, only patients with data on CTC status before and 2 years after chemotherapy were included. Patient outcomes in terms of overall survival (OS) and disease-free survival (DFS) were analyzed by univariate log-rank tests and multivariate Cox regressions adjusted for age, menopausal status, tumor stage, nodal stage, grading, histological type, hormone receptor status and HER2 status. Survival time was measured beginning with the date of follow-up CTC assessment two years after chemotherapy. Results: Data on CTC status before and 2 years after chemotherapy were available for 1103 (29.4 %) of 3754 randomized patients. The CTC status 2 years after chemotherapy was positive in 204 (18.5%) patients. The median follow-up time was 37 months. Multivariate Cox regressions including CTC status before chemotherapy showed significant independent prognostic role for CTC status 2 years after chemotherapy on OS (hazard ratio (HR) 3.95, 95% confidence interval (CI) 2.13 – 7.32, p Conclusion: The presence of CTCs two years after chemotherapy analyzed during routine breast cancer follow-up care was associated with decreased survival. According to these results, persisting CTCs during long term follow-up independently predict patients9 outcome and may serve as surveillance marker. Citation Format: Janni W, Rack B, Fasching P, Haeberle L, Friedl T, Tesch H, Lorenz R, Neugebauer J, Koch J, Jaeger B, Fehm T, Mueller V, Schneeweis A, Lichtenegger W, Beckmann M, Scholz C, Pantel K, Trapp E. Persistence of circulating tumor cells in high risk early breast cancer patients during follow-up care suggests poor prognosis – Results from the adjuvant SUCCESS A trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S2-03.