Christof Bernemann

Expression of AR-V7 in Circulating Tumour Cells Does Not Preclude Response to Next Generation Androgen Deprivation Therapy in Patients with Castration Resistant Prostate Cancer

The androgen receptor splice variant AR-V7 has recently been discussed as a predictive biomarker for nonresponse to next-generation androgen deprivation therapy (ADT) in patients with castration-resistant prostate cancer. However, we recently identified one patient showing a response from abiraterone despite expression of AR-V7 in his circulating tumour cells (CTC). Therefore, we precisely assessed the response in a cohort of 21 AR-V7 positive castration-resistant prostate cancer patients who had received therapy with abiraterone or enzalutamide. We detected a subgroup of six AR-V7 positive patients showing benefit from either abiraterone or enzalutamide. Their progression free survival was 26 d (censored) to 188 d. Four patients displayed a prostate-specific antigen decrease of >50%. When analysing prior therapies, we noticed that only one of the six patients had received next-generation ADT prior to CTC collection. As a result, we conclude that AR-V7 status in CTC cannot entirely predict nonresponse to next generation ADT and AR-V7-positive patients should not be systematically denied abiraterone or enzalutamide treatment, especially as effective alternative treatment options are still limited. PATIENT SUMMARY A subgroup of patients can benefit from abiraterone and/or enzalutamide despite detection of AR-V7 splice variants in their circulating tumour cells.

Influence of secreted frizzled receptor protein 1 (SFRP1) on neoadjuvant chemotherapy in triple negative breast cancer does not rely on WNT signaling

BackgroundTriple negative breast cancer (TNBC) is characterized by lack of expression of both estrogen and progesterone receptor as well as lack of overexpression or amplification of HER2. Despite an increased probability of response to chemotherapy, many patients resistant to current chemotherapy regimens suffer from a worse prognosis compared to other breast cancer subtypes. However, molecular determinants of response to chemotherapy specific to TNBC remain largely unknown. Thus, there is a high demand for biomarkers potentially stratifying triple negative breast cancer patients for neoadjuvant chemotherapies or alternative therapies.MethodsIn order to identify genes correlating with both the triple negative breast cancer subtype as well as response to neoadjuvant chemotherapy we employed publicly available gene expression profiles of patients, which had received neoadjuvant chemotherapy. Analysis of tissue microarrays as well as breast cancer cell lines revealed correlation to the triple negative breast cancer subtype. Subsequently, effects of siRNA-mediated knockdown on response to standard chemotherapeutic agents as well as radiation therapy were analyzed. Additionally, we evaluated the molecular mechanisms by which SFRP1 alters the carcinogenic properties of breast cancer cells.ResultsSFRP1 was identified as being significantly overexpressed in TNBC compared to other breast cancer subtypes. Additionally, SFRP1 expression is significantly correlated with an increased probability of positive response to neoadjuvant chemotherapy. Knockdown of SFRP1 in triple negative breast cancer cells renders the cells more resistant to standard chemotherapy. Moreover, tumorigenic properties of the cells are modified by knockdown, as shown by both migration or invasion capacity as well reduced apoptotic events. Surprisingly, we found that these effects do not rely on Wnt signaling. Furthermore, we show that pro-apoptotic as well as migratory pathways are differentially regulated after SFRP1 knockdown.ConclusionWe could firstly show that SFRP1 strongly correlates with the triple negative breast cancer subtype and secondly, that SFRP1 might be used as a marker stratifying patients to positively respond to neoadjuvant chemotherapy. The mechanisms by which tumor suppressor SFRP1 influences carcinogenic properties of cancer cells do not rely on Wnt signaling, thereby demonstrating the complexity of tumor associated signaling pathways.

Genomic Profiling in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is defined by a lack of hormone receptor expression as well as lack of over-expression/amplification of HER2/neu. Patients with TNBC show a significantly worse prognosis compared to patients with other breast cancer subtypes. TNBC, however, is a heterogeneous entity both with regard to clinical/pathological characteristics and molecular biology. This review summarizes the current data on TNBC with a particular focus on mutational and gene expression profiling and the association between TNBC and breast cancer stem cells.

Novel AR-V7 detection in whole blood samples in patients with prostate cancer: not as simple as it seems

performed with EpCAM (epithelial cell adhesion molecule) based approaches [12]. This might be even more interesting since more insights are gained into biology of non-EpCAM positive tumor cells—cells which might have shifted to a mesenchymal rather than an epithelial phenotype and thus, might escape an EpCAM enrichment strategy [13]. Moreover, focussing on AR-V7 mRNA from CTCs origin only presumably excludes significance of AR-V7 transcripts originated from extracellular vesicles, which might exhibit clinical biomarker validity as well [14]. Another advantage of the whole blood RNA extraction method is the missing necessity of special processing, presumably allowing higher reproducibility among different laboratories. However, results of these studies are highly diverse. Therefore, we would like to sound a note of caution. In the study of Liu et al. the authors separated the blood sample by using CD45-antibody coupled magnetic beads to discriminate between leukocytes which do not express AR-V7 (CD45+) as well as the remaining cell population in which AR-V7 positive tumor cells are supposed to be present (CD45−). However, the authors detected AR-V7 in CD45+ samples and explained this phenomenon by cross contamination due to non-specific binding of the CD45 antibody to tumor cells, aberrant expression of CD45 by tumor cells or leukocyte/CTC clusters. In the study by Todenhöfer et al. the authors used blood samples from pre-treated CRPC patients (n = 64) as well as healthy donors of similar sex and age comparable to prostate cancer patients, i.e. men ≥50 years of age which had serum PSA value <1 ng/ml as well as no clinical signs of prostate cancer [10]. The authors performed RNA extraction followed by cDNA synthesis and gene-specific pre-amplification. Subsequently, they used quantitative real time RT-PCR (qPCR) to detect AR-V7 as well as other prostate cancer marker genes, e.g. Dear Editor,

Reply from Authors re: Emmanuel S. Antonarakis, Howard I. Scher. Do Patients With AR-V7–Positive Prostate Cancer Benefit from Novel Hormonal Therapies? It All Depends on Definitions. Eur Urol 2017;71:4–6: Unsplicing a Conflict

In their Platinum Priority editorial, Drs. Antonarakis and Scher [1] — two of the leaders in the field — raise questions regarding the conclusions of our recent study [2]. The intent of our response is to clarify, because the editorial could be perceived as a conflict between two competing groups. Here, we want to empower readers to make their own informed decision about the clinical utility of being identified as AR-V7–positive. Our recent findings emphasize that a sizeable fraction of prostate cancer patients who are currently deemed unresponsive (by AR-V7 status) may actually benefit from an otherwise safe medication (arbiraterone or enzalutamide) [3,4]. Regarding the difference in assays, we provide a direct comparison in Figure 1A. After capture of circulating tumor cells and isolation of processed RNA using the same commercially available kit, both assays use a reverse transcription–polymerase chain reaction approach specific for AR-V7 transcripts. The basic principle of the TaqMan design is that it requires close proximity of the three oligonucleotides ( 50–150 bp), which increases specificity [5,6]. In brief, the Taq polymerase extends the primer and synthesizes the nascent strand whereby the 50–30 exonuclease activity degrades the probe (orange in Fig. 1A) that is subsequently detected using fluorescence [5]. We agree that there are currently limitations to computational search tools for templates of exonspanning oligonucleotides.

Reply from Authors re: Emmanuel S. Antonarakis, Howard I. Scher. Do Patients With AR-V7–Positive Prostate Cancer Benefit from Novel Hormonal Therapies? It All Depends on Definitions. Eur Urol. In press. http://dx.doi.org/10.1016/j.eururo.2016.08.038: Unsplicing a Conflict

In their Platinum Priority editorial, Drs. Antonarakis and Scher [1] — two of the leaders in the field — raise questions regarding the conclusions of our recent study [2]. The intent of our response is to clarify, because the editorial could be perceived as a conflict between two competing groups. Here, we want to empower readers to make their own informed decision about the clinical utility of being identified as AR-V7–positive. Our recent findings emphasize that a sizeable fraction of prostate cancer patients who are currently deemed unresponsive (by AR-V7 status) may actually benefit from an otherwise safe medication (arbiraterone or enzalutamide) [3,4]. Regarding the difference in assays, we provide a direct comparison in Figure 1A. After capture of circulating tumor cells and isolation of processed RNA using the same commercially available kit, both assays use a reverse transcription–polymerase chain reaction approach specific for AR-V7 transcripts. The basic principle of the TaqMan design is that it requires close proximity of the three oligonucleotides ( 50–150 bp), which increases specificity [5,6]. In brief, the Taq polymerase extends the primer and synthesizes the nascent strand whereby the 50–30 exonuclease activity degrades the probe (orange in Fig. 1A) that is subsequently detected using fluorescence [5]. We agree that there are currently limitations to computational search tools for templates of exonspanning oligonucleotides.

Abstract 5580: Wnt signaling as chemotherapy sensitivity marker of triple negative breast cancer (TNBC).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Triple negative breast cancer is characterized by an unfavorable prognosis. While patients do not benefit from endocrine or Her2 targeted therapies except chemotherapy, there is an increased response (pCR) to taxane-/anthracycline-containing therapy compared to other breast cancers. However, molecular determinants of chemotherapy specifically in TNBC remain largely unknown. Current research suggests an increased Wnt Pathway activity in TNBC while Wnt pathway activity has been shown to play an important role in breast cancer pathogenesis. Material and Methods: Differentially expressed genes in TN vs. non-TN breast cancers of patients treated with neoadjuvant taxane-/anthracycline-containing chemotherapy were identified and validated in an independent AffymetrixU133A gene chip dataset (Hess et al. 2006). Genes were tested for correlation with relapse-free survival, response to neoadjuvant chemotherapy and Ki67 expression. The effects of siRNA mediated knockdown of SFRP1 and treatment with small molecule XAV939 (specific inhibitor of Wnt signaling) were analyzed in TNBC cell lines MDA-MB 468, MDA-MB 231, HCC 1806 and luminal cell line MCF7 on chemo sensitivity. Results: Our list of over expressed genes in TNBC vs. non-TNBC contains several genes which are already discussed as potential markers for TNBC like αB-crystallin and transcription factor FOXC1. Furthermore, a few Wnt pathway associated genes (i.e. SFRP1, TCF7L1, TCF7L2, SOX10) occur in this list, which was validated in an independent dataset. While SFRP1 expression was not associated with relapse-free survival in TNBC, it was significantly correlated with a pCR. Importantly, no correlation to Ki67 expression could be demonstrated. mRNA knockdown of Wnt pathway inhibitor SFRP1 in two different TNBC cell lines (MDA-MB 468 and HCC 1806) resulted in an increased resistance to paclitaxel, doxorubicin and cisplatin. Treatment with Wnt inhibitor XAV939 resulted in TNBC cell lines MDA-MB 231, MDA-MB 468 and in luminal cell line MCF7 increased chemo sensitivity to paclitaxel and doxorubicin. Conclusion: We suggest Wnt Signaling and/or SFRP1 expression as a novel maker of chemotherapy sensitivity to taxane-, anthracycline and/or platinum-containing chemotherapy in TNBC in vitro that is independent of Ki67 expression. Citation Format: Carolin Huelsewig, Christof Bernemann, Christian Ruckert, Ludwig Kiesel, Lajos Pusztai, Cornelia Liedtke. Wnt signaling as chemotherapy sensitivity marker of triple negative breast cancer (TNBC). [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 5580. doi:10.1158/1538-7445.AM2013-5580

Performance comparison of two androgen receptor splice variant 7 (AR‐V7) detection methods

To compare the performance of two established androgen receptor splice variant 7 (AR‐V7) mRNA detection systems, as paradoxical responses to next‐generation androgen‐deprivation therapy in AR‐V7 mRNA‐positive circulating tumour cells (CTC) of patients with castration‐resistant prostate cancer (CRPC) could be related to false‐positive classification using detection systems with different sensitivities.

Overexpression of nuclear AR-V7 protein in primary prostate cancer is an independent negative prognostic marker in men with high-risk disease receiving adjuvant therapy

BACKGROUND Overexpression of the androgen receptor (AR) splice variant 7 (AR-V7) has recently been reported to be associated with resistance to antihormonal therapy. Herein, we address the question whether tumor cells with AR-V7 expression can be detected at the time of radical prostatectomy, that is, before long-term hormonal manipulation and castration resistance, and what the potential prognostic impact on the biochemical recurrence (BCR)-free survival may be. METHODS An anti-AR-V7 antibody was first validated in a training set of prostate cancer specimens by a comparison of AR-V7 protein to AR-V7 mRNA expression. We then analyzed nuclear AR-V7 protein expression in the primary tumors and lymph node metastases from 163 predominantly high-risk patients (cohort I) as well as the primary tumors from patients of a second, consecutive patient cohort (n = 238, cohort II) not selected for any clinicopathological features. Staining results were correlated to patient characteristics and BCR-free patient survival. RESULTS High nuclear AR-V7 protein expression was detected in approximately 30%-40% of patients in cohort I and II at the time of radical prostatectomy. High baseline expression of nuclear AR-V7 protein was associated with an unfavorable BCR-free survival in the high-risk patient cohort I but not in the unselected consecutive cohort II. Remarkably, AR-V7 was an independent negative prognostic factor in high-risk prostate cancer patients of cohort I who were selected to receive adjuvant treatment. CONCLUSIONS Prostate cancer cells with high nuclear AR-V7 protein expression can be detected in a substantial proportion of tumors at the time of radical prostatectomy. The presence of AR-V7-positive tumor cells is associated with an unfavorable prognosis for BCR-free survival in a high-risk patient cohort including a subgroup of patients selected to receive adjuvant therapy, in which AR-V7 was an independent negative prognosticator. Overexpression of nuclear AR-V7 protein hence identifies a subset of tumors with remarkably aggressive growth characteristics among clinically and histologically high-risk patients at the time of radical prostatectomy.

Abstract LB-101: The antiandrogen drug dutasteride sensitizes triple negative breast cancer cells to chemotherapy via HIF-1α / VEGF-signaling

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Triple negative breast cancer is characterized by a lack of expression of both estrogen and progesterone receptor as well as lack of amplification of HER2. Patients with triple negative breast cancer carry an unfavorable prognosis compared to other breast cancer subtypes given that endocrine or HER-2 targeted therapies are not effective, rendering chemotherapy the sole effective treatment option to date. In a previous work by Liedtke et al. ((Liedtke et al., Cancer Res 2009) we generated a list of genes, which showed higher expression in triple negative breast cancer and, in addition, are known to encode targets for known non-oncologic drugs. These gene products might represent targets for future therapies of triple negative breast cancer. Results: We could identify SRD5A1, which encodes the type-1 isoform of the steroid-5-alpha-reductase, showing a higher expression in triple negative breast cancer both in vivo and in vitro. Dutasteride is a dual blocker of both the type-1 and type-2 isoform of SRD5A1 and is indicated in the treatment of benign prostate hyperplasia. Treatment of triple negative breast cancer cell lines with dutasteride was associated with a dose-dependent decrease in cell viability, altered protein expression of VEGF and HIF-1α and increased chemosensitivity. Conclusion: Our results demonstrate that firstly, using differential gene expression analysis in clinically relevant breast cancer subtypes, potential new drug targets can be generated. Secondly, we identified the SRD5A1-corresponding anti androgenic drug dutasteride as a combinatorial therapeutic option besides standard chemotherapy in highly aggressive triple negative breast cancer. Citation Format: Marie-Kristin von Wahlde, Carolin Huelsewig, Christian Ruckert, Martin Gotte, Ludwig Kiesel, Cornelia Liedtke, Christof Bernemann. The antiandrogen drug dutasteride sensitizes triple negative breast cancer cells to chemotherapy via HIF-1α / VEGF-signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-101. doi:10.1158/1538-7445.AM2014-LB-101